INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY ENVIRONMENTAL HEALTH CRITERIA 68 HYDRAZINE This report contains the collective views of an international group of experts and does not necessarily represent the decisions or the stated policy of the United Nations Environment Programme, the International Labour Organisation, or the World Health Organization. Published under the joint sponsorship of the United Nations Environment Programme, the International Labour Organisation, and the World Health Organization World Health Orgnization Geneva, 1987 The International Programme on Chemical Safety (IPCS) is a joint venture of the United Nations Environment Programme, the International Labour Organisation, and the World Health Organization. The main objective of the IPCS is to carry out and disseminate evaluations of the effects of chemicals on human health and the quality of the environment. Supporting activities include the development of epidemiological, experimental laboratory, and risk-assessment methods that could produce internationally comparable results, and the development of manpower in the field of toxicology. Other activities carried out by the IPCS include the development of know-how for coping with chemical accidents, coordination of laboratory testing and epidemiological studies, and promotion of research on the mechanisms of the biological action of chemicals. ISBN 92 4 154268 3 The World Health Organization welcomes requests for permission to reproduce or translate its publications, in part or in full. Applications and enquiries should be addressed to the Office of Publications, World Health Organization, Geneva, Switzerland, which will be glad to provide the latest information on any changes made to the text, plans for new editions, and reprints and translations already available. (c) World Health Organization 1987 Publications of the World Health Organization enjoy copyright protection in accordance with the provisions of Protocol 2 of the Universal Copyright Convention. All rights reserved. The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the Secretariat of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. The mention of specific companies or of certain manufacturers' products does not imply that they are endorsed or recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters. CONTENTS ENVIRONMENTAL HEALTH CRITERIA FOR HYDRAZINE 1. SUMMARY 2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, ANALYTICAL METHODS 2.1. Identity 2.2. Physical and chemical properties 2.3. Analytical methods 3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE 3.1. Natural occurrence 3.2. Man-made sources 3.2.1. Industrial production 3.2.2. Methods of transport 3.2.3. Disposal of waste 3.3. Use pattern 4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION AND TRANSFORMATION 4.1. Transport and distribution between media 4.2. Abiotic degradation 4.3. Biodegradation 4.4. Interactions with soil 5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE 5.1. Environmental levels 5.2. General population exposure 5.3. Occupational exposure 5.4. Populations at special risk 6. KINETICS AND METABOLISM 6.1. Absorption and distribution 6.2. Metabolism and excretion 6.3. Reaction with body components 7. EFFECTS ON ORGANISMS IN THE ENVIRONMENT 7.1. Aquatic organisms 7.2. Microorganisms 7.3. Plants 8. EFFECTS ON EXPERIMENTAL ANIMALS 8.1. Single exposures 8.2. Short-term exposures 8.2.1. Inhalation exposure 8.2.2. Other routes of exposure 8.3. Biochemical effects and mechanisms of toxicity 8.3.1. Effects on lipid metabolism 8.3.2. Effects on carbohydrate and protein metabolism 8.3.3. Effects on mitochondrial oxidation 8.3.4. Effects on microsomal oxidation 8.3.5. Effects on the central nervous system 8.4. Reproduction, embryotoxIcity, and teratogenicity 8.5. Mutagenicity and related end-points 8.5.1. DNA damage 8.5.2. Mutation and chromosomal effects 8.5.3. Cell transformation 8.6. Carcinogenicity 8.6.1. Inhalation exposure 8.6.2. Oral exposure 9. EFFECTS ON MAN 9.1. Poisoning incidents 9.2. Occupational exposure 9.2.1. Inhalation exposure 9.2.2. Skin and eye irritation; sensitization 9.2.3. Mortality studies 10. EVALUATION OF HUMAN HEALTH RISKS AND EFFECTS ON THE ENVIRONMENT 10.1. Evaluation of human health risks 10.2. Evaluation of effects on the environment 11. RECOMMENDATIONS FOR FURTHER STUDIES 12. PREVIOUS EVALUATIONS BY INTERNATIONAL BODIES REFERENCES WHO TASK GROUP ON HYDRAZINE Members Dr B. Gilbert, CODETEC, University City, Campinas, Brazil Professor P. Grasso, Robens Institute, University of Surrey, Guildford, Surrey, United Kingdom Mr M. Greenberg, Environmental and Criteria Assessment Office, US Environmental Protection Agency MD-52, Research Triangle Park, North Carolina, USA Professor M. Ikeda, Department of Environmental Health, Tohoku University School of Medicine, Sendai, Japan (Chairman) Dr N.N. Litvinov, A.N. Sysin Institute of General and Community Hygiene, USSR Academy of Medical Science, Moscow, USSR (Vice-Chairman) Dr G.B. Maru, Carcinogenesis Division, Cancer Research Insti- tute, Tata Memorial Center, Parel, Bombay, India Professor M. Noweir, Occupational Health Research Centre, High Institute of Public Health, University of Alexandria, Alexandria, Egypt Dr E. Rauckman, Carcinogenesis and Toxicological Evaluation Branch, National Institute of Environmental Health Sciences, National Toxicology Program, Research Triangle Park, North Carolina, USA Professor D.J. Reed, Environmental Health Sciences Center, Oregon State University, Corvallis, Oregon, USA Dr E. Rosskamp, Institute for Water, Soil and Air Hygiene of the Federal Ministry of Health, Berlin (West) Dr S. Susten, Document Development Branch, Division of Stan- dards Development and Technology Transfer, National Institute for Occupational Safety and Health, Cincinnati, Ohio, USA (Rapporteur) Professor J.A. Timbrell, University of London, School of Pharmacy, Toxicology Unit, London, United Kingdom Observers Dr P. Schmidt (European Chemical Industry Ecology and Toxico- logy Centre), Bayer AG, Leverkusen-Bayerwerk, Federal Republic of Germany Dr D. Steinhoff (European Chemical Industry Ecology and Toxico- logy Centre), Bayer AG, Institute for Toxicology, Wuppertal, Federal Republic of Germany Secretariat Professor F. Valic, Andrija Stampar School of Public Health, University of Zagreb, Zagreb, Yugoslavia (Secretary) a Dr T. Ng, Office of Occupational Health, World Health Organ- ization, Geneva, Switzerland Ms F. Ouane, International Register of Potentially Toxic Chemicals, United Nations Environment Programme, Geneva, Switzerland Dr T. Vermeire, National Institute of Public Health and Environmental Hygiene, Bilthoven, Netherlands (Temporary Adviser) Mr J. Wilbourn, Unit of Carcinogen Identification and Evaluation, International Agency for Research on Cancer, Lyons, France ------------------------ a IPCS Consultant. NOTE TO READERS OF THE CRITERIA DOCUMENTS Every effort has been made to present information in the criteria documents as accurately as possible without unduly delaying their publication. In the interest of all users of the environmental health criteria documents, readers are kindly requested to communicate any errors that may have occurred to the Manager of the International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland, in order that they may be included in corrigenda, which will appear in subsequent volumes. * * * A detailed data profile and a legal file can be obtained from the International Register of Potentially Toxic Chemicals, Palais des Nations, 1211 Geneva 10, Switzerland (Telephone no. 988400 - 985850). ENVIRONMENTAL HEALTH CRITERIA FOR HYDRAZINE A WHO Task Group on Environmental Health Criteria for Hydrazine met in Geneva from 25 to 30 August, 1986. Professor F. Valic opened the meeting on behalf of the Director- General. The Task Group reviewed and revised the draft criteria document and made an evaluation of the health risks of exposure to hydrazine. The draft of this document were prepared by DR T. VERMEIRE of the National Institute of Public Health and Environmental Hygiene, Bilthoven, the Netherlands. The efforts of all who helped in the preparation and finalization of the document are gratefully acknowledged. * * * Partial financial support for the publication of this criteria document was kindly provided by the United States Department of Health and Human Services, through a contract from the National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA - a WHO Collaborating Centre for Environmental Health Effects. The United Kingdom Department of Health and Social Security generously supported the costs of printing. 1. SUMMARY Anhydrous hydrazine is a caustic, fuming, hygroscopic liquid at ordinary temperature and pressure. The odour perception threshold is 3 - 9 mg/m3. It decomposes on heating or when exposed to ultraviolet radiation to form ammonia, hydrogen, and nitrogen. This reaction may be explosive, especially when catalysed by certain metals and metal oxides. Spontaneous ignition can occur in contact with porous materials. Hydrazine hydrate, the principal compound produced, contains 64% by weight hydrazine. Hydrazine is basic and is a strong reducing agent. In 1981, the world production capacity of hydrazine was estimated to be in excess of 35 000 tonnes. Sensitive analytical methods have been developed for the determination of hydrazine in air, water, biota, food, drugs, and cigarette smoke. Minimum detection limits reported are 2 µg/m3 air (gas chromatography), 5 µg/litre water (colori- metry), 1 µg/litre blood, plasma, or urine (gas chromato- graphy/mass spectrometry), and 3000 µg/kg drug (gas chromato- graphy). Hydrazine is not known to occur naturally, except perhaps in the tobacco plant. Currently, the primary uses of hydrazine hydrate are as a raw material in the manufacture of agricultural chemicals, blowing agents, polymerization catalysts, and pharm- aceutical products, and as a corrosion inhibitor in boiler water. Both the hydrate and anhydrous hydrazine are used as propellant fuels. Emission factors for loss of hydrazine into the atmosphere, estimated for the Federal Republic of Germany, amounted to 0.06 - 0.08 kg/tonne of hydrazine produced and 0.02 - 0.03 kg/tonne of hydrazine during handling and further processing. Accidental discharges into air, water, and soil can result from bulk storage, handling, transport, and improper waste disposal. At production facilities, data show that concentrations of up to 0.35 mg/m3 and, occasionally, up to 1.18 mg/m3, can occur during production under normal conditions. During handling of the fuel, concentrations of up to 0.25 mg/m3 have been measured under normal conditions, exceptionally rising to 2.59 mg/m3. A level of 800 mg/m3 was measured at the site of a leak in an industrial plant. The general population may be exposed to hydrazine vapour via accidental discharge. Evaporation of hydrazine from a liquid spill can be sufficient to generate an atmospheric concentration as high as 4 mg/m3, 2 km downwind of the spill. Hydrazine is degraded rapidly in air through reactions with ozone, hydroxyl radicals, and nitrogen dioxide. In polluted air, the life-time of hydrazine is estimated to be of the order of minutes. In a clean atmosphere, the life-time will be approximately 1 h. In soil, aqueous hydrazine is adsorbed and decomposed on clay surfaces under aerobic conditions. The rate of degradation of hydrazine in water is highly dependent on factors such as pH, temperature, oxygen content, alkalinity, hardness, and the presence of organic material and metal ions. The compound is biodegradable by microorganisms in activated sludge. However, at concentrations above 1 mg/litre, it is also toxic for these microorganisms. The blue algae Microcystis aeruginosa was the most sensitive aquatic species tested with respect to hydrazine; the 10-day toxicity threshold was reported to be 0.00008 mg/litre. Fish species showed LC50 values of between 0.54 and 5.98 mg/litre. A test to assess damage to the embryo showed a lowest-observed- adverse-effect level of 0.1 mg/litre for the fathead minnow. In view of the low octanol/water partition coefficient of hydrazine and its ready degradation, it will not bioaccumulate. Hydrazine is toxic for plants and can inhibit germination. Hydrazine is absorbed rapidly through the skin or via other routes of exposure. It is rapidly distributed to, and eliminated from, most tissues. In mice and rats, part of the absorbed hydrazine is excreted unchanged, and part as labile conjugates or as acid-hydrolysable derivatives via the urine. When hydrazine is metabolized, a significant amount of nitrogen is produced, which is excreted via the lungs. In human beings, hydrazine is irritating to the skin, eyes, and respiratory tract. It is a strong skin sensitizer. In a number of cases of accidental exposure, severe adverse effects were observed, principally in the central nervous system, liver, and kidneys. In experimental animals, in addition to the above effects, common observations following single exposure include loss of body weight, anaemia, hypoglycaemia, fatty degeneration of the liver, and convulsions. Continuous exposure of mice, rats, monkeys, and dogs to levels of 0.26 and 1.3 mg/m3 for 6 months resulted in adverse effects in all species at 1.3 mg/m3 and in mice (fatty liver) and rats (decrease in growth), also at 0.26 mg/m3. When rats were treated with hydrazine in the drinking- water at 0.0003 - 0.3 mg/kg body weight per day, no adverse effects were observed at levels of 0.003 mg/kg or less. This is the only study in which a no-observed-adverse-effect level by the oral route in rats has been reported. No data are available for establishing a no-observed-adverse-effect level by the inhalation route. Data are lacking concerning the effects of hydrazine on the human embryo or fetus. In studies on rats and mice, hydrazine administered by injection, orally, or through inhalation pro- duced adverse effects on embryos and fetuses, when administered at doses that were toxic for the mother. The adverse effects observed in these studies included increased resorptions, reduced fetal weight, increased perinatal mortality, and increased incidences of litters and fetuses with abnormalities. The abnormalities observed were primarily supernumerary and fused ribs, delayed ossification, moderate hydronephrosis, and moderate dilation of brain ventricle. These abnormalities were considered to be minor by the authors. On the basis of these studies, it was concluded that, in the absence of human data, it is prudent to assume that hydrazine would have an adverse effect on the human embryo or fetus at levels near those producing toxic effects in the mothers. Such exposures may occur from accidental spillages. Hydrazine caused increased DNA damage and repair in vitro. No increased unscheduled DNA synthesis was observed in the germ cells of mice after exposure in vivo. Hydrazine induced indir- ect methylation of O6 and N7 of guanine in the liver DNA of rodents after in vivo exposure to toxic doses. It also induced gene mutations and chromosome aberrations in a variety of test systems including plants, phages, bacteria, fungi, Drosophila, and mammalian cells in vitro . However, in gene mutation assays using bacteria, there were variable responses following the addition of rat liver metabolic activation systems. Hydrazine was found to transform hamster and human cells in vitro . It did not induce chromosome aberrations, micronuclei, or dominant lethals in mice in vivo, but chromosomal aberrations were reported in rats in vivo . Hydrazine vapour induced nasal tumours, most of which were benign, in Fischer 344 rats, following inhalation exposure for 12 months to concentrations of 1.3 or 6.5 mg/m3 with subsequent observation for 18 months, and in Syrian golden hamsters exposed to 6.5 mg/m3 with subsequent observation for 12 months. Such effects were not seen in C57BL/6 mice exposed to concentrations of 0.06, 0.33, or 1.3 mg/m3 for 12 months followed by 15 months of observation, except for an increased incidence of lung adenomas of borderline significance at 1.3 mg/m3. In several limited gavage and drinking-water studies, hydrazine induced an increased incidence, in some cases dose-related, of multiple pulmonary tumours in various mouse strains and in Cb/Se rats. In CBA/Cb/Se and BALB/c/CB/Se mice, an increased incidence of hepatocarcinomas was also induced. A very low, but increased, incidence of hepatocarcinomas was observed in male Cb/Se rats. No tumours were observed in hamsters. On the basis of the carcinogenicity studies on experimental animals, there is evidence that hydrazine is an animal carcino- gen. Human data are inadequate. Hydrazine induced DNA damage in vitro, methylation of DNA guanine in vivo, and positive results in in vitro mutagenesis assays. Making an overall evaluation, hydrazine can be regarded as posing little hazard for the general population at normal ambient levels. However, in the work-place and under conditions of accidental exposure, hydrazine can present a significant health hazard. Human data are limited but show that hydrazine may affect the central nervous system, liver, and kidneys. In addition, it may produce skin and eye irritation and skin sensi- tization. The results of animal studies suggest that effects on human beings may also include embryotoxicity at levels near those producing toxic effects in the mothers and adverse effects on the respiratory system. On the basis of the evidence of carcinogenicity in animals and positive results in short-term tests, it would be prudent to consider hydrazine to be a possible human carcinogen and, therefore, the levels in the environment should be kept as low as feasible. It can also be concluded that hydrazine may present a hazard for the aquatic environment and plant life. Further studies are needed on: (a) dose-response in relation to DNA alkylation, damage to the nasal epithelium, and pulmonary effects; (b) skin sensitization, focusing on cross-reactivity with hydrazine derivatives; (c) dermal irritation-promotion; (d) dose-response in sensitive fish species in relation to diet; (e) metabolism with regard to effects on DNA; and (f) effects of continuous low-level exposure on reproduction in sensitive rodent species. 2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, ANALYTICAL METHODS 2.1. Identity Chemical formula: N2H4 Chemical structure: H H | | N - N | | H H Relative molecular mass: 32.05 Common name: hydrazine Common synonyms: diamide, diamine, anhydrous hydra- zine, hydrazine base Common trade names: Aerozine-50 (a 1:1 w/w fuel mixture (of mixtures) of anhydrous hydrazine and 1,1- dimethylhydrazine); Hydrazine hydrate (N2H4 H2O) (a 1:1 molar mixture of anhydrous hydrazine and water; Levoxin (a 15-64% aqueous solution); SCAV-OX (a 35-64% aqueous solution); Zerox (a 15-64% aqueous solution) CAS chemical name: hydrazine CAS registry number: 302-01-2 Conversion factors: 1 ppm = 1.31 mg/m3 at 25 °C and 101.3 kPa (760 mmHg) 1 mg/m3 = 0.76 ppm Hydrazine exposures are always expressed as exposure to free base N2H4. The actual compound used is given in parentheses (sections, 6, 8, 9). 2.2. Physical and Chemical Properties Anhydrous hydrazine is a caustic, fuming, highly polar, weakly basic, hygroscopic liquid at ordinary temperature and pressure. It is a combustible substance, burning with a blue flame. The pure compound decomposes on heating or when exposed to ultraviolet radiation to form ammonia, hydrogen, and nitro- gen. This reaction may be explosive, especially when catalysed by certain metals and metal oxides. Hydrazine can ignite spon- taneously in air, when in contact with porous materials. In aqueous solution, considerable hydrogen bonding takes place. Hydrazine and water form a constant boiling mixture that con- tains 68% by weight of hydrazine and boils at 120.5 °C. Hydra- zine and water also form the compound hydrazine monohydrate, which contains 64% hydrazine by weight. Hydrazine solutions in water have basic properties. Hydrazine is a powerful reducing agent. Autooxidation occurs in alkaline solutions and is strongly catalysed by metal ions, notably copper, yielding hydrogen peroxide as a by-product. Decomposition of aqueous hydrazine occurs in the presence of metal catalysts such as platinum or Raney nickel. Some physical and chemical properties of hydrazine and its hydrate are given in Table 1. 2.3. Analytical Methods A selection of analytical methods for the determination of hydrazine in air, water, biota, drugs, and smoke is presented in Table 2. A review of methods can be found in Schmidt (1984). Gas chromatographic methods are the most specific assays available providing that the chromatographic behaviour of hydrazine is improved by derivatization, for example, by reaction with p-dimethylaminobenzaldehyde or 2,4-pentanedione. Hydrazine can be determined simultaneously with its derivatives using these methods. Colorimetric and titrimetric methods are subject to interference, especially by hydrazine derivatives. Direct-reading papers or indicating tubes, based on these colorimetric methods, are available commercially with reported detection limits of 65 µg/m3 for tapes and 330 µg/m3 for tubes (US NIOSH, 1978; Schmidt, 1984). The instability of hydrazine can present a problem in aqueous samples. Usually, acidification of the samples with sulfuric acid will prevent degradation of hydrazine. Table 1. Some physical and chemical properties of hydrazine and its hydrate --------------------------------------------------------------------------- Property Anhydrous hydrazine Hydrazine hydrate (100% N2H4) (64% N2H4) --------------------------------------------------------------------------- Physical state liquid liquid Colour colourless colourless Odour ammoniacal and pungent ammoniacal and pungent Odour perception 3 - 9 mg/m3a 3 - 9 mg/m3 Melting point 2 °C -51.5 °C Boiling point 113.5 °C 120.1 °C (azeotrope) Flash point 38 °C (open cup) 75 °C (open cup) Flammable limits 1.8 - 100% 3.4 - 100% Vapour pressure 1.39 kPa (10.4 mmHg) 1 kPa (7.5 mmHg) at 20 °C at 20 °C Density 1008 g/litre 1032 g/litre at 20 °C at 20 °C Relative vapour density 1.1 log n-octanol/water -3.08 partition coefficient Solubility in water infinite infinite Surface tension 66.7 dyne/cm at 25 °C 74.2 dyne/cm at 25 °C ----------------------------------------------------------------------------- a From: Jacobson et al. (1955, 1958). Table 2. Sampling, preparation, analysis --------------------------------------------------------------------------------------------------------- Medium Sampling method/ Analytical method Detection Comments Reference pretreatment limit --------------------------------------------------------------------------------------------------------- Air trapping in dilute colorimetry after reac- 20 µg/m3 sample size 100 litre; US NIOSH hydrochloric acid tion with p-dimethyl- suitable for personal and (1977a) aminobenzaldehyde area monitoring; rec- ommended range is 589 - 3440 µg/m3 Air trapping on sulfuric gas chromatography with 2 µg/m3 sample size 96 litre; US NIOSH acid-coated silica- flame-ionization detec- suitable for personal and (1977b) gel; desorption tion after derivatiza- area monitoring; rec- with water tion with 2-furaldehyde ommended range is and extraction into 2 - 60 000 µg/m3 ethyl acetate Air trapping in chilled gas chromatography of 5 µg/m3 sample size 2 litre; Holtzclaw acetone acetone derivative with suitable for area et al. a nitrogen detector monitoring (1984) Water sample must be acidi- colorimetry after re- 5 µg/ recommended range is ASTM (1981); fied when not anal- action with p-dimethyl- litre 5 - 150 µg/litre Velte ysed immediately aminobenzaldehyde (1984) Water polarography after re- 50 µg/ Slonim & action with 5-nitro- litre Gisclard salicylaldehyde (1976) Water sample adjusted to gas chromatography with 100 µg/ recommended range is Dee (1971) pH 6 - 9 flame-ionization detec- litre 100 - 50 000 µg/litre tion after derivatiza- tion with 2,4-pentane- dione Urine, sample adjusted to gas chromatography with 400 µg/ p-bromobenzaldehyde Timbrell water pH 3 nitrogen detection after litre used as internal standard et al. derivatization with p- (1977) chlorobenzaldehyde and extraction with methylene chloride --------------------------------------------------------------------------------------------------------- Table 2. (contd.) --------------------------------------------------------------------------------------------------------- Medium Sampling method/ Analytical method Detection Comments Reference pretreatment limit --------------------------------------------------------------------------------------------------------- Blood blood pretreated colorimetry after re- 200 µg/ detectable range 200 - Reynolds & with trichloroacetic action with p-dimethyl- litre 2900 µg/litre; a serum Thomas acid to precipitate aminobenzaldehyde blank should be included (1965); protein Springer et al. Urine urine adjusted to pH 3 (1981) Blood fluorimetry after reac- 5 µg/ Lewalter tion with p-dimethyl- litre et al. aminobenzaldehyde (1984) Drugs sample dissolved gas chromatography with 3000 µg/kg the method was used in Matsui et in water and nitrogen detection after drug or the analysis of isoniazid al. (1983) centrifuged derivatization with ben- formul- and hydralazine zaldehyde and extraction ation into n-heptane Cigar- trapping in penta- gas chromatography with 0.002 µg/ the method was also used Liu et al. ette fluorobenzaldehyde electron-capture detec- 20 cigar- for the analysis of (1974) smoke in methanol tion after extraction ettes tobacco with ether and enrich- ment by thin-layer chroma- tography with elution by ether Plasma, gas chromatography/mass 1 µg/ 15 N -hydrazine used as Timbrell biolo- spectrometry after deriv- litre the internal standard et al. gical atization with pentafluoro- (1982); media benzaldehyde, adsorption Blair on silica, elution with et al. hexane (1985) --------------------------------------------------------------------------------------------------------- 3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE 3.1. Natural Occurrence The only natural occurrence of hydrazine reported was in the tobacco plant (Liu et al., 1974). Model system studies have indicated that nitrogenase-bound hydrazine may be an inter- mediate in biological nitrogen fixation (Jackson et al., 1968; Mitchell & Scarle, 1972; Thorneley et al., 1978). 3.2. Man-Made Sources Hydrazine can be released into the atmosphere during venting operations, storage, and transfer. In the Federal Republic of Germany, the emission factor for the production of hydrazine is estimated to be 0.06 - 0.08 kg/tonne. It is estimated that 0.02 - 0.03 kg of hydrazine is lost to the environment for each tonne of hydrazine subjected to handling and further processing (Brugger, 1983). Accidental discharge into water, air, and soil can result from bulk storage, handling, transport, and improper waste disposal. 3.2.1. Industrial productiona Most production methods are based on the ketazine process, a variation of the Raschig process, in which ammonia is oxidized by chlorine or chloramine in the presence of aliphatic ketones, usually acetone. The resulting ketazine is then hydrolysed to hydrazine. In a recent method, hydrogen peroxide is used to oxidize ammonia in the presence of a ketone. A production process of minor importance involves the reaction between urea and sodium hypochlorite (Schiessl, 1980; Schmidt, 1984). The world production capacity was estimated to be about 36 000 tonnes in 1981, not including countries with planned economies (Schmidt, 1984). In addition to hydrazine hydrate, a small amount of anhydrous hydrazine is produced. In 1964, domestic consumption in the USA was approximately 7000 tonnes (Raphaelian, 1966). In 1974, the total production in the USA was reported to be 17 000 tonnes (US NIOSH, 1978; Schmidt, 1984). A more recent estimate for the USA is a production capacity of 17 240 tonnes in 1979. In the same year, the production capacity was 6400 tonnes in the Federal Republic of Germany, 3200 tonnes in France, 6500 tonnes in Japan, and 1900 tonnes in the United Kingdom (Schiessl, 1980; Schmidt, 1984). 3.2.2. Methods of transport In 1978, it was reported that the US Department of Energy Management annually transported an average of 600 tonnes of hydrazine fuel and 900 tonnes of hydrazine-1,1-dimethylhydrazine fuel (Aerozine-50) by rail, road, and ship. These fuels were transported in aluminium tank cars, stainless steel tank trailers, or in stainless steel drums (Watje, 1978). Current international regulations require the transport of hydrazine hydrate and its aqueous solutions in metal containers with polyethylene liners, in plastic canisters, or in stainless steel containers. 3.2.3. Disposal of waste Hydrazine has been disposed of by dilution with water to form at least a 400 g/litre solution, followed by neutralization with dilute sulfuric acid and drainage into a sewer with abundant water (IRPTC, 1985). However, it should be noted that even very dilute solutions of 0.1 mg/litre can be toxic for aquatic life (section 7.1). Alternatively, hydrazine has been burnt in an open pit after the addition of a hydrocarbon solvent (IRPTC, 1985). A better procedure is to dilute with abundant water and then oxidize the diluted solution (to below 20 g/litre) with hydrogen peroxide, calcium hypochlorite, or sodium hypochlorite before draining into a sewer (NEPSS, 1975). Hydrazine vapour emissions can be controlled by scrubbing, using water as the scrubbing liquid, or by the direct flame of catalytic incineraton (Gordon & Lewandowski, 1980). Hydrazine sulfate, a commonly-used derivative, may be disposed of by incineration (IRPTC, 1985). 3.3. Use Pattern The first important use of hydrazine was as a rocket propellant. In 1964, 73% of the hydrazine consumed was used for this purpose in the USA. The remainder was mainly used as an intermediate in the synthesis of agricultural chemicals such as maleic hydrazine, blowing agents for plastics, drugs such as the antitubercular isoniazid and the antihypertensive hydralazine, and the solder fluxes, hydrazine bromide and hydrazine chloride. Aqeous hydrazine was in use at that time as a corrosion inhibitor in boiler water (Raphaelian, 1966). This use pattern has shifted towards a relatively greater use of hydrazine as a chemical intermediate. For the year 1977, it was estimated that only 5% of the world production of hydrazine was used as fuel, while 19% was used for boiler water treatment, 32% for the synthesis of agricultural chemicals, and 34% for the synthesis of blowing agents (Schiessl, 1980). Currently, the main use of hydrazine hydrate as a raw material is in the manufacture of agricultural chemicals (40%), blowing agents (33%), polymeriza- tion catalysts, and pharmaceutical products. Use as a corrosion inhibitor in boiler water (15%) continues, and there are appli- cations as a chemical reducing agent in the metal-plating, metal --------------------------------------- a All production capacities and consumption figures were calculated for anhydrous hydrazine, although actual production and consumption was of hydrazine hydrate or of more dilute aqueous mixtures. recovery, and photographic industries, and as an ignitor in explosives (Schiessl, 1980; Schmidt, 1984). Smaller amounts are used as a rocket propellant and emergency fuel (Pitts et al., 1980; Wald et al., 1984). A very small amount of anhydrous hydrazine is used as a monopropellant in space vehicles and satellites (Schmidt, 1984). 4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION, AND TRANSFORMATION 4.1. Transport and Distribution Between Media Pure hydrazine has a low vapour pressure and is highly soluble in water. Nevertheless, the evaporation rate from a liquid spill can be sufficient to generate an atmospheric concentration of 4 mg/m3, 2 km downwind under unfavourable meterological conditions. Dilution with large amounts of water reduces the evaporation rate significantly (MacNaughton et al., 1981). 4.2. Abiotic Degradation Alkaline solutions of hydrazine in water can be subject to autooxidation by dissolved oxygen. Hydrogen peroxide is an important intermediate (Audrieth & Ogg, 1951). In acidic solutions or in the absence of metal ions, notably copper, no appreciable degradation was observed in aerated distilled water (Gormley & Ford, 1973; MacNaughton et al., 1981). Gormely & Ford (1973) measured a rapid oxygen depletion from 0.02 to 0.1% alkaline solutions of hydrazine in the presence of copper ions and developed a mathematical expression relating the aqueous degradation rate of hydrazine to the concentrations of hydra- zine, copper ions, and oxygen at constant pH and temperature. Degradation rates for dilute hydrazine solutions were highly variable (Slonim & Gisclard, 1976; MacNaughton et al., 1981). Hydrazine was almost completely degraded within one day in muddy river water, sampled directly after a rain storm. However, in softened, filtered water at the same temperature and with the same dissolved oxygen content, but a lower initial pH, little degradation occurred within 4 days. The main factors that favour abiotic hydrazine degradation are the presence of certain metal ions, organic material, in general, and organic oxidizers, in particular, increased hardness, and high pH (Slonim & Gisclard, 1976). In air, hydrazine can be oxidized in a number of different ways. There is no information on how hydrazine in the atmo- sphere is degraded. The destruction of hydrazine by ozone and by hydroxyl radicals has been experimentally investigated (Hack et al., 1974; Harris et al., 1979, Pitts et al., 1980). The rate of hydroxyl radical reaction with hydrazine was found to be a linear function of the hydrazine concentration, independent of temperature and pressure. Assuming an average hydroxyl radical concentration of 106 radicals/cm3 for the lower troposphere, the half-life of hydrazine with respect to this radical is estimated to be about 3 h (Harris et al., 1979; Pitts et al., 1980). Assuming an average level of 80 µg ozone/m3 air (Singh et al., 1978), the lifetime of hydrazine with respect to ozone would be approximately 1 h. Nitrogen dioxide also reacts with hydrazine (Pitts et al., 1980; Tuazon et al., 1982). In a polluted atmo- sphere, the lifetime would be of the order of minutes (Pitts et al., 1980; Tuazon et al., 1982; Schmidt, 1984). Diazene, hydro- gen peroxide, and small amounts of nitrous oxide and ammonia have been identified as products of these reactions (Tuazon et al., 1982). 4.3. Biodegradation Hydrazine has been shown to be co-metabolized mainly to nitrogen gas by the nitrifying bacterium Nitrosomonas (Kane & Williamson, 1983). Preliminary studies have also indicated that hydrazine can be reduced to ammonia by nitrogenase isolated from the nitrogen-fixing bacterium Azobacter vinelandii (Davis, 1980). When hydrazine was added continously to a waste-water treatment plant, only concentrations below 1 mg/litre ensured complete absence of the compound from the effluent, without inhibiting treatment efficiency (Farmwald & MacNaughton, 1981). 4.4. Interactions with Soil Heck et al. (1963) found that dilute hydrazine was adsorbed in a column of soil or decomposed, if the soil contained a moderate amount of clay. Probably, decomposition on clay particles was more important than adsorption. Another factor influencing adsorption is the organic content of the soil (Isaacson et al., 1984). Dilute hydrazine leached completely through a column of sand (Heck et al., 1963). 5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE 5.1. Environmental Levels No data on environmental levels of hydrazine are available. This is because degradation is so rapid that measureable levels are not normally encountered (Schmidt, 1984). 5.2. General Population Exposure Hydrazine levels of 23 - 43 ng/cigarette were found in cigarette smoke by Liu et al. (1974). Traces of hydrazine have been found in samples of commercial maleic hydrazide, one of the uses of which is to inhibit sucker growth on tobacco. However, the amount of hydrazine measured in tobacco that had been treated with maleic hydrazide (12 - 51 ng/cigarette) was not very much different from that measured in untreated tobacco (14 - 22 ng/cigarette), indicating another source of hydrazine in tobacco (Liu et al., 1974). It has been reported that analyses of hydrazine-treated boiler water and the condensate of steam, which could have been in contact with food, confirmed the presence of hydrazine (US FDA, 1979). District heating water has been mentioned as an additional potential route of accidental human exposure. This water may contain a low concentration of hydrazine as a corrosion inhi- bitor. If this water is used to heat tap water and there is a leak inside the heat-exchanger at the user end, the tap water may be contaminated. Cases have been reported in which hot water became contaminated with levels of up to 10.72 mg/litre and drinking-water, up to 0.47 mg/litre (Bodenschatz, 1986). 5.3. Occupational Exposure Workers may be exposed to hydrazine at facilities producing hydrazine itself and those producing its salts and derivatives, at propulsion testing and rocket launching sites, and at locations where aircraft using hydrazine as an emergency fuel are assembled or refueled. Workers at plants using high- pressure boilers are potentially exposed to relatively dilute solutions of hydrazine. The number of workers and levels of exposure for sites in the USA are given in Table 3 (US NIOSH, 1986). Earlier and essentially similar data are reported by US NIOSH (1978) and Suggs et al. (1980). Workers normally exposed to anhydrous or concentrated hydrazine are provided with respiratory and skin protection. The difference between air levels outside and inside protective masks was illustrated by Cook et al. (1979) who found levels of 0.29 - 2.59 mg/m3 outside the masks at a rocket propellant- handling facility, and levels below the detection limit of 0.013 mg/m3 inside the masks. Much higher levels (800 mg/m3) were observed at the site of a leak (Suggs et al., 1980). Table 3. Occupational exposure to hydrazine in the USA --------------------------------------------------------------------- Site Approximate numbers Measured levels (mg/m3) exposed Normal Exceptional Normal Potential --------------------------------------------------------------------- A Rocket testing 10 100 0.01-0.02 0.14a B Production 100 800 < 0.13 0.13-0.26b C F-16 fighter 32 - 0.04-0.05 station D Rocket testing 10 300 no data no data E F-16 assembly 51 16 500 0.04-0.25 no data F Space-craft no data 14 000 no data no data launching G Derivative manu- < 25 no data < 0.13 ca. 0.13 facturer H Production no data 1100 < 0.35 < 1.18 --------------------------------------------------------------------- a Level measured during aeration of the waste-water holding pond. b Short-term samples during specific operations. 5.4. Populations at Special Risk Although not strictly an environmental occurrence of hydrazine, the presence of this compound in inadequately purified or aged medicinal drugs can expose a section of the human population to hydrazine. Two drugs that exemplify this exposure risk are isoniazid (Spinkova, 1971; Matsui et al., 1983; Blair et al., 1985) and hydralazine (Matsui et al., 1983; Blair et al., 1985). Hydrazine can also be formed during the metabolism of these drugs (Noda et al., 1978; Timbrell & Harland, 1979). Recently, hydrazine was detected in the plasma of 8 healthy male volunteers taking isoniazid for 2 weeks and in the plasma of 8 out of 14 hypertensive patients treated with, among others, hydralazine. After 2 weeks of dosing with isoniazid, the average level of acid-labile hydrazine in men of a slow acetylator phenotype was 2.7 times higher than in men of a rapid acetylator phenotype (Blair et al., 1985). 6. KINETICS AND METABOLISM 6.1. Absorption and Distribution When undiluted hydrazine (free base) was applied to the uncovered skin of dogs, the compound was detectable in plasma within 30 seconds. Maximum concentrations were reached 1 - 3 h after application. The concentration of hydrazine in blood increased with dose (Smith & Clark, 1972). An aqueous solution (700 g/litre) was administered dermally at a dose of 12 mg hydrazine (free base)/kg body weight to groups of 4 rabbits by fixing a piece of fibre glass screen to an area of shaved skin. The area was not covered, but corrections were made for evaporation loss. Hydrazine was rapidly detectable in serum and reached a maximum concentration of 10 mg/litre approximately 1 h after application. The half- life of disappearance from serum was 2.3 h. The apparent volume of distribution was determined to be 630 ml/kg body weight. It was calculated that 55% of the applied hydrazine was absorbed percutaneously (Andersen & Keller, 1984). Following intraperitoneal (ip) injection of 32 mg hydra- zine/kg body weight in rats or mice (free base and hydrazine sulfate, respectively), peak concentrations of hydrazine in blood of approximately 10 mg/litre occurred almost immediately, and then the hydrazine disappeared rapidly from the blood (Springer et al., 1981; Nelson & Gordon, 1982). A half-life of 44 min was observed in the blood of the rats during the first 3 h following exposure, followed by a slower phase with a half- life of 27 h (Springer et al., 1981). When rats were exposed to hydrazine vapour at concentrations of between 0 and 40 mg/m3 (free base), the blood concentration of hydrazine increased with exposure. After 6 h of exposure to a hydrazine concentration of 20 - 25 mg/m3, a blood concentration of 0.64 mg/litre was measured (Dost et al., 1981). Hydrazine was distributed rapidly in most tissues of mice and rats after ip or subcutaneous (sc) exposure. Elimination from these tissues also occurred rapidly (Dambrauskas & Cornish, 1964; Nelson & Gordon, 1982; Kaneo et al., 1984). For example, 24 h after the injection of 30 mg (ip to mice, hydrazine sulfate) or 60 mg (sc to rats, free base) hydrazine/kg body weight, less than 15% of the hydrazine present in the various tissues at 2 h was retained in these tissues at 18 h (Dambrauskas & Cornish, 1964; Nelson & Gordon, 1982). The highest levels of hydrazine were measured in the kidneys of both rats (Dambrauskas & Cornish, 1964; Kaneo et al., 1984) and mice (Nelson & Gordon, 1982), levels in other tissues being much lower. In rats, the greater part of sc-administered hydrazine, recoverable from tissues and blood (approximately 75%), was recovered from skin and muscles (Dambrauskas & Cornish, 1964). 6.2. Metabolism and Excretion A significant part of hydrazine (free base), administered sc, ip, or intravenously (iv), was excreted unchanged or as acetylhydrazine in the urine of dogs (McKennis et al., 1955) and rabbits (McKennis et al., 1959). After acid hydrolysis, small quantities of 1,2-diacetylhydrazine were identified in the urine of rabbits, but not in that of dogs (McKennis et al., 1959). Dambrauskas & Cornish (1964) administered 60 mg hydrazine (free base)/kg body weight, sc, to mice and rats and recovered 48.3 and 27.3% of the dose, respectively, in the urine, as hydrazine or acetylhydrazine, while almost none was left in the body. Approximately 14% of an ip dose of 5 mg/kg body weight (hydra- zine hydrate) was recovered in the urine of rats as hydrazine (10.3%), acetylhydrazine (2.2%), and diacetylhydrazine (1.2%) (Wright & Timbrell, 1978). In a similar study on rats, 30% of sc doses of 2.6 and 5.1 mg/kg body weight (hydrazine monohydro- chloride) was recovered in the urine as hydrazine (19%), acetylhydrazine (10%), and small quantities of diacetylhydrazine (Perry et al., 1981). When rats were injected sc with 9.9 mg hydrazine (hydrazine sulfate), the percentages of the dose recovered as acetylhydrazine and diacetylhydrazine were 2.9 and 2.5%, respectively, in 48-h urine samples (Kaneo et al., 1984). In spite of the variation in these results, it is clear that the major part of the hydrazine administered is not accounted for. Noda et al. (1985b) studied the effects of microsomal enzyme inducers on hydrazine disposition in rats. After iv administra- tion of 1.2 mg hydrazine (hydrazine sulfate)/kg body weight to rats, the plasma half-life was decreased from 1.69 to 1.2 h and 1.03 h by pretreatment of the animals with rifampicin and pheno- barbital, respectively. Urinary excretion of hydrazine was significantly decreased by phenobarbital pretreatment from 21.3% to 14.6% of the dose. In vitro studies revealed that both oxyhaemoglobin in erythrocytes and liver microsomal oxygenases can catalyse the oxidation of hydrazine to nitrogen (Clark et al., 1968; Springer et al., 1981; Nelson & Gordon, 1982). Diazene (C4H4N2) has been proposed as a probable intermediate (Nelson & Gordon, 1982). Rat liver cytochrome P-450 has been implicated in the formation of a free radical intermediate that must be a precursor of diazene during microsomal oxidation of hydrazine (Noda et al., 1985a). Tracer balance studies with 15N-labelled hydrazine have shown that rats and mice can convert hydrazine to nitrogen gas, which is excreted via the lungs. The results of these studies are summarized in Table 4. Table 4. Tracer-balance studies with 15N-labelled hydrazine ---------------------------------------------------------------------------------------- Species Route Dose Medium Metabolites % of Reference (mg/kg dose body weight) ---------------------------------------------------------------------------------------- Rat ip 32 expired air 15N-nitrogen 25 Springer (free et al. base) urine hydrazine, 30 (1981) acetylhydrazine; acid-hydrolysable 20 derivativesa; 15N-ammonia NDb bile hydrazine and acid- < 1 hydrolysable derivatives Mouse ipc 32 expired air, 15N-nitrogen 30 - 35 Nelson & (hydrazine Gordon sulfate) urine hydrazine or labile 15 (1982) conjugates; acid-hydrolysable 25 derivatives ---------------------------------------------------------------------------------------- a Excluding acetylhydrazine. b ND = not detected. c sc and iv injection resulted in minor differences in conversion. Nelson & Gordon (1982) reported the identification of some of the acid-hydrolysable derivatives, as shown in Fig. 1. They postulated that, when administered in vivo , hydrazine is rapidly oxidized to nitrogen gas by haem constituents, including oxy- haemoglobin and cytochrome P-450, and to a free radical of hydrazine leading to diazene, which spontaneously decomposes to nitrogen gas. After this initial release of nitrogen during the first 15 - 30 min, nitrogen release is much slower, and acetyl- ation and carbonyl group reactions are the dominant processes leading to urinary products (Fig. 1). About 20 - 30% of the hydrazine dose is expired as nitrogen gas in the first 2 h in both rats and mice (Springer et al., 1981; Nelson & Gordon, 1982). Approximately 25% of the hydrazine dose remains unaccounted for. Ammonia was found in the blood of dogs without significantly elevated blood-urea nitrogen (Floyd, 1980). Springer et al. (1981) did not find labelled ammonia in the urine of rats exposed to 15N-hydrazine (Table 4). Therefore, the ammonia in dogs was probably not derived from hydrazine (section 8.3.2), but was the result of an effect on metabolic pathways (Floyd, 1980; Springer et al., 1981). 6.3. Reaction With Body Components No adduct formation between hydrazine and DNA in vivo has been reported (Shank, 1983). Under non-physiological conditions, hydrazine can react with pyrimidine bases. These reactions were reviewed by Kimball (1977). Indirect methylation of guanines in DNA following hydrazine exposure has been demonstrated and will be discussed in section 8.5.1. 7. EFFECTS ON ORGANISMS IN THE ENVIRONMENT 7.1. Aquatic Organisms A summary of acute toxicity data is presented in table 5. It should be realized that the rate of decay of hydrazine in the aquatic environment depends on the conditions (section 4.2). When the concentration of hydrazine is not monitored during exposure, it should be noted that the toxic effects observed must have occurred at concentrations lower than the nominal ones, due to degradation of the compound. the increased toxicity of hydrazine for guppies in soft water at a pH just below 7 compared with the toxicity in hard water at a pH of approximately 8, as found by Slonim (1977), is at least partly explained by the increased persistence of hydrazine in soft and non-alkaline water. Taking into account the decay of hydrazine, increases in water temperature were found to enhance the toxicity of the compound for bluegills (hunt et al., 1981). Teratogenicity and toxicity screening were reported using the South African clawed toad (Greenhouse, 1976a,b), fathead minnow (Henderson et al., 1981), and rainbow trout (Henderson et al., 1983). Eggs of the South African clawed toad in the cleavage were exposed to hydrazine until hatching. Survival and development into normal larvae occurred at exposures below 10 mg/litre. At 10 mg/litre, 35% of the embryos were malformed at hatching. The effect was dose-related. Additional studies revealed that teratogenic effects appeared during neurulation (Greenhouse, 1976a). When larvae of the South African clawed toad were exposed to 1.0 mg hydrazine/litre water, for 120 h, all died in 24 - 48 h following exposure. No significant effects on survival and development were observed after exposure to 0.1 mg/litre, the next lower concentration tested (Greenhouse, 1976b). Eggs of fathead minnows at the mid-cleavage stage were exposed to hydrazine for 24 or 48 h. Embryos, exposed for 24 h, to 0.1 mg/litre, showed several defects, such as slightly or moderately subnormal heart beat, haemoglobin levels, body movement amount of eye pigment. From 1 mg/litre upwards, the responses were generally stronger; in addition, body pigment was absent and developmental arrest was observed. Embryos exposed to a hydrazine concentration of 1.0 mg/litre for 48 h appeared to have little chance of survival. Surviving embryos showed severe deformities and larvae exhibited reduced growth (Henderson et al., 1981). Table 5. Acute aquatic toxicity of hydrazine --------------------------------------------------------------------------------------------------------- Organism Tempera- pH Hardness Flow/ Parameter Concentration Reference ture (mg CaCO3 stata (mg/litre) (°C) /litre) --------------------------------------------------------------------------------------------------------- Bacteria Pseudomonas putida 20 stat 16-h TT 0.019 Bringmann & Kühn (1980)b Protozoa Entosiphon sulcatum 25 6.9 stat 72-h TT 0.93 Bringmann & Kühn (1981)b Uronema paraduczi 25 6.9 stat 22-h TT 0.24 Bringmann & Kühn (1981)b Chilomenas paramecium 20 6.9 stat 48-h TT 0.002 Bringmann & Kühn (1981)b Algae Green algae (Chlorella pyrenoidosa) 23 6.8 75 stat 48-h EC50 ca.10c Heck et al. 48-h EC100 ca.100c (1963)d Crustacea Water flea 20 8.0 stat 24-h EC50 2.3c Bringmann & (Daphnia pulex) Kühn (1982) 8.2 stat 24-h LC50 1.16c Heck et al. (1963) 20 7.1-7.2 stat 24-h LC50 0.51 and 1.01 Velte (1984)e 48-h LC50 0.16 and 0.19 Amphibia South African clawed toad (Xenopus laevis) eggs 8.2-8.7 stat LOEL 10c Greenhouse (1976a)f larvae stat 120-h LOEL 1.0c Greenhouse 120-h NOEL 0.1c (1976b)g Table 5. (contd.) --------------------------------------------------------------------------------------------------------- Organism Tempera- pH Hardness Flow/ Parameter Concentration Reference ture (mg CaCO3 stata (mg/litre) (°C) /litre) --------------------------------------------------------------------------------------------------------- Fish (fresh-water) Guppy (Lebistes reti- 22-24 7.8-8.2 400-500 stat 96-h LC50 3.85c Slonim (1977) culatus) 22-24 6.3-6.9 20-25 stat 96-h LC50 0.61c Fathead minnow (Pimephales promelas) eggs 21 7.0-7.5 150 flow 48-h LOEL 0.1 Henderson et 48-h NOEL 0.001 al. (1981)h adults 20 192 stat 96-h LC50 4.5c Cowen et al. (1981) adults 20 6.9 flow 96-h LC50 5.98 Velte (1984)e Bluegill sunfish 23-24 7.2-8.4 240-292 stat 96-h LC50 1.08 Fisher et al. (Lepomis macrochirus) (1980) 23-24 7.8-7.9 164 flow 96-h LOEL 0.43 Fisher et al. (1980)i 23-24 7.1-7.9 239 stat 96-h LOEL 0.1 Fisher et al. (1980)i 10 6.7-8.0 160-190 flow 96-h LC50 1.6 Hunt et al. 15.5 1.0 (1981) 21 1.2 Goldfish (Carassius 8.2-8.5 stat 48-h LC50 2.8c Heck et al. auratus) (1963)j 19 8.1-8.5 135 stat 24-h LC50 0.95 Proteau et al. (1979) Roach (Rutilus 19 8.1-8.5 135 stat 24-h LC50 0.54 Proteau et al. rutilus) (1979) Zebra fish (Brachydanio rerio) 5-day-old 26 7.8 110 stat 24-h LC50 0.75 Proteau et al. (1979) 3-month-old 20 7.6-8.2 110 stat 24-h LC50 2.03 Proteau et al. (1979) Table 5. (contd.) --------------------------------------------------------------------------------------------------------- Organism Tempera- pH Hardness Flow/ Parameter Concentration Reference ture (mg CaCO3 stata (mg/litre) (°C) /litre) --------------------------------------------------------------------------------------------------------- Green sunfish (Leptomis 8.2-8.5 stat 48-h LC50 5.1c Heck et al. (1963)j Large mouth bass 8.2-8.5 stat 48-h LC50 3.6c Heck et al. (1963)j Channel catfish 8.2-8.5 stat 48-h LC50 1.6c Heck et al. (1963)j Fish (marine species) Stickle back (Gaster- 14- 7.6-8.0 stat 96-h LC50 3.4c Harrah osteus aculeatus) 15.5 (1978)k --------------------------------------------------------------------------------------------------------- a Flow-through or static test. b TT = toxicity threshold. c No analysis for hydrazine during exposure was reported. d EC50 and EC100 for 50% and 100% growth inhibition, measured by reading optical density. e Soft water. f LOEL = lowest-observed-adverse-effect level for teratogenicity. Exposure of eggs in cleavage stage until hatching. g LOEL = lowest-observed-adverse-effect level for lethality. NOEL = no-observed-effect level for lethality and development. h LOEL and NOEL = lowest-and-no observed-adverse-effect level for toxicity and teratogenicity. i LOEL = lowest-observed-adverse-effect level for dorsal light response (at a non-lethal concentration). j Standard reference water. k Salinity, 1.8%. Henderson et al. (1983) also exposed eggs of rainbow trout (Salmo gairdneri) for 48 h, to hydrazine in continuous-flow tests at 11.5 - 12 °C, a pH of 7 - 7.5, and a water hardness of 15 mg calcium carbonate/litre. During exposures up to 5 mg/litre a dose-related increase was observed in the incidence of poorly fitting jaws, pronounced mouth gape, and absence of body movement. However, no effects were observed on mortality, heart beat, hatching rate, or hatching period. Reduced growth and abnormal development of larvae were observed at 1 and 5 mg/litre. Poor muscular development and poor bone growth were observed; the authors postulate that this is a result of calcium binding by hydrazine. 7.2. Microorganisms The toxicity of hydrazine for a number of species of bacteria, algae, and protozoa was measured by Bringmann (1975) & Bringmann & Kuhn (1980, 1981). Some very low toxicity thresholds were reported, for example, 0.005 mg/litre for a 7-day exposure for the algae Scenedesmus quadricauda and 0.00008 mg/litre for a 10-day exposure for the blue algae Microcystis aerogenosa. This is a very sensitive test. London et al. (1983) described the toxicity of hydrazine for the soil heterotroph Enterobacter cloacea. Hydrazine caused a concentration-dependent increase in the lag time of this organism. In a medium containing 10 mg/litre, this did not affect the growth rate and final growth yield after the lag period. At 100 mg/litre, the bacteria were not viable. Although relatively high concentrations of hydrazine in water have been recorded as inhibiting, either completely or partially, the activities of Nitrosomonas, Nitrobacter, and other bacteria in culture media (Yoshida & Alexander, 1964) in waste-water treatment (Tomlinson et al., 1966; Farmwald & MacNaughton, 1981; Kane & Williamson, 1983), the highest continuously maintained tolerable level in waste water is of the order of 1 mg/litre, as stated in section 4.3 (Farmwald & MacNaughton, 1981). 7.3. Plants Heck et al. (1963) studied the effects of hydrazine on the germination of seeds and seedling growth after application as a hydroponic culture contaminant and an air fumigant. Seeds of summer brush squash (Cucurbita pepo), peanut (Arachis hypogaea), and corn (Zea mays) were soaked for 48 h in water containing hydrazine at levels of between 0 and 1000 mg/litre. The temperature was 30 °C. At the highest concentra- tion, germination of peanut and corn seed was inhibited. Seed- ling growth was inhibited from 10 mg/litre for squash, 100 mg/litre for corn, and 1000 mg/litre for peanut. Sixteen-day-old seedlings of cotton (Gossypium hirsutum) in a hydroponic culture were exposed to hydrazine in the growth medium for 9 days at concentrations of between 0 and 1000 mg/litre and a temperature of between 22 and 29 °C. Plants died within 48 h of exposure to 300 mg/litre and within 30 h at the higher concentrations. Injury was first noted as foliar dehydration, without chlorosis or necrosis, after 9 days of exposure to 50 mg/litre or within 24 h of exposure to 300 mg/litre or more. Several plants were also exposed for 4 h to hydrazine vapour at concentrations of between 0 and 100 mg/m3 air. Species tested were soybean (Glycine max), cow pea (Vigna sinensis), pinto bean (Phaseolus vulgaris), cotton (Gossypium hirsutum), endive (Cichorium endivia), alfalfa (Medicago sativa), and squash (Cucurbita pepo). Wilting of leaves in all species was observed within 2 - 24 h of exposure to 30 mg/m3, followed by wilting of the whole plant. Death occurred in pinto beans and endive plants at this exposure level. At higher concentrations, plants of soybean and alfalfa also died. Six days after exposure, all surviving plants started to recover. 8. EFFECTS ON EXPERIMENTAL ANIMALS In section 8, all doses have been expressed in terms of the free base; however, the form of hydrazine used in each study has been indicated when possible. 8.1. Single Exposures The toxicology of hydrazine has been reviewed by Krop (1954), Clark et al. (1968), and US NIOSH (1978). LD50 values for rats and mice after oral, iv, and ip expo- sure were not significantly dependent on the route of exposure and ranged from 55 to 64 mg/kg body weight for rats and from 57 to 82 mg/kg body weight for mice (free base or hydrazine hydrate) (Witkin, 1956; O'Brien, 1964; Yaksctat, 1969; Azar et al., 1970). Oral LD50 values for hydrazine (hydrazine hydrate) in guinea-pigs and rabbits were 26 and 35 mg/kg body weight, respectively (Yaksctat, 1969). Dogs and rabbits appeared more sensitive, LD50 values following iv injection being 25 and 26 mg/kg body weight, respectively. The dermal LD50 for the rabbit was 93 mg hydrazine (free base)/kg body weight (Rothberg & Cope, 1956; Witkin, 1956). When doses between 96 and 481 mg/kg body weight (free base) were applied to the skin of dogs, 10 out of 25 animals died within the 6-h observation period; a dose-effect relationship was not observed (Smith & Clark, 1972). When rats and mice inhaled hydrazine (free base) for 4 h, the LC50s were 750 and 330 mg/m3, respectively (Jacobson et al., 1955). Death occurred quickly in both species. Lethal doses of hydrazine usually induced convulsions, excitement or inactivity, and other effects on the central nervous system. Rats and mice inhaling lethal concentrations of hydrazine (free base) also showed dyspnoea (Comstock et al., 1954; Jacobson et al., 1955; O'Brien, 1964). Spontaneous motor activity depression was noted in rats at ip doses of 39 and 52 mg/kg body weight (hydrazine sulfate) (Pradhan & Ziecheck, 1971). Dogs receiving a sublethal iv dose (free base) did not exhibit convulsions but showed increased neuromuscular activity, salivation, diarrhoea, vomiting, and hyperventilation (Wong, 1966). Few pathological changes have been reported following acute lethal doses. Some rats that died after inhaling hydrazine (free base), showed lung oedema with localized damage to the bronchial mucosa (Comstock et al., 1954). Wells (1908) observed fatty changes in the liver in 24 h following sublethal doses in many species. Fatty changes have also been observed in the kidneys of rats (free base or hydrazine hydrate) (Dominguez et al., 1962; Scales & Timbrell, 1982). In rats, accumulation of lipid, swelling of mitochondria, and an increased number of microbodies were observed in the liver and in the proximal tubules of the kidneys, 24 h after an ip dose of 20 or 30 mg/kg body weight (hydrazine hydrate). Similar changes were observed within 1 h, after doses of 40 or 60 mg/kg body weight (Scales & Timbrell, 1982). In addition, nuclear and nucleolar enlargement and hypertrophy of the smooth endoplasmic reticulum were observed in the liver of rats, 2 or more hours after a single intraperitoneal dose of 64 mg/kg body weight (hydrazine sulfate) (Ganote & Rosenthal, 1968). Studies on the mechanisms by which hydrazine causes these effects will be discussed later (section 8.3) together with other effects on the intermediary metabolism, notably hypoglycaemia and lipid peroxidation, and effects on the central nervous system, such as an increase in gamma- aminobutyrate levels in the brain. In dogs given a sublethal dose of hydrazine (free base), degeneration of the proximal convoluted tubules of the kidneys was accompanied by decreased creatinine clearance and increased glucose reabsorption by the tubules. The glomerular filtration rate was decreased because of decreased renal blood flow (Van Stee, 1965; Wong, 1966). In rhesus monkeys treated intravenously with 2.5 - 9.8 mg hydrazine (hydrazine sulfate)/kg, liver function tests were generally within normal limits up to 72 h after dosing. A dose of 80 mg/kg caused fatty liver, but no necrosis (Warren et al., 1984). 8.2. Short-Term Exposures 8.2.1. Inhalation exposure In a 6-month inhalation study, groups of 50 male Sprague Dawley rats, 40 female ICR mice, 8 male Beagle dogs, and 4 female rhesus monkeys were exposed to 0.26 or 1.3 mg hydrazine (free base)/m3 air, continuously, or 1.3 or 6.5 mg hydrazine/m3 air, for 6 h/day, 5 days/week. Exposure concentrations were monitored. Control groups contained the same number of animals. The exposure regimen was chosen in such a way that the weekly doses received by the continuously exposed groups were approxi- mately equal to the weekly doses of hydrazine received by the intermittently exposed groups. An increased mortality rate was only seen in mice at the 2 higher exposure levels. In rats, there was a dose-related decrease in body weight gain, while body weights of dogs were decreased at the 2 higher exposure levels. In dogs, the reduced weights were at least partly due to reduced food consumption. Weights of the exposed monkeys were comparable with those of controls. Organ weights were unaffected by the exposure in rats, dogs, and monkeys. Organ and body weights of mice were not recorded. Central nervous system effects observed included lethargy in mice at the 2 higher exposure levels, and tonic convulsions in 1 dog exposed continuously to 1.3 mg/m3. Monkeys exhibited slight eye irrita- tion at the 2 higher exposure levels. Fatty changes of the liver were observed in mice at all exposures and in dogs at the 2 higher exposure levels. The livers of exposed monkeys showed slight-to-moderate fat accumulation. However, this was also seen, to some extent, in control animals. Livers of rats were normal. Finally, dogs exhibited reduced red blood cell counts, haematocrit, and haemoglobin values at the 2 higher exposure levels, together with an increased resistance to osmotic haemo- lysis at all exposure levels. Haematological variables were normal in rats and monkeys and were not measured in mice. In dogs, the effects on the liver and the haematological variables appeared reversible (Haun & Kinkead, 1973). Decreases in red blood cell count and haematocrit were also observed in 20 female Swiss mice exposed to 130 mg hydrazine (free base)/m3 air for 1 h/day, 6 days/week, for 4 weeks. In this study, a decreased osmotic resistance to haemolysis was noted in exposed mice (Cier et al., 1967). Groups of 10 - 30 male Wistar rats were exposed to hydrazine (free base) at average concentrations of 6, 18, 26, 70, or 295 mg/m3 air, for 5 days/week, 6 h/day, over periods ranging from 5 to 40 days at the 3 highest exposure levels to approximately 6 months at the 2 lowest exposure levels. The control group consisted of 10 rats. Increased mortality was observed at all exposure levels but not in controls, and body weights were decreased at the 3 highest exposure levels. Rats became sluggish during the 6-month exposure, while at the 3 highest exposure levels, an initial restlessness was followed by a tendency to sleep. In some cases, pathological examination revealed lung oedema with local damage to the bronchial mucosa at the 3 highest exposure levels. Fatty livers were observed in many rats after 5 days of exposure at 295 mg/m3 (Comstock et al., 1954). 8.2.2. Other routes of exposure Groups of 25 male Sprague Dawley rats were dosed ip with 10 or 20 mg hydrazine (free base)/kg body weight, 5 times per week, for 5 weeks. The control group consisted of 15 rats. Mortality was increased at the dose of 20 mg/kg body weight; 10/25 rats died after 8 - 21 doses. Body weight was lost in a dose-related manner; 4.4 and 25.7% of the initial body weight was lost in 10 days in the 10 and 25 mg/kg groups, respectively. At 20 mg/kg body weight, rats also displayed weakness and lethargy, and 2 rats exhibited convulsions. Pathological examination revealed hyperaemia and oedema in the lungs of 4 rats and slight fatty vacuolation in the liver of 7 rats at 20 mg/kg body weight. At both doses, the haematocrit values were maximally decreased after 13 injections (Patrick & Back, 1965). Patrick & Back (1965) treated 12 rhesus monkeys ip with hydrazine (free base), 5 times per week. Six monkeys received 5 mg/kg body weight for 4 weeks; two of these monkeys received a further 8 doses of 10 mg/kg body weight, followed by 4 or 5 doses of 20 mg/kg body weight. A group of 6 monkeys received only 4 or 5 doses of 20 mg/kg body weight. The control group consisted of 10 monkeys. No monkey died, but all exposed monkeys showed decreased body weights. Lethargy, weakness, and vomiting were see in 7 of the 8 monkeys exposed to 20 mg/kg body weight, while tremors were seen in 1 of these monkeys. Fatty changes were observed in the liver, proximal tubules of the kidneys, heart, and skeletal muscles at 20 mg/kg body weight, and occasionally at 5 mg/kg body weight. Extensive periportal necrosis was found in the liver of one of the dosed monkeys. The level of bilirubin was increased and the serum was icteric. Haematocrit and haemoglobin values, measured at the lower dose only, dropped slightly, relative to control values (Patrick & Back, 1965). The pathological effects on the liver were also investigated microscopically, in groups of 20 - 29 male DDY mice and 10 male Wistar rats, after administration of 5, 10, or 20 mg hydrazine (free base)/kg powdered diet, for 3 - 10 days. No animals died. Animals of both species exhibited weakness. Megamitochondria or fatty vacuolation with moderately swollen mitochondria and focal proliferation of the smooth endoplasmic reticulum were induced in rats and mice at dose levels of 10 and 20 mg/kg feed. The induction of megamitochondria was a reversible process (Waka- bayashi et al., 1983). Noda et al. (1983) observed centrilobular hepatic necrosis in male rabbits dosed for 5 days with between 14.6 and 32.3 mg hydrazine (hydrazine monohydro-chloride)/kg body weight per day, iv. In other studies, hydrazine (hydrazine hydrate) was given orally in drinking-water to albino rats and guinea-pigs for 7 months at levels providing 0.3, 0.03, 0.003, and 0.0003 mg/kg body weight per day. At the two highest doses, adverse effects were observed in the CNS (changes in conditioned reflexes), liver (increased I131 excretion, changes in enzyme activity, protein dystrophia), and blood (symptoms of haemoloytic anaemia). The dose of 0.003 mg/kg body weight was reported to be the no-observed-adverse-effect level (Yaksctat, 1969). 8.3. Biochemical Effects and Mechanisms of Toxicity All of the studies described in this section were performed with doses considered to be toxic. 8.3.1. Effects on lipid metabolism Hydrazine caused a dose-dependent increase in hepatic triglyceride levels in rats, the threshold dose for a single ip injection being 10 - 20 mg/kg body weight. The maximal effect, an increase of 7 times the control value, was observed after a dose of 40 or 60 mg hydrazine hydrate/kg body weight. At these dose levels, the effect was measurable 4 h after injection (Timbrell et al., 1982). Other authors have also reported accumulation of triglycerides in the liver of rats exposed to single doses of hydrazine via injection routes (Amenta & Dominguez, 1965a; Clark et al., 1970; Lamb & Banks, 1979). Several mechanisms have been proposed: 1. Increased mobilization of free fatty acids from adipose tissue (particularly observed at low plasma- glucose levels) leading to an increased uptake of free fatty acids, followed by increased triglyceride synthesis in the liver (Trout, 1965, 1966; Clark et al., 1970). This mobilization of free fatty acids might be caused by the effects of hydrazine on the sympathetic nervous system and on levels of adrenal steroid hormone, possibly in response to the hypo- glycaemia induced by hydrazine (Amenta & Dominguez, 1965a). Cooling et al. (1979) found elevated concentrations of circulating corticosterone and decreased concentrations of insulin in the serum of rats exposed to hydrazine. Decreased blood-insulin levels were also measured in rats by Aleyassine & Lee (1971). 2. Increased synthesis of triglycerides caused by increased enzymatic activity of phosphatidate phospho- hydrolase (EC 184.108.40.206) in hepatocytes both in vivo and in vitro was reported by Lamb & Banks (1979). It has been suggested that this was a result of increased corticosterone levels (Cooling et al., 1979). In addition, Marshall et al. (1983) found increased fatty acid synthesis in the liver of rats after hydrazine administration. 3. Triglycerides could accumulate in hepatocytes as a result of a decreased secretion of lipoproteins from liver to plasma (Amenta & Dominguez, 1965a; Clark et al., 1970). This could be explained by a decreased lipid-binding capacity of lipoproteins following an observed alteration in the proportion of phospholipids and cholesterol (Clark et al., 1970) or by increased lipid peroxidation (Di Luzio et al., 1973; Kopylova et al., 1982). The protein moiety of lipoproteins could also be subject to change (section 8.3.2). 8.3.2. Effects on carbohydrate and protein metabolism Rats and dogs with starvation-induced depletion of glycogen stores showed rapidly declining plasma-glucose levels with a concomitant rise in lactate and pyruvate levels after exposure to single intravenous hydrazine doses of 64 (free base or hydra- zine sulfate) and 25 mg (free base)/kg body weight, respectively. In well-fed dogs, hyperglycaemia and depletion of glycogen stores preceded hypoglycaemia. Acidosis developed slowly as a result of an increased lactate-pyruvate ratio (Fortney, 1966; Fortney et al., 1967; Ray et al., 1970). It has been postulated that hydrazine inhibits glyconeo- genesis (Fortney, 1966; Fortney et al., 1967). This could occur via inhibition of pyridoxal phosphate-dependent aminotrans- ferases and decarboxylases. It has been shown that hydrazine interferes with pyridoxal phosphate synthesis in vitro (McCormick & Snell, 1961) and in vivo (Chatterjee & Sengupta, 1980) (section 8.3.5). Inhibition of transaminases would also explain the increase in free amino acids observed in the plasma, liver, brain, and muscle of rats (Cornish & Wilson, 1968; Banks, 1970) and in the plasma and urine of dogs (Korty & Coe, 1968). It could further explain several observations in rats, such as the depressed conversion of amino acids to carbon dioxide (Amenta & Dominguez, 1965b; Dost et al., 1971), the depressed incorporation of amino acids in plasma-glucose (Fortney et al., 1967), and the enhanced incorporation of amino-labelled acids in liver proteins, 24 h following hydrazine exposure (Banks, 1970). Inhibition of protein synthesis was also observed in rat livers up to 8.5 h after exposure (Lopez-Mendoza & Villa-Trevino, 1971). Hydrazine treatment of rats resulted in inhibited activity of specific aminotransferases and decarboxylases including: liver aspartate aminotransferase (EC 220.127.116.11) (Stein et al., 1971), brain gamma-aminobutyrate aminotransferase (EC 18.104.22.168) and glutamate decarboxylase (EC 22.214.171.124) (Medina, 1963; Perry et al., 1981), and liver ornithine 2-oxo-acid aminotransferase (EC 126.96.36.199) (Roberge et al., 1971). The activity of rat liver ornithine decarboxylase (EC 188.8.131.52) increased following hydrazine exposure (Springer et al., 1980). Inhibition of phosphoenolpyruvate carboxykinase (ATP) (EC 184.108.40.206), an enzyme involved in gluconeogenesis, was also measured in vitro . Hydrazine increased the levels of citrate, malate, and oxaloacetate in the rat liver (Ray et al., 1970). Hydrazine affects the urea cycle. Decreased specific activity of ornithine 2-oxo-acid aminotransferase, caused by administration of hydrazine to rats (Roberge et al., 1971), provoked an increase in ornithine in the liver (Banks, 1970), brain, and plasma (Perry et al., 1981). The concentrations of citrulline and urea in the liver, kidneys, brain, and blood were increased, as was the activity of argininosuccinate lyase (EC 220.127.116.11) (Roberge et al., 1971). 8.3.3. Effects on mitochondrial oxidation Swelling of hepatic mitochondria was observed in rats and mice following hydrazine administration (Ganote & Rosenthal, 1968; Scales & Timbrell, 1982; Wakabayashi et al., 1983) (sections 8.1 and 8.2.2). in vitro studies on the effects of high concentrations of hydrazine on the functional status of mitochondria showed decreased oxidation of keto-acids (Von Krulik, 1966). Oxidative phosphorylation measured as the P/O ratio was either not affected or decreased independently of the hydrazine concentration (Von Krulik, 1966; Fortney et al., 1967). Inhibition of beef heart cytochrome a by hydrazine was also reported (Takemori et al., 1960). After administration of a single intraperitoneal dose of hydrazine to rats, slightly stimulated mitochondrial oxidation of succinate and glutamate was observed with a slight increase in P/O ratio, respiration control rate, and phosphorylation rate. ATPase (EC 18.104.22.168) activity was not affected (Higgins & Banks, 1971). When mice received hydrazine (free base) in the diet (10%) for 3 days and rats received hydrazine in the diet (20%) for 8 days, oxidation of succinate and glutamate, coupling efficiency, P/O ratio, and the activities of ATPase (EC 22.214.171.124) and cytochrome c oxidase (EC 126.96.36.199) were slightly decreased in liver megamitochondria, while the activity of monoamine oxidase (EC 188.8.131.52) was moderately decreased (Wakabayashi et al., 1983). 8.3.4. Effects on microsomal oxidation Proliferation of the smooth endoplasmic reticulum was observed in the liver of rats and mice following hydrazine administration (Ganote & Rosenthal, 1968; Wakabayashi et al., 1983) (sections 8.1 and 8.2.2). A single dose of 55 mg hydrazine (free base)/kg body weight in rats decreased the hepatic cytochrome P-450 content (Gorshtein & Kopylova, 1983). Rats exposed for 4 days to 12 mg hydrazine (hydrazine sulfate)/kg body weight per day did not show any effect on cytochrome P-450 levels in the microsomal fraction of the liver, but a slightly decreased level of cytochrome b 5, inhibition of benzopyrene hydroxylase (EC 184.108.40.206), and increased parahydroxylation of aniline (Akin & Norred, 1978). 8.3.5. Effects on the central nervous system The relationship between the effects of hydrazine on the central nervous system, especially the occurrence of convul- sions, and changes in levels of gamma-aminobutyric acid, an inhibitory neurotransmitter, in the brain of rats and mice has been investigated. An increase in the level of gamma-amino- butyric acid was observed in the whole brain of rats after: (a) a single intraperitoneal dose of 51 mg hydrazine (free base)/kg body weight (Medina, 1963); (b) a single intravenous dose of 5 mg hydrazine (hydrazine sulfate)/kg body weight (Matsuyama et al., 1983); or (c) after a daily subcutaneous dose of 2.6 mg hydrazine (hydrazine monohydrochloride)/kg body weight over 109 days (Perry et al., 1981). In the whole brain of mice, an increase was observed following a single intramuscular dose of 54 mg hydrazine (free base)/kg body weight (Wood et al., 1980). The changes in the concentration of this amino acid are caused by inhibition of pyridoxal phosphate requiring gamma- aminobutyrate aminotransferase (EC 220.127.116.11) and glutamate decarboxylase (EC 18.104.22.168) (Medina, 1963; Perry et al., 1981). Hydrazine treatment of rats also caused a general amino acid imbalance in the brain (Perry et al., 1981). A relationship was suggested between the excitable state of the brain and the gamma-aminobutyric acid contents of nerve endings (decreased) rather than the whole brain contents of gamma-aminobutyric acid (increased) (Wood et al., 1980; Geddes & Wood, 1984). 8.4. Reproduction, Embryotoxicity, and Teratogenicity When groups of 26 Wistar rats were exposed to 0 or 8 mg hydrazine (hydrazine monohydrochloride)/kg body weight per day, sc, from day 11 to day 20 of gestation, the exposed dams showed a 20% decrease in body weight and 2 dams died. Reduced number of viable fetuses were found in 9 dams killed on day 21 of gestation (63/172 versus 142/179 in controls), while the number of implants per litter was not affected. The fetuses had reduced weights and appeared pale and oedematous, but did not exhibit any major malformations. In the rats allowed to deliver (12), perinatal mortality was 100% in treated rats and 20% in controls (Lee & Aleyassine, 1970). These results agree with those of another study in which groups of 6 - 27 Fisher 344 rats received 0, 2.5, 5, or 10 mg hydrazine (free base)/kg body weight per day, ip, from day 6 to day 15 of gestation. Body weight gains in dams were decreased and the number of resorptions per dam were increased in a dose- related manner. The differences between treated and control animals were statistically significant at doses of 5 or 10 mg/kg, but not at 2.5 mg/kg for both variables. The numbers of implants per dam and fetal weights were not affected at any dose. The incidence of litters or fetuses with abnormalities was not significantly increased at any dose; however, at 10 mg/kg, only one out of the 6 females produced a viable litter, and only 6 fetuses were examined. In a subsequent study, 27 rats were untreated and 11 rats were treated with 10 mg hydrazine/kg per day, during what appeared to be the most susceptible period of gestation (days 7 - 9). The incidence of litters or fetuses with abnormalities in the 10 mg/kg group (6 litters out of 8; 8 fetuses out of 16) was significantly higher than that in the control group of the preceeding study (8 litters out of 27; 11 fetuses out of 181). The abnormalities observed were mainly supernumerary and fused ribs, delayed ossification, moderate hydronephrosis, and moderate dilation of brain ventricles (Keller et al., 1982). Subtle postnatal changes were reported in the offspring of 24 female Syrian golden hamsters exposed orally to 0 or 170 mg hydrazine (hydrazine hydrate)/kg body weight on the 12th day of gestation. The pups of exposed dams did not exhibit cleft palate formation, but showed effects on the development of intestinal brush border enzymes. No other end-points were investigated (Schiller et al., 1979). Groups of ICR mice were treated ip with 0, 4, 12, 20, 30, or 40 mg hydrazine (free base)/kg body weight per day, from day 6 to day 9 of gestation. Some dams administered the highest dose died. Body weights were reduced at doses of 12 mg/kg body weight or more. While at the lower doses the number of resorptions per litter was unchanged compared with controls, embryotoxicity was evident at 30 and 40 mg/kg body weight. At 12 and 20 mg/kg body weight, 17-day-old fetuses showed reduced weights, and there was a dose-related increased incidence of litters with abnor- malities, mainly exencephaly, hydronephrosis, and supernumerary ribs (Lyng et al., 1980). Savchenkov & Samoilova (1984) studied the adverse effects on reproductive function (fertility of females, numbers of newborn, and resorption of embryos) of female and male albino rats exposed by gavage to hydrazine (hydrazine nitrate) at a dose of 13 mg/kg body weight, once a day, for 30 days prior to mating. The development of the surviving litters did not differ from that of controls. Albino rats (10/group and 20 controls) of both sexes were exposed to hydrazine (free base) (99.5% purity) in the drinking- water at concentrations of 0.82, 0.018, or 0.002 mg/litre (0.016 mg/kg, 0.0014 mg/kg, or 0.00016 mg/kg, respectively, nominal dose, assuming water consumption 20 ml per day and animal weights of 250 g). The duration of the study was 6 months. The number of animals studied and the scheme and time of mating were not reported. The female rats exposed to the highest concen- trations had fewer live embryos and more resorptions, as well as pre- and post-implantation deaths, than the controls. No effects were observed in animals administered 0.002 mg/litre. Developmental abnormalities were not reported in any of the 293 embryos from all exposed animals. Destruction of gonadal epithelium was observed in male rats after 6 months of oral exposure to hydrazine at concentrations of 0.82 and 0.018 mg/litre (Duamin et al., 1984). In the same study, albino rats were exposed to hydrazine (free base) at concentrations of 0.85, 0.13, or 0.01 mg/m3 (0.10 mg/kg body weight, 0.016 mg/kg, or 0.0012 mg/kg, respectively, nominal dose, assuming inhalation of 6 litres air per h and animal weights of 200 g), for 5 h per day, 5 days/week, for 4 months. At the two highest concentra- tions, embryotoxic effects of the same severity were seen as when hydrazine was administered orally at the two highest doses. No abnormalities were observed among 315 embryos. No gonado- toxic effects occurred in male rats under the conditions of the study (Duamin et al., 1984). Additional information relating to reproductive end-points is presented in section 8.5.1 (Sotomayer et al., 1982). 8.5. Mutagenicity and Related End-Points 8.5.1. DNA damage Hydrazine was found to react with pyrimidine bases under non-physiological conditions. These reactions were reviewed by Kimball (1977). No hydrazine-DNA adducts have been reported to be formed in vivo. A single oral or intraperitoneal dose of hydrazine (free base or hydrazine sulfate) administered to rats, mice, guinea-pigs, and hamsters resulted in the rapid formation of N7-methylguanine and O6-methylguanine in liver DNA, which was not detected in controls (Becker et al., 1981; Quintero-Ruiz et al., 1981; Bosan & Shank, 1983; Shank, 1983). Methylation was detectable at toxic doses and a sharp increase in the methyl- ation of guanine in liver-DNA was observed at oral doses exceeding 60 mg/kg body weight in rats and 45 mg/kg body weight in hamsters. In both species, maximum methylation approximated 80 N7-methylations and 7 O6-methylations per 100 000 residues of guanine, 6 h after oral exposure to 90 mg hydrazine/kg body weight. Elimination of 7-methylguanine from DNA began about 24 h after exposure with a half-life of 40 - 50 h. Elimination of O6- methylguanine from DNA began about 24 h after exposure in rats and about 50 h after exposure in hamsters, with a half-life of 13 h and 17 h, respectively (Becker et al., 1981; Bosan & Shank, 1983). The source of the methyl group was S -adenosyl-methionine (Becker et al., 1981; Quintero-Ruiz et al., 1981). Single-strand breaks were detected by the alkaline elution assay in rat liver cells exposed in vitro (Sina et al., 1983), and in the liver and lung cells of mice injected ip with 50 or 100 mg hydrazine (hydrazine hydrate)/kg body weight (Parodi et al., 1981). Hydrazine was reported to induce lambda phage in Escherichia coli (Heinemann, 1971); however, no such induction was found by Thomson (1981), nor did hydrazine induce HPlcl phage in Haemo- philus influenzae (Balganesh & Setlow, 1984). Repair of DNA lesions induced by hydrazine was observed in vitro. In the International Collaborative Programme for the Evaluation of Short-Term Tests for Carcinogenicity, 5 bacterial DNA repair tests, using Bacillus subtilis or E. coli, were all found to produce weak to medium positive responses. Only one assay required rat liver microsomal fraction. The other positive responses were reduced in magnitude by microsomal fraction (Ashby & Kilbey, 1981). Increased unscheduled DNA synthesis was observed in 1 out of 2 tests using human fibro-blasts (Agrelo & Amos, 1981; Robinson & Mitchell, 1981). No increase in unscheduled DNA synthesis was observed in the germ cells of mice, 16 days after a 5-day exposure to doses of hydrazine (hydrazine dihydrochloride) of up to 120 mg/kg body weight per day (Sotomayer et al., 1982). 8.5.2. Mutation and chromosomal effects The results of tests for gene mutations and chromosome damage induced by hydrazine or its salts are summarized in Table 6. Hydrazine induces gene mutations and/or chromosome aberrations in a variety of test systems including plants, phage phi 80, bacteria, fungi, Drosophila melanogaster, and mammalian cells in vitro . In a few cases, microsomal activation was an absolute requirement for a positive effect (Gupta & Goldstein, 1981; Perry & Thomson, 1981). In most cases, an effect could be observed, both with and without microsomal activation, with a stronger effect in some tests with activation and in other tests without activation. These inconsistencies were evaluated by Ashby (1981). Some negative results in the forward mutation tests with mammalian cells invitro can be traced back to the high locus specificity of hydrazine also observed in plants. Duamin et al. (1984) observed increased chromosomal aberrations in bone marrow cells (4.12 ± 0.65% versus 2.48 ± 0.46% in controls) of albino rats (N = 10) exposed to hydrazine (free base) at 0.85 mg/m3, 5 h per day, 5 days/week, for 4 months. No increased incidence of nuclear aberrations, micronuclei, dominant lethals, and sperm-head abnormalities were observed in hydrazine-treated mice (free base or hydrazine sulfate). Table 6. Tests for gene mutations and chromosome damage induced by hydrazine or its salts --------------------------------------------------------------------------------------------------------- Test description System description Result Reference Organism Species/strain/cell type --------------------------------------------------------------------------------------------------------- Forward mutations transforming Bacillus subtilis + Freese et al. (1967)a G DNA + Bresler et al. (1968)a E Forward mutations plant tomato + Jain et al. (1968)b + Chandra Sekhar & Reddy (1971)b N rice + Reddy & Reddy (1972)b barley + Kak & Kaul (1975)b E wheat + Khamankar & Jain (1978)b broad bean + Vishnoi & Gupta (1980)b Reverse mutations virus phage phi 80 + Chu et al. (1973)c M Reverse mutations bacteria Salmonella typhimurium TA 1530 + Rosenkranz & Poirier (1979);Tosk et al. (1979) U Salmonella typhimurium TA 1535 + Purchase et al. (1978); Herbold (1978); T Rosenkranz & Poirier (1979); Bridges et al. (1981);d,e; A Parodi et al. (1981); Rogan et al. (1982); T Braun et al. (1984); De Flora et al. (1984) I Salmonella typhimurium TA 1537 - Bridges et al. (1981)d,e; Parodi et al. (1981); O Rogan et al. (1982) N Salmonella typhimurium TA 1538 + Bridges et al. (1981)d,e Purchase et al. (1978); S Herbold (1978); Rosenkranz & Poirier (1979); Parodi et al. (1981) Salmonella typhimurium TA 100 + Purchase et al. (1978); Herbold (1978); Bridges et al. (1981)d,e --------------------------------------------------------------------------------------------------------- Table 6. (contd.) --------------------------------------------------------------------------------------------------------- Test description System description Result Reference Organism Species/strain/cell type --------------------------------------------------------------------------------------------------------- Reverse mutations bacteria Salmonella typhimurium TA 100 - Bridges et al. (1981)d,e; G Parodi et al. (1981) E Salmonella typhimurium TA 98 -, + Herbold (1978); Bridges et al. (1981)d,e; Parodi et al. (1981) N + Purchase et al. (1978) E Salmonella typhimurium G 46 + Braun et al. (1984) Reverse mutations bacteria Salmonella typhimurium G 46 + Röhrborn et al. (1972) (host-mediated (mouse) (NMRI) assay) M Reverse mutations bacteria Escherichia coli + Von Wright & Tikkanen (1980); Bridges et al. (1981)d,e U Haemophilus influenzae + Kimball & Hirsch (1975); T Kimball (1976) A Reverse mutations fungi Saccharomyces cerevisiae + Mehta & Von Borstel (1981)e; Vasudeva & Vashishat (1985) T Forward mutations fungi Saccharomyces cerevisiae + Lemontt (1977, 1978) Saccharomyces pombe + Loprieno (1981)e I Sex-linked visibles insect Drosophila melanogaster + Jain & Shukla (1972); O Shukla (1972); Vijaykumar & Jain (1979) N Sex-linked lethals insect Drosophila melanogaster + Shukla (1972) S Forward mutations hamster ovary cells in vitro + Gupta & Goldstein (1981)e - Carver et al. (1981)e; Hsie et al. (1981)e mouse lymphoma cells in vitro + Rogers & Back (1981) + Amacher et al. (1980) --------------------------------------------------------------------------------------------------------- Table 6. (contd.) --------------------------------------------------------------------------------------------------------- Test description System description Result Reference Organism Species/strain/cell type --------------------------------------------------------------------------------------------------------- C Breaks plant horse bean primary root + Gupta & Grover (1970) H Breaks, deletions, plant horse bean primary root + Heindorff et al. (1984) translocations R Aberrationsf plant chick pea root + Farook & Nizam (1979) O Aberrations rat epithelial liver cells - Dean (1981)e M in vitro O Aberrations rat bone marrow cells in vivo + Duamin et al. (1984) S Breaks, gaps, hamster ovary cells in vitro + Natajaran & Van Kesteren- exchanges Van Leeuwen (1981)e O Sister chromatid hamster ovary cells in vitro + MacRae & Stich (1979); Perry & Thomson (1981)e M exchanges - Natarajan & Van Kesteren- Van Leeuwen (1981)e; Baker et al. (1983) E lung cells in vitro + Baker et al. (1983) V-79 cells in vitro + Speit et al. (1980) Nuclear aberrations mouse epithelial colon cell - Wargovich et al. (1983) (oral exposure)g --------------------------------------------------------------------------------------------------------- Table 6. (contd.) --------------------------------------------------------------------------------------------------------- Test description System description Result Reference Organism Species/strain/cell type --------------------------------------------------------------------------------------------------------- D Micronuclei (ip mouse polychromatic erythrocytes + Salomone et al. (1981)e exposure) - Kirkhart (1981)e; A Tsuchimoto & Matter (1981)e M Dominant lethals mouse germ cells - Epstein et al. (1972) (ip exposure) A G E ------------------------------------------------------------------------------------------------------------------------ a Inactivation of transforming DNA was observed, which was inhibited by catalase, EDTA, or nitrogen gas treatment. b Hydrazine appears to be a mutagen that is highly specific for certain loci. Moreover, it not only produced mutants in the M2 generation but also homozygous recessive mutants in the M1 generation (plants, raised from treated seeds). c Inactivation of virus observed. Inactivation and, at lower concentrations, mutations were reduced by catalase treatment. d Hydrazine sulfate was tested in the International Collaborative Programme for the Evaluation of Short-Term Tests for Carcinogenicity (de Serres & Ashby, 1981). In the summary on the assay performance of bacterial mutation assays, it was reported that hydrazine sulfate was mutagenic in most of a total of 20 laboratories. However, there were inconsistencies in the strains in which the effect was seen and the requirements for S9 mix. Salmonella typhimurium TA 1535 was the strain in which mutagenic activity was most commonly observed, but it was also seen in all laboratories that used Escherichia coli strains. In some laboratories, hydrazine sulfate was also mutagenic in Salmonella typhimurium TA 100. Two laboratories reported a marginal mutagenic activity for strain TA 98, and TA 1538 was positive in another, while no mutagenic activity was observed with TA 1537. e Tested in the International Collaborative Programme for the Evaluation of Short-Term Tests for Carcinogenicity. In the final assessment, it was stated that hydrazine was a genotoxic agent in bacteria, yeast, and higher eukaryotes in vitro . Hydrazine did not appear to be genotoxic in vivo in higher eukaryotic cells (de Serres & Ashby, 1981). f Stickiness and clumping at metaphase, bridges and fragments at anaphase, laggards, micronuclei, and delayed cytokinesis at telophase, tripolar spindles, prophase, and metaphase in one cell. g Micronuclei, pyknotic nuclei, karyorrhetic nuclei, cytolysosomes. 8.5.3. Cell transformation Hydrazine increased the transformation of baby hamster kidney cells (Purchase et al., 1978; Daniel & Dehnel, 1981) and human liver cells (Purchase et al., 1978). While Purchase et al. (1978) observed an increased transformation of baby hamster kidney cells, both with and without metabolic activation, Daniel & Dehnel (1981) found a much lower increased transformation frequency only with metabolic activation. Hydrazine also caused enhancement of transformation of mouse 3T3 cells by Herpes simplex virus in vitro (Johnson, 1982). Hydrazine induced transformation of human fibroblasts. Transformed cells were able to induce undifferentiated mesenchymal tumours after sc injections in pre-irradiated nude mice, while control cells did not induce tumours (Milo et al., 1981). 8.6. Carcinogenicity 8.6.1. Inhalation exposure Groups of 100 Fischer 344 rats of each sex, 400 female C57BL/6 mice, and 200 male golden Syrian hamsters were exposed to the vapour of hydrazine (free base) for 12 months, 6 h per day, for 5 days per week, and observed for a further 18, 15, and 12 months, respectively (Table 7). Rats were exposed at 0.06, 0.33, 1.3, or 6.5 mg/m3, mice at 0.06, 0.33, or 1.3 mg/m3, and hamsters at 0.33, 1.3, or 6.5 mg/m3 air. Control groups contained 150 rats of each sex, 800 female mice, and 200 male hamsters, respectively. The hydrazine vapour was monitored throughout the exposure period. A reduction in body weight gain, seen in exposed rats throughout the entire study, was greatest in males at 6.5 mg/m3. The mortality rate was not affected by the exposures. Inflammatory changes were observed in the upper respiratory tract of both sexes and in the uterus and oviduct, especially at the highest exposure. At 6.5 mg/m3, the incidence of squamous metaplasia was increased in the nose, larynx, and trachea, while the incidence of epithelial hyperplasia was increased in the nose and lungs. An increased incidence of hyperplasia was also observed in the lymph nodes and uterus of females at 6.5 mg/m3 and in the liver of females at 1.3 and 6.5 mg/m3. A dose-related increased incidence of benign epithelial tumours of the nose, mainly adenomatous polyps, was observed in both sexes at the 2 highest exposures, while at the highest exposure (6.5 mg/m3), the incidence of malignant epithelial tumours was also increased (2/98 in males and 5/95 in females). The latency time exceeded 87 weeks in males and 97 weeks in females. The incidence of lung tumours was not significantly increased, but, in males, 3 bronchial adenomas and, in one female, 1 bronchial adenoma, were observed at 6.5 mg/m3 compared with none in the controls. The total incidence of adenomas and adenocarcinomas of the thyroid in males was not increased, but hydrazine exposure at 6.5 mg/m3 increased the fraction of malignant tumours. Table 7. Tumour incidence in mice, rats, and hamsters following hydrazine exposure --------------------------------------------------------------------------------------------------------- Route Species/ Exposure Daily dose Tumour incidenceb Reference strain period (mg/kg Nose or lung liver (months) body weight)a Male Female Male Female (life-time observation) --------------------------------------------------------------------------------------------------------- Inhalation mouse 12 0 8/385 - Vernot et al. (1985) (C57BL/6) 4/378 0.34c 12/379 - rat 12 0 0/146 0/145 - - Vernot et al. (1985) (Fischer 0.16c 11/97 4/94 - - 344) 0.8d 75/98 38/95 - - hamster 12 0 1/181 - Vernot et al. (1985) (golden 0.93d 16/160 - Syrian) Oral mouse 6 - 9 0 1/37 4/47 3/30 1/29 Biancifiori (1969, (gavage) (CBA) 0.98 2/26 10/25 1/26 0/25 1970a) 2.0 4/25 16/25 7/25 2/25 3.9 5/25 21/24 12/25 16/24 8.0 16/21 19/21 15/25 15/24 rat 17 0 0/28 0/22 0/19 0/14 Severi & Biancifiori (Cb/Se) 14.6/9.7 3/14 5/18 4/13 0/18 (1968) Oral mouse 24 - 28 0 11/110 14/110 2/110 3/110 Toth (1969, 1972a)e (drinking- (Swiss) 2.0/1.6 24/50 27/50 2/50 1/50 water) 5.1/4.6 25/50 24/50 3/50 1/50 --------------------------------------------------------------------------------------------------------- a Calculated doses based on a weight of 300 g and a minute volume of 100 ml for rat, a weight of 35 g and a minute volume of 25 ml for mice, and a weight of 140 g and a minute volume of 54 ml for hamsters. b - = not found; open space = not tested (nose in inhalation studies with rats and hamsters, lung in the other studies. c 1.3 mg/m3, 6 h/day, for 5 days/week. d 6.5 mg/m3, 6 h/day, for 5 days/week. e Liver tumours were hepatomas (1/50 in males at both exposure levels) and angiomas. A slightly increased mortality rate seen in mice right up to the end of the study was not related to dose. Body weights were not affected during exposure, but all exposed groups gained less weight than controls during the post-exposure period. The incidence of non-neoplastic lesions was similar in treated and control groups. The only neoplastic change observed was a slightly increased incidence of lung adenomas at the highest exposure. A background incidence of 2 - 3% for pulmonary adenoma in C57BL/6 mice was reported to be common in the laboratory concerned. Almost double the number of deaths occurred during exposure in each exposed group of hamsters compared with the control group, but the final mortality rates were similar in all groups, including the control group. During exposure to 6.5 mg/m3 and for 10 months after exposure, body weights were depressed. For several months, an unexplained decrease in body weight occurred in all groups, including the controls. Dose-related degenerative changes were observed. These were characterized by amyloidosis in several organs and issues, mineralizaton in kidneys, and haemosiderosis in the liver. Increased incidences of senile atrophy and aspermatogenesis were observed in the testes at the highest exposure level. In addition, the incidence of bile-duct hyperplasia was increased at all exposures. The incidence of benign nasal polyps was increased at 6.5 mg/m3. The only other striking tumours observed were 3 adenocarcinomas, 1 leiomyoma, and 1 papilloma in the colon, 1 basal cell carcinoma in the stomach, and 4 parafollicular cell adenomas in the thyroid at 6.5 mg/m3. No such tumours were found in the controls, but the increased incidence of each tumour type was not statistically significant. The authors concluded from these studies "that hydrazine is capable of inducing nasal tumours, primarily benign, in rats and hamsters after 1-year intermittent exposure" (Vernot et al., 1985). 8.6.2. Oral exposure The oral carcinogenicity studies on hydrazine have been either preliminary or limited in scope (inadequate limited histopathology, use of one dose or one drinking-water concentra- tion, small treatment groups, lack of adequate controls, and treatment of only one sex); therefore, only the more relevant studies have been described in this section. Most studies have concentrated on various mouse strains. Groups of approximately 25 CBA/Cb/Se mice of each sex received, by gavage, 150 daily doses of hydrazine sulfate in water, equivalent to 0.03, 0.07, 0.14, or 0.28 mg hydrazine in 25 weeks (these doses are expressed in Table 7 as 0.98, 2.0, 3.9, or 8.0 mg/kg body weight). The animals were observed for their life-time. Control groups containing 30 males and 29 females were not treated. Liver, lung, various endocrine glands, and tissues suspected of having lesions were examined microscopically. No statistical analysis was reported. No deaths occurred during the exposure period, but later the mortality rate was increased in animals that had been exposed daily to levels of 2.0 mg/kg upwards. Brown degeneration of the adrenals in treated females was mentioned as a marked non-neoplastic lesion. An apparent dose-related increased incidence of hepatocarcinomas was observed in male and female mice. The average latency time for this tumour did not decrease as the dose increased (Biancifiori, 1970a). The historical control incidence of hepatocarcinomas in the laboratory concerned was 8% (Severi & Biancifiori, 1968). Multiple pulmonary tumours, which were also observed, are described in another report (Biancifiori, 1969). In this report (Biancifiori, 1969), the 3 lowest dose groups, containing 21 - 26 males and 21 - 25 females, were treated in the same way as in the study above. The data for the control group and the highest dose groups were taken from earlier reports (Biancifiori et al., 1964; Severi & Biancifiori, 1968). The untreated control groups contained 37 males and 47 females. The highest dose group comprising 21 mice of each sex, received 250 daily doses of hydrazine sulfate in water equivalent to 0.28 mg hydrazine in 36 weeks. In the 1969 report, the treatment schedule was reported to be 150 daily doses of 0.28 mg hydrazine in 25 weeks, which was contrary to the original data. Lungs with trachea, liver, various endocrine glands, and other tissues suspected of having lesions were examined microscopically. No deaths occurred during the exposure period, but the mortality rate after the treatment period was increased from a dose level of 2.0 mg/kg per day upwards. Common non-neoplastic lesions in the liver at 0.28 mg per day included areas of diffuse cell regeneration. Necrosis, nodular regeneration, sclerosis, and bile-duct proliferation were also observed. The occurrence of hepatomas has already been discussed. The incidence and multiplicity of pulmonary tumours were increased at all dose levels in an apparently dose-related manner and more so in females than in males (Table 7). The average latency time for these tumours was also shorter in females than in males. At the highest dose level, approximately 80% of the tumours were characterized as adenomas in both sexes, the remainder being described as "anaplastic adenomas and adenocarcinomas" (Biancifiori, 1969). Induction of lung tumours by hydrazine sulfate after oral exposure by gavage was also reported in other mouse strains (Milia et al., 1965; Roe et al., 1967; Kelly et al., 1969; Biancifiori, 1970b; Bhide et al., 1976; Maru & Bhide, 1982). Liver tumour induction was reported in BALB/c/Cb/Se mice (Biancifiori, 1970b). Severi & Biancifiori (1968) also exposed 14 male and 18 female Cb/Se rats, by gavage, for 68 weeks, to daily doses of hydrazine sulfate in water, equivalent to 4.4 mg hydrazine (14.6 mg/kg body weight) per day for males and 2.9 mg hydrazine (9.7 mg/kg) per day for females. Controls (28 males and 22 females) were not treated. The animals were observed for their life-time. Lungs, liver, and other organs showing gross lesions were examined microscopically. No statistical analysis was reported. Exposed rats showed an increased mortality rate. Pulmonary tumours, adenomas, and adenocarcinomas were observed in both sexes (Table 7). Carcinomas (2) and spindle cell sarcomas (2), observed in the livers of exposed males, were not seen in the controls. Groups of 23 and 85 golden hamsters of both sexes received hydrazine sulfate by gavage as 60 and 100 daily doses, equiv- alent to 0.74 and 0.68 mg hydrazine over 15 and 20 weeks, respectively. The control group contained 56 hamsters. Animals were observed for their life-time and examined as described above for rats. The mortality rate increased in an apparently dose-related manner, and toxic effects were observed in treated animals (liver lesions, reticuloendothelial cell proliferation, cirrhosis, bile-duct proliferation, degenerative fibrous cells in hyalinized tissue). No tumours were observed, but, because of toxic effects, the treated animals were not observed for the same period of time as the controls (Biancifiori, 1970a). Groups of 50 Swiss mice of each sex received doses of hydra- zine sulfate in drinking-water, equivalent to 0.056 mg hydrazine (2 mg/kg body weight, males; 1.6 mg/kg, females) per day for their life-time up to a maximum of 113 weeks (Toth, 1972a) or 0.18 mg (5.1 mg/kg body weight) per day (males) and 0.16 mg (4.6 mg/kg) per day (females) for a maximum of 95 weeks (Toth, 1969). The untreated control group, containing 110 animals of each sex, was tested concurrently with the groups receiving 0.16 or 0.18 mg per day, but not with those receiving 0.05 mg per day (Toth, 1969). Liver, kidneys, spleen, lungs, and organs with gross lesions were examined microscopically. No statistical analysis was reported. Drinking-water solutions containing hydrazine were prepared 3 times per week. There was no indication that hydrazine concentrations were measured in the solutions during the studies. At the highest dose level, body weights were not affected, but the mortality rate in females was increased. Numerous lung tumours were observed in both sexes with a comparable incidence (Table 7) and degree of malignancy at both dose levels. The average latency time for this tumour was decreased at both dose levels. At the high dose level, the latency period for malignant lymphomas, which are characteristic for the strain, and the incidence of adenocarcinomas of the mammary gland appeared to be decreased. Fifty Syrian golden hamsters of each sex also received, for their life-time, up to a maximum of 110 weeks, doses of hydra- zine sulfate in drinking-water, equivalent to 0.57 mg hydrazine per day. Untreated non-concurrent control groups contained 100 hamsters of each sex. The protocol was similar to that described above for rats. Body weights of exposed hamsters were slightly reduced, but no tumours or other effects were noted. The authors considered that there was no treatment-related tumour incidence and that the mortality rate was not affected by the exposure, though the mean survival period was slightly reduced (Toth, 1972b). Groups of 34 male and 29 female Swiss mice were administered hydrazine sulfate by gastric intubation at doses equivalent to 0.27 mg hydrazine per mouse per day, 5 days per week, for their lifetime. Lung adenocarcinomas occurred in 30 out of 34 male mice and in 21 out of 29 female mice compared with 1 out of 20 controls. The earliest tumour appeared after 14 months of exposure. An increased incidence of lung tumours (20/22) occurred in F1 animals, which were obtained from treated mothers, and received hydrazine sulfate post weaning from the age of 11 weeks. No control mice were mated. An increase in lung tumours (16/35) was also observed in F2 animals obtained from F1 animals that had been treated with hydrazine throughout gestation and lactation (Menon & Bhide, 1983). 9. EFFECTS ON MAN 9.1. Poisoning Incidents Several incidents of systemic poisoning have been reported, mainly showing effects on the central nervous system, respira- tory system, and stomach. After a laboratory technician had drunk 20 - 30 ml of a 6% aqueous solution of hydrazine (free base), he immediately vomited. Four hours later, weakness, somnolence, and arrhythmia were observed. Laboratory findings showed a slight but persis- tent leukocytosis. The serum-albumin fraction was decreased with an increase in the fraction of globulin. Two days after exposure, slightly elevated body temperature and a few red blood cells in the urine were noted, while the patient showed irregular breathing. Five days after exposure, the patient had recovered (Drews et al., 1960). A case was reported of a man who drank between a mouthful and a cupful of hydrazine in concentrated solution. He immediately vomited, became unconscious, and flushed. Vomiting ceased about 12 h after admission to the hospital. The patient showed sporadic violent behaviour and later developed ataxia with a lateral nystagmus, a decreased sense of vibration, and paraesthesia in the arms and legs. Oliguria was also noted. Pyridoxine treatment was administered, but it was not clear from the report whether he benefited from this. The patient's final condition was not reported (Reid, 1965). A 24-year-old man accidentally ingested "a mouthful" of hydrazine and immediately became confused, lethargic, and restless. On admission to hospital, a complete chemistry profile was normal, but, 3 - 4 days later, hepatotoxicity was evident as indicated by elevated levels of aspartate amino-transferase (EC 22.214.171.124), lactate dehydrogenase (EC 126.96.36.199), and total bilirubin. At this time, the patient was treated with pyridoxine and recovered completely within 5 days. Peripheral neuropathy, however, developed subsequently, which resolved over the next 6 months. The authors attributed this neuropathy to the megadose pyridoxine therapy (Harati & Niakan, 1986). Another man sustained a chemical burn during an industrial hydrazine explosion. Exposure occurred probably both via the skin and by inhalation. The burns on his skin covered 22% of the body surface. On admission to the hospital, the neuro- logical status of the patient was normal but, 14 h after exposure, he became comatose. When the coma persisted for 60 h, he was treated with pyridoxine, and the neurological disorders cleared within the next 12 h. Other findings were persistently elevated glucose levels, haematuria, and respiratory difficulties. Several biochemical indicators of liver malfunction were elevated from day 3 after exposure and returned to normal levels over the next 5 weeks (Kirklin et al., 1976). Two men were exposed to Aerozine 50 vapour (50% hydrazine and 50% 1,1-dimethylhydrazine by volume) via a leak in a fuel line. The first man was exposed for about 90 min via a defective gas mask. He reported headache, nausea, and a shaky feeling, a sensation of burning of the face, a sore throat, and tightness in the chest. The second man had inhaled the vapour 3 times before evacuation from the contaminated area. He was dyspnoeic, trembling, and weak. Both men showed neurological disorders including twitching of extremities and clonic movements in the first victim and hyperreactive reflexes in both men. All symptoms cleared after treatment with pyridoxine. From clinical signs, it was concluded that pulmonary oedema developed in both men and was treated successfully. Four other cases of poisoning by Aerozine after a spill were mentioned in this report. These 4 men showed nausea and vomiting. Both symptoms disappeared within 20 min following pyridoxine treatment (Frierson, 1965). One case of systemic lupus erythaematosus-like disease due to occupational exposure to hydrazine (free base) has been described. The patient, a laboratory technician, had recurrent episodes of joint pain, photosensitive rash, fatigue, and fever following hydrazine exposure. A challenge patch test suggested that hydrazine caused the illness. On the basis of detailed examinations of the patient and her family, it was concluded that hydrazine could cause the disease in a person with a genetic predisposition. Some of the genetic factors identified were slow acetylation phenotype, HLA DR2,3, and immune system responsiveness such as antinuclear antibodies, antibodies to single-stranded and native DNA, and inhibition of pokeweed mitogen-stimulated immunoglobulin G synthesis in lymphocytes (Reidenberg et al., 1983). 9.2. Occupational Exposure 9.2.1. Inhalation exposure One case was reported of a man who had handled hydrazine (hydrazine hydrate) once a week for an unknown number of hours over a period of 6 months. In simulated conditions, only 0.071 mg hydrazine/m3 air was measured, but probably skin exposure had also occurred. The man experienced conjunctivitis, tremor, and lethargy after each exposure. Following the last exposure, he was feverish, vomited, and showed diarrhoea. In a hospital, 6 days later, many disorders were noted: conjunctivitis, stomatitis, arrhythmia, upper abdominal pain, enlarged abdomen, icterus, a tender and palpable liver, black faeces, incoherence, and oliguria. X-ray examinations showed pleural effusion and lung shadowing. Laboratory findings comprised elevated bilirubin and creatinine levels in the blood, and protein and red blood cells in urine. Treatments administered included haemodialysis and B-vitamins, which brought only temporary relief. The man died 21 days after the last exposure. Autopsy revealed pneumonia, severe renal tubular necrosis and nephritis, and mild hepatocellular damage (Sotaniemi et al., 1971). 9.2.2. Skin and eye irritation; sensitization Two cases of skin irritation (Frierson, 1965; Kirklin et al., 1976) and one case of eye irritation (Sotaniemi et al., 1971) have already been cited in sections 9.1 and 9.2.1, respectively. Dermatitis of the hands without systemic effects was observed in 2 men who were occupationally exposed to hydra- zine hydrate over a period of 5 months. Both men had had similar experiences previously, after handling of hydrazine hydrate (Evans, 1959). No patch tests were carried out, but the allergic nature of the contact dermatitis that can develop after skin exposure to hydrazine, has been reported earlier. In 1959, an outbreak of dermatitis was reported among 12 out of 34 female workers in a factory where hydrazine hydrochloride was used as a solder flux. Four months after the use of the solder flux had stopped, 6 out of 12 workers showed strong positive reactions towards hydrazine sulfate, while 30 controls did not show any reaction (Frost & Horth, 1959). In an electronics plant, 35 workers, constituting half the work force, developed contact dermatitis from a solder flux containing hydrazine hydrochloride, over a period of 5 years. The symptoms did not reappear when contact with hydrazine was avoided. Patch tests on 5 female workers of this group were all clearly positive for hydrazine fluxes, while 3 controls did not react (Wheeler et al., 1965). Another 7 workers in a chemical workshop, where solder flux containing hydrazine hydrochloride was used, showed allergic contact dermatitis towards hydrazine, as did 5 workers in a pharmaceutical firm producing isoniazid. The results of the patch tests were strongly positive for the 12 subjects, but a control group was lacking (Zina & Bonu, 1962). Hydrazine induced allergic contact dermatitis in 4 men when used as a corrosion inhibitor (Schultheiss, 1959; Hovding, 1967; Von Keilig & Speer, 1983). A negative control group was tested in only one of these studies (Hovding, 1967). Allergic contact dermatitis developed in 15 workers in 2 industries producing hydrazine sulfate. In one of these industries, patch tests on 15 workers were clearly positive for hydrazine, while 13 controls from the same industry did not show any reaction. Symptoms did not reappear when contact with hydrazine sulfate was avoided (Brandt, 1960). In the other industry, 10 persons produced a positive reaction towards hydrazine. No negative controls were tested (Sonneck & Umlauf, 1961). Two cases of allergic contact dermatitis were reported following exposure to hydrazine in stain removers with strong positive reactions towards the chemical in patch tests. Five controls did not react (Van Ketel, 1964). Systemic effects were not noted in any of these studies. Cross sensitization to hydrazine derivatives, such as isoniazid, phenylhydrazine, and hydralazine, was found by several investigators (Schultheiss, 1959; Zina & Bonu, 1962; Van Ketel, 1964; Hovding, 1967; Von Pevny & Peter, 1983). 9.2.3. Mortality studies The causes of death in a cohort of 427 male workers, employed for a minimum of 6 months in a hydrazine manufacturing plant between 1945 and 1971, were studied retrospectively and followed until July 1982. The plant ceased production in 1971. The men were divided according to 3 categories of exposure. The first category contained 78 men who might have been exposed to concentrations of hydrazine ranging from 1.3 to 13 mg/m3 air. Categories 2 and 3 (a total of 375 men) included those who might have been exposed to concentrations between 0.6 and 1.3 mg/m3 and those who had little or no exposure, respectively. A further subdivision in the first category distinguished between men with more than 10 years since the first exposure and those with less. Some men were covered by more than one category. The cohort was also exposed to other unspecified organic chemicals. The total number of deaths in 406 men who could be traced was 49 against 61.47 expected. Five men died from lung cancer against an expected 6.65. Two of these men belonged to the most highly exposed category. Another 7 cases of cancer were detected (9.27 expected). Further details were not given, except for the remark that no nasal tumours were involved. The authors noted that the number of men exposed to hydrazine in this study was small. It was concluded that the results were encouraging in that no obvious hazard had yet appeared. A note of caution is introduced by the observation of 2 deaths from lung cancer in men who had first been exposed in the heaviest category more than 10 years previously against 1.61 expected (Wald et al., 1984). The cohort is being followed up. A seemingly unusually high incidence of myocardial infarc- tion prompted further study among workers in a plant manufac- turing hydrazine. The population at risk was limited to every on-line hydrazine worker who had worked for 3 months or more between 1953 and 1978. The number of man years at risk was 534, and the average employment period was 8 years. Only a superficial examination of the population at risk and the working environment was completed. Reference data were provided by the US Air Force men's rates for the years 1975-77 and by men's rates from a study over the past 30 years by the US National Heart Institute (Framingham study). There was a close compar ison between the 2 sets of reference data. Rates of occurrence of myocardial infarction (death and non-death) per thousand men per year within 5-year age groups were calculated. A total of 5 cases of myocardial infarction, concurrent with the employment period, were identified among the hydrazine workers. Their average employment period was 16 years. A total of 1.2 cases was expected. The difference is highly significant statistically. Four additional cases were identified in ex- hydrazine workers. However, as it was impossible to trace all ex-hydrazine workers, the population at risk was limited to the "concurrent cases". The author warned that the findings should be treated with due caution because of the small numbers involved. He also stated that the strength of the findings rendered a negative hypothesis almost untenable without further investigation into the cause of the adverse effect found (Hamill, 1978). The Task Group recognized that exposure concentrations were not reported and that there was no indication that confounding variables, e.g., smoking history or exercise habits, had been taken into account. 10. EVALUATION OF HUMAN HEALTH RISKS AND EFFECTS ON THE ENVIRONMENT 10.1. Evaluation of Human Health Risks Exposure of human beings to hydrazine may occur occupation- ally or accidentally, through the ingestion of hydrazine-based drugs, or through the use of tobacco. Despite the high react- ivity of this compound and its wide industrial use, few systematic studies have been carried out on its adverse effects on man. Hydrazine is rapidly absorbed through the skin, lungs, and gastrointestinal tract and rapidly distributed throughout the body (section 6). In cases of acute human poisoning, vomiting, severe irrita- tion of the respiratory tract with the development of pulmonary oedema, central nervous system depression, and hepatic and renal damage have been reported. Data are not available from which the levels of hydrazine inhaled can be estimated in cases of acute poisoning by the respiratory route. However, from reports of poisoning by the oral route, it would appear that ingestion of amounts of the order of 20 - 50 ml causes severe intoxication and may be lethal (section 9.1). It is not possible to estimate a no-observed-adverse-effect level from the available human data. Most of the effects in human beings exposed to hydrazine have also been observed in experimental animals. In addition, loss of body weight, anaemia, hypoglycaemia, and fatty liver have frequently been reported. Fatty liver in mice and reduced growth in rats were reported when the animals were exposed through inhalation to the lowest (0.26 mg/m3) of three dose levels. Monkeys and dogs were unaffected at this dose. In only one study, a no-observed-adverse-effect level of 3 µwg/kg was reported, when hydrazine was administered by the oral route to rats. No data are available on the basis of which a no-observed- adverse-effect level by inhalation can be established (section 8.2). Embryotoxicity, fetotoxicity, and minor fetal abnormalities were observed in rats and mice exposed to hydrazine at doses toxic for the mother. At such doses, perinatal mortality is high. These effects may also occur at doses just below the maternally toxic dose (section 8.4). No data are available on the effects of hydrazine on the human fetus. In the absence of human data, it would be prudent to assume that hydrazine might have adverse effects on the human embryo or fetus at exposure levels approximating those producing toxicity for the mother. Such levels may occur during accidental spillage but would be unlikely to occur in the ambient environment because atmospheric concentrations are low. Skin and eye irritation have been observed in human beings who have come into contact with hydrazine, but the data are insufficient to establish a no-observed-effect level for irritation. Hydrazine is a strong skin sensitizer in man and cross-reacts with hydrazine derivatives (section 9.2.2). Slight eye irritation was observed in monkeys at hydrazine concentra- tions of 1.3 and 6.5 mg/m3, but none occurred in animals exposed to 0.26 mg/m3 (section 8.2.1). No skin irritancy studies on animals have been reported. The available data indicate that hydrazine induces gene mutations and chromosome aberrations in a variety of test systems including plants, phages, bacteria, fungi, Drosophila, and mammalian cells in vitro. Hydrazine induced indirect alkylation in the liver DNA of rodents after in vivo exposure to toxic doses, and it also caused DNA damage in vitro . Hydrazine transformed human and hamster cells in vitro. No increased unscheduled DNA synthesis was observed in the germ cells of mice in vivo . It did not induce chromosome aberrations, micronuclei, or dominant lethals in mice in vivo , but chromosomal aberrations were reported in rats in vivo (section 8.5). Hydrazine vapour induced nasal tumours, most of which were benign, in F-344 rats and Syrian golden hamsters, after 12 months of treatment and life-time observation. At the exposure levels at which most of the tumours occurred, there were signs of severe mucosal irritation. In several studies on mice, hydrazine induced an increased incidence of pulmonary tumours, when administered by the oral route. Hepatic tumours were also reported in two studies. In some of the studies, the pulmonary tumour incidence was related to the dose administered. Although in these reports there is little information about the toxic effects of the compound, it is probable that the doses admin- istered in some of the studies were at, or near, the toxic levels. No tumours were observed in hamsters treated orally with hydrazine (section 8.6). On the basis of the carcinogenicity studies on experimental animals, there is evidence that hydrazine is an animal carcinogen. Human data are inadequate to assess its carcinogenicity in man. In the absence of human data, and taking into account the mutagenicity data as well as the carcinogenicity data in animals, it would be prudent to consider hydrazine as a possible human carcinogen. Thus, exposure to human beings should be kept as low as feasible. Since measurable levels are not normally encountered in the general environment, it can be concluded that, except in cases of accidental exposure, hydrazine does not pose a significant practical hazard for the general population. 10.2. Evaluation of Effects on the Environment Hydrazine can be released into the atmosphere during venting operations, storage, and transfer. The total emission has been estimated to be nearly 0.01% of the hydrazine produced (production > 35 000 tonnes in 1981). Accidental discharges into water, air, and soil can result from bulk storage, handling, transport, and improper waste disposal. Evaporation of hydrazine after a spill can generate an atmospheric concentration as high as 4 mg/m3, 2 km downwind. Hydrazine is degraded rapidly in air through reactions with ozone, hydroxyl radicals, and nitrogen dioxide. In water, it is degraded rapidly, especially under aerobic conditions, and in the presence of organic material and/or in alkaline or hard water. It is more persistent in soft, metal-free water. In soil, it is adsorbed and decomposed on clay surfaces, under aerobic conditions. However, available data are inadequate to describe the nature of hydrazine behaviour in the soil. Because of the rapid degradation of hydrazine in the environment, measurable levels are not normally encountered. Hydrazine can be toxic for aquatic life, even at very low concentrations. Fish species showed LC50 values of between 0.54 mg/litre (roach) and 5.98 mg/litre (fathead minnow). Nitrifying bacteria in activated sludge are inhibited by hydrazine levels higher than 1 mg/litre. Some other microorganisms are more sensitive and show toxicity thresholds at levels reported to be as low as 0.00008 mg/litre. Hydrazine in both air and water is toxic for plants; in water, it can inhibit plant germination. On the basis of these data, it can be concluded that hydra- zine may present a significant hazard for the aquatic environ- ment and plant life. 11. RECOMMENDATIONS FOR FURTHER STUDIES 1. Dose-response studies pertaining to: (a) DNA alkylation (adduct identification); (b) damage of nasal epithelium; and (c) pulmonary effects. 2. Skin sensitization studies with focus on cross-reactivity with hydrazine derivatives. 3. Dose-response studies on sensitive, commercially important fish species and their food supplies. 4. Metabolic studies in conjunction with effects on DNA. 5. Dermal initiation-promotion studies. 6. Reproduction toxicity studies in sensitive rodent species at continuous low exposure levels. 12. 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See Also: Hydrazine Hydrazine (IARC Summary & Evaluation, Volume 71, 1999)