### MINISTERIO DE SANIDAD YCONSUMO DIRECCION GENERAL DE SALUD PUBLICA SUBDIRECCION GENERAL DE SANIDADAMBIENTAL 

# << 4 

# 061 P. 1

Subject : EU-Regulation 793/93, draft risk assessment report

ACETONITRILE (CAS 75-05-8) . Dear colleague, Please find enclosed, the first draft of the risk assessment acetonitrile, (first priority list), prepared by the Spanish, C .A . The report will be discussed at the next Technical Meeting, on April 28-30 and at the SIAM 6 in Paris .

Yours sincerely,

Madrid, 4th March 1997

Fdo . : Dr . Francisco Vargas Marcos . (For Spain Competent Authority)

To al SIDS ontact Points ~~~ D To al As for Reg 93/793 p i'~ Mr . P . Mc . Cutcheon, CEC, DGXI ~ ~ t uU Mr . B .Hansen, CEC, DG XI, ECB

### MINISTERIO DE SANIDAD Y CONSUMO DIRECCION GENERAL DE SALUD PUBLICA SUBDIRECCION GENERAL DE SANIDADAMBIENTAL



# RISK ASSESSMENT OF ACETONITRILE

DRAFT REPORTCONTENTS Page 0 OVERAL CONCLUSIONS OF THE RISK ASSESSMENT 1 1 GENERAL SUBSTANCE INFORMATION 2 1 .1 Identification of the substance 2 1 .2 Purity/impurities, additives 2 1 .3 Physico-chemical properties 2 1 .3 .1 Physical state (at ntp) 3 1 .3 .2 Freezing point 3 1 .3 .3 Boiling point 3 1 .3 .4 Relative density 3 1 .3 .5 Vapour pressure 3 1 .3 .6 Solubility 3 1 .3 .7 Partition coefficient 3 1 .3 .8 Flash point 3 ( 1 .3 .9 Autoflammability 3

1 .3 .10 Flammability 4 1 .3 .11 Explosive Properties 4 1 .3 .12 Surface tension 4 1 .3 .13 Dissociation constant 4 1 .4 Classification 6 2 GENERAL INFORMATION ON EXPOSURE 7 2 .1 Production 7 2 .2 Use 7 3 ENVIRONMENT 3 .1 Exposure assessment 3 .1 .0 General discussion

3 .1 .0 .1 Releases into the environment

3 .1 :0 .1 .1 Releases from production 3 .1 .0 .1 .2 Releases from use 3 .1 .0 .1 .3 Releases from other sources 3 .1 .0 .2 Degradation

3 .1 .0 .2 .1 Abiotic degradation 3 .1 .0 .2 .2 Biodegradation 3 .1 .0 .3 Distribution 3 .1 .0 .4 Accumulation 3 .1 .1 Aquatic compartment (incl . sediment)

3 .1 .1 .1 Calculation of Predicted Environmental

Concentrations 3 .1 .1 .2 Measured levels 3 .1 .1 .3 Comparison of PECs with measured levels 3 .1 .2 Terrestrial compartment

3 .1 .2 .1 Calculation of Predicted Environmental

Concentration 3 .1 .2 .2 Measured levels 3 .1 .3 Atmosphere

3 .1 .3 .1 Calculation of Predicted Environmental

Concentrations 3 .1 .3 .2 Measured levels . 3 .1 .3 .3 Comparison of PECs with measured levels 9 9 9 9 9 11 13 15 15 16 18 18 18 18 22 22 23 23 23 24 24 25 254 .1 .2 .8 Carcinogenicity 72 4 .1 .2 .8 .1 Studies in animals 72 4 .1 .2 .8 .2 Studies in humans 75 4 .1 .2 .8 .3 Summary of carcinogenicity of

acetonitrile 76 4 .1 .2 .9 Toxicity for reproduction 76

4 .1 .2 .9 .1 Studies in animals 76 4 .1 .2 .9 .2 Studies in humans 81 4 .1 .2 .9 .3 Summary of toxicity for

reproduction 82 4 .1 .3 Risk caracterisation 82

4 .1 .3 .0 General aspects 82 4 .1 .3 .1 Workers 86

4 .1 .3 .1 .1 Manufacture 86 4 .1 .3 .1 .2 End uses 87 4 .1 .3 .2 Consumers 87 4 .1 .3 .3 Man exposed indirectly via the environment 88 4 .2 Human health (physico-chemical properties 88 4 .2 .1 Exposure assessment 88

4 .2 .1 .1 Workers 88 4 .2 .1 .2 Consumers 89 4 .2 .1 .3 Man exposed indirectly via the environment 89 4 .2 .2 Effects assessment 89

4 .2 .2 .1 Explosivity 89 4 .2 .2 .2 Flammability 89 4 .2 .2 .3 Oxidizing potential 89 4 .2 .3 Risk characterizacion (physico-chemical properties) 89 5 CONCLUSIONS 90 6 REFERENCES 91 Appendix A

### 0 . OVERALL CONCLUSIONS OF THE RISK ASSESSMENT CAS N° : 75-05-8 EINECS N° : 200-835-2 Substance Name (IUPAC Name) : ACETONITRILE 

### (i) There is a need for further information and/or testing . 

### Sensitizati on No data is available on sensitization of acetonitrile. Therefore, there is need for testing for skin sensitization in accordance with the EU-Annex V-standard. Mutageniitv Acetonitrile did no induce gene mutations neither in bacteria nor in mammalian cells in culture. Showed week clastogenic activity in cultured mammalian cells. However, was found to be a inducer of aneuploid in a diploid strain of Saccharomyces cerevisae and in Drosophila melanogaster. In addition, positive results, in vivo, were reported in micronucleus test. Additional well-validated, in vivo, study in order to detect chromosome damage is required, before further conclusions can be made with regard to the risk assessment . Workers This conclusion is reached because the highest levels of exposure obtained in the "end uses" scenario have limited meaningfulness since the processes, work practices and the activities involved are not described and information on the duration and frequency of exposure are not available. (ii) There is at present no need for further information and/or testing or for risk reduction measures beyond those being applied already. 

### This conclusion applies to: The environmental risk, with respect to aquatic compartment, including water, sediments and biological processes of WWTP . Terrestrial environment, at the regional and continental levels, as well as non compartment specific effects relevant to the food chain (secondary poisoning) . This conclusion also applies to risk to workers involved in the acetonitrile manufacture, consumers, risk from indirect exposure of man via the environment and from physic-chemical properties . -1-

### 1 . GENERAL SUBSTANCE INFORMATION 1 .1 . Identification of the substance IUPAC name: ACETONITRILE Synonyms: Cyanomethane, Ethanenitrile, Ethyl nitrile, Methanecarnitrile, Methyl cyanide EEC N4: 200-835-2 EINECS N° : 200-835-2 CAS N4 : 75-05-8 

### N4 INDEX (Annex I) : 608-001-00-3 Molecular formula: C21-13N Molecular weight: 41 .05 Structural formula:

H H-C-C--- N H 1 .2. Purity/impurities, additives Degree of purity: >_ 99.5 % Identity and percentage of impurities:

- Propionitrile (0.02 %) - Water, distilled, conductivity or of similar purity (0.01%) 

### 1 .3. Physico-chemical properties Acetonitrile is a volatile, colourless liquid with a sweet, etherlike odour. It is infinitely soluble in water and readily miscible with ethanol, ether, acetone, chloroform, carbon tetrachloride and ethylene chloride. It is immiscible with many satured hydrocarbons (petroleumfractions) . Acetonitrile has high dielectric constant and high polarity and strongly reactive (Hawley's Dictionary) . -2-Acetonitrile produces hydrogen cyanide when heated to decomposition or when reacted with acids or oxidizing agents (Reynolds, 1982) . 

### 1 .3.1 . Physical state at normal temperature and pressure (ntp) Acetonitrile is a volatile, colourless liquid with a sweet etherlike odour. 1 .3.2. Melting point Grayson, (1985) gives a value of -45.7°C. Verschuerenk, (1983), the Merck Index. 11th Edition (1989) and Aldrich, catalogue handbook (1994-1995) give values between: -41 °C - -48°C . 

### 1 .3.3 . Boiling point Merck Index, (11 th Edition) and Howard Ph (1993) give the figure 81 .6°C at 1013.25 hPa

. 

### 1 .3.4. Relative density IUCLID presents a density of 0 .786 at 20oC (Aldrich, 1994-1995) . 

### 1 .3.5. Vapour pressure The ca. value provided in the IUCLID is between 94.51-98.64 hPa at 20°C (Verschueren K

., 1983, Envirofate, 1994) . 

### 1 .3.6. Water solubility Acetonitrile is infinitely soluble in water . (Clayton & Clayton, 1982) . Sax N .I . (1989) gives a value for water solubility 999999-999 mg/I at 25°C . 

### 1 .3.7. Partition coefficient (log n-octanol/water) IUCLID provides a value of -0.34 (Leo A . et al ., 1971) . 1 .3.8. Flash point Two values 5°C and 5.6°C are given as flash point (open cup) (Aldrich, 1994- 1995, Reynolds, 1982) . In closed cup the value is 12.8°C (Reynolds, 1982) . These values conclude in the R-phrase: R11 . 1 .3.9. Autoflammability Sax. N .I ., 1989 ; provides a value of 524°C . -3-

### 1 .3.10. Flammability Acetonitrile is highly flammable, with a lowerflammability limit of 4.4%in volume and an upper flammability limit of 16% in volume. These data are obtained from Grayson M

. (1985) and Sax N

.I . (1989) . 

### 1 .3.11 . Explosive properties Sax. N .I ., (1989) ; presents that acetonitrile forms explosive mixtures with air. The lower explosive limit is 3.05% in volume and the upper explosive limit 17% in volume. 

### 1 .3 .12. Surface tension 29.04 dynes/cmat 20°C (Merck Index, 11th Edition, 1989) . 1 .3.13. Dissociation constant pKa = 29.1 (Howard, P.H., 1993) .

### Table 1 .3.- Physico-Chemical Properties of Acetonitrile PROPERTY VALUE REMARKS Physical state Liquid - Colourless with a sweet, (at ntp) etherlike odour . - Produces hydrogen cyanide when heated or when reacted with acids or oxidizing agents. Melting point -41 - -48 'C Boiling point 81 .6 At 1013.25 hPa °C Relative density (d4Z°) 0 .786 Vapour pressure 94.51-98.64 At 20°C hPa Water solubility 999999.999 Infinitely soluble in water (at 25°C) mg/I Partition coefficient 0 .34 Log n-octanoVwater Flash point 5-5.6 (open cup) °C 12.8 (dosed cup) Autoflammability 524 °C Flammability 4.4 Lower flammability limit (LFL) 9b in volume 16 Upper flammability limit (UFL) Explosive properties 3.05 Lower explosive limit (LEL) % in volume 17 Upper explosive limit (UEL) Conversion factor 1 .68 At 25°C mg/m' Henry's Law

Constant 2.04x10'5-2.93x10-5 At 25°C atm .m'/mol Relative vapour density 1 .42 (air=1) Refractive index 1 .34604 At 15°C 1 .33934 At 30°C Surface tension 29.04 At 20°C dynes cm Dissociation constant 29.1 pKa

-5-

### 1 .4. Classification and labelling Classification according to Annex I of Directive 67/548/EEC Symbol and indication of danger : F: Highly flammable T: Toxic Risk phrases: R11 : Highly flammable 

### R23/24/25: Toxic by inhalation, in contact with skin and if swallowed Labelling: F, T, R11-23/24/25

S16-27-45 

### Specific concentrations limits: C>_2096 T ; R23/24/25 Specific concentrations limits: 3%<_C<20% Xn; R20/21/22

### 2. GENERAL INFORMATION ON EXPOSURE 2 .1 . Production Acetonitrile is obtained as a by-product of acrylonitrile synthesis, by a method known as the SOHIO(Standard Oil Company of Ohio) process, which involves a high temperature catalytic reaction between propylene and ammonia and produces crude acrylonitrile containing acetonitrile, hydrogen cyanide and carbon oxides as the main impurities. The reported ratio of acetonitrile/acrylonitrile in this product varies between 3% (Lowenheim and Moran, 1975; Kirk-Othmer 1978) and 109'a (Riegel, 1974; Reinders 1983) . Acetonitrile is obtained from the reaction product, after cooling, by fractional distillation . The main organic impurity in commercial acetonitrile is propionitrile together with small amounts of allyl alcohol (Kirk-Othmer, 1978; USEPA, 1992) . Other potential routes of acetonitrile synthesis are the dehydration of acetamide, the reaction of ammonia and acetylene, the ammonolysis of glacial acetic acid and the catalytic reaction of ethanol and ammonia (Merck, 1989; USEPA, 1980) . 

### Acetonitrile is produced in EU by 2 companies in 2 european countries and one company imports this product fromoutside, Table 2.1 . 2.2. Use Acetonitrile is used in different processes in the chemical industry and in research laboratories. The use pattern of this compound is shown in Table 2.2. This compound is used as a starting material for the synthesis of many chemicals, pharmaceutical and pesticides and in the manufacturing of photographic film . Acetonitrile is used as a solvent in various extraction processes like butadiene extraction fromC, streams and isoprene fromC5 streams, dissolution of cationic textile dyes, recrystallization of steroids, extraction of fatty acids fromanimal and vegetable oils and removal of tars, phenols and colouring matter from petroleumhydrocarbons. Acetonitrile is widely used in research and analytical laboratories. It is used as a solvent for genetic engineering research and in the analytical determination of a great number of chemicals by high performance liquid chromatography (HPLC) .I n addition, it is used as an inert medium in physico-chemical investigations and as a solvent in non-aqueous titrations.

### Table 2.1 . Acetonitrile production and import in EU in 1993. Country Company Quantity (tonnes/year) produced or imported Germany' PCK AG Schwedt 1000-5000 Italy Enichem S.p.A. 1000-5000 United Kingdomb BP Chemicals Ltd. 1000-5000 

### g1-990data b imported fromoutside, 1992 data 

### Table 2 .2. Use pattern of acetonitrile

Use Approximative percentage Pharmaceutical industry 50 Extraction solvent 15 Analytical laboratories (HPLC) 7 Photographic film 7 Pesticide manufacture 3 Other uses 18 Merck, 1989; USEPA, 1980 ; BP, 1992; Hawley, 1971 ; Borman, 1990

### 3. Environment 3.1 . Exposure assessment 3 .1 .q. General discussion Sources of acetonitrile release to the environment can be anthropogenic or natural . Acetonitrile occurs in coal tar in small amounts, has been detected in volcanic gases and quantified in emissions from the combustion of wood and other biomass. Acetonitrile is also released during its manufacture and use, from some industrial operations like shale oil retorting and coal gasification and fromcombustion processes in gas turbines, ignition engines and automobile exhaust . 

### 3.1 .0 .1 . Releases into the environment 3.1 .0 .1 .1 . Releases fromproduction Emission factors for acetonitrile during production have been reported by several authors. Bouscaren et al . (1986) quoted an emission factor of 2kg acetonitrile/tonne acrylonitrile, Reinders (1983) reported a range of factors, for acetonitrile release, between 1 and 7 kg acetonitrile/tonne acrylonitrile and USEPA (1980) reported a factor of 7.02 kg/tonne acrylonitrile produced . The emission factor to air, as well as to water and soil, has been estimated by using default values from the TGD and assuming that acetonitrile is in category Ic (intermediates stored off-site) . Table 3 .1 shows the calculated emissions factors and releases of acetonitrile to air, water and soil compartments during production . The estimated emission factor to air (5 kg/ tonne acetonitrile) is lower than those previously reported (2-7.02 kg/tonne acrylonitrile) considering, as indicated in section 2 .1, that the reported ratio of acetonitrile/acrylonitrile in crude acrylonitrile varies from 3 to 10% . Site specific information on releases of acetonitrile to air during production has been provided for one site and the total emission (fugitive and point emission) reported for two production plants was 77 tonnes/year in 1993, which corresponds to about 256 .6 kg/day per site and 128 .3 kg/day per production plant . These values are higher than the calculated value using TGD default values, probably due to the fugitive (accidental) emissions, and 256.6 kg/day will be considered as a worst case release value.c O : . a c w

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### 3.1 .0.1 .2 . Releases fromuse a) Use in the pharmaceutical industry Approximately 509'0 of acetonitrile produced is used by the pharmaceutical industry to manufacture different products like insulin, antibiotics and vitamins, Table 2 .2. In the manufacture of these products, the solvent is handled in close reaction and distillation sections, purified and recycled a number of times and the waste solvent is disposed of by licensed waste contractors. No site specific information is available regarding releases from this use. The emissions factors to the environment have been calculated by using default values fromthe TGD for industrial category 3 (chemical industry: chemicals used in synthesis) at processing, and main category Ic, Table 3 .2. 

### Table 3.2. Release of acetonitrile fromuse In the pharmaceutical Industry Environment Compartment Emission factor Production

Volume (t/a) Release

(kg/day) Local Air 0 .001 2,500 8 .3 Regional " " " 8 .3 

### Continental " " 7,500 25 Local Water 0 .007 2,500 58.3 

### Regional " " " 58.3 Continental " " 7,500 175 Local Soil 0 .0001 2,500 0 .8 Regional " " " 0.8 Continental " " 7,500 2 .5 

### b) Use in butadiene production Approximately 15% acetonitrile is used as extraction solvent in the obtention of 1-3-butadiene from the mixed C4-hydrocarbon fraction in petrochemical cracking processes for ethylene manufacture. The emission factors reported by Reinders (1983) ranged from 0 - 0.1 kg acetonitrile /tonne butadiene produced to air and the same range of emission factors to water. If it is assumed that acetonitrile is used exclusively in the butadiene extraction process and considering a butadiene production volume of 195,128 tonnes for the UK in 1990 (Chem-Fats 1991), the maximum release possible, using these emission factors, is about 65 kg acetonitrile / day for each compartment, air and water, for the production volumes of 1990. -11-



# 16

### The emission factors to the different environmental compartments have been

calculated by using default values from the TGD for industrial category 3 and main category Ic, Table 3.3. Table 3 .3 . Release of acetonitrile fromuse in butadiene production Environment Compartment Emission factor Production

Volume (t/a) Release

(kg/day) Local Air 0.001 750 2.5 Regional " " " 2.5 Continental " " 2,250 7 .5 

### Local Water 0 .007 750 17.5 Regional " 10 17.5 

### Continental " " 2,250 52.5 Local Soil 0 .0001 750 0 .25 Regional " " " 0 .25 Continental " " 2,250 0 .75 Information on emissionsdata fromacompany have been provided during 1993 and aqueous discharge was 4 .70 tonnes/year, corresponding to 15.7 kg/day, which is in agreement with local water release shown in Table 3 .3, although no information was given on the volume of acetonitrile used in butadiene production. Air point emissions of 2.93 tonnes acetonitrile/year (9.8 kg/day) were reported together with accidental discharges of 38.40 tonnes/year (128 kg/day) . 

### c) Other uses Acetonitrile is also used in the manufacture of photographic film, in the synthesis of some pesticides and in analytical and research laboratories, Table 2.2. In these processes, acetonitrile is used as a solvent in closed reaction or processing systems. When used in HPLCthe solvent should be collected after use and disposed of by licensed waste contractors, although this may not always be accomplished.

### Table 3

.4. Release of acetonitrile fromother uses Environment Compartment Emission factor Production

Volume (t/a) Release

(kg/day) Local Air 0 .001 1,750 5 .8 

### Regional 5.8 Continental " " 5,250 17.5 

### Local Water 0.007 1,750 40 .8 Regional 40.8 ; Continental " " 5,250 122.5 

### Local soil 0.0001 1,750 0.6 Regional " " " 0 .6 Continental " " 5,250 1 .8



### Information on the emissions for acetonitrile for these processes have not been found. Nevertheless, it is possible to estimate these emission factors assuming that 359'0 of acetonitrile produced is used in these processes, Table 2.2, and using default values fromthe TGD for main category Ic, Table 3.4. 3 .1 .0.1 .3 . Releases fromother sources a) Biomass burning Biomass burning is widespread, particularly in the tropics and in developing countries, and serves various purposes such as clearing of forest, control of pests, nutrient mobilization, production of charcoal and energy production for cooking and heating (Crutzen and Andreae, 1990) . These authors estimated in 3 - 6 x 10'5 g the biomass carbon burned annually, corresponding to 24 - 57 x 10'2 g of biomass nitrogen. Lobert et al (1990), based in these estimates, reported an acetonitrile emission from biomass burning to the atmosphere equivalent to 410,000 - 2,220,000 tonnes/year. Hamm and Warneck (1990) estimated an emission of 850,000 tonnes/year, which is in the same range, while the emission reported by Arijs and Brasseur (1986) of 24,000 - 174,000 tonnes/year is smaller. According with these estimates, a global acetonitrile release from biomass burning of 24,000 - 2,200,000 tonnes/year can be estimated. Assuming that 10% of this release can take place in Europe, a continental estimate of 2,400 - 222,000 tonnes of acetonitrile release per year from biomass burning can be considered. This corresponds to about 8 - 740 tonnes/day for the EU. The release at regional level can be estimated in one tenth, which corresponds to 0 .8 - 74 tonnes/day. -13-b) Automobile exhaust Various nitrogen compounds, including acetonitrile and hydrocyanic acid, have been identified as combustion products (Schuchmann and Laidler, 1972) . The global release of acetonitrile from automobile exhaust has been estimated in 80,000 tonnes/year by Arijs and Brasseur (1986) . Dulson (1978) reported that exhaust gases fromgasoline-powered engines contained 1 .32% by mass acetonitrile . Assuming an emission of 617,400 VOC tonnes/year at one area (UK) and a content of 1 .39'o by mass acetonitrile, the release of acetonitrile from motor exhaust would be 6,359 tonnes/year, equivalent to 21 tonnes/day. The overall amount for the EU, considering a population ratio of 6, would be 126 tonnes/day. These releases from motor exhaust are likely to be overestimates, due to the use of diesel in a significant number of cars and the use of new technologies, such as catalytic converters, utilized to reduce emissions from automobiles.

### Table 3.5. Summary of release estimates Source Main

compartment Amount/site kg/day Amount/regional

kg/day Amount/EU kg/day Production Air 83/256.6' 83.3/256.6' 250/769.82

W .Water 50 50 150 Soil 1 .7 1 .7 5 Use Pharm .lnd. Air 8.3 8 .3 25 W .Water 58.3 58.3 175 Soil 0

.8 0

.8 2 .5 Use Butad. Prod. Air 2 .5 2

.5 7 .5 W .Water 17.5 17.5 52 Soil 0

.25 0

.25 0 .75 Other uses Air 5 .8 5

.8 17.5 W .Water 40.8 40.8 122 .5 Soil 0

.6 0

.6 1 .8 Disp. sourc. : Biomass bur. Motor exh. Air Air -

- 800-74,000

21,000 8,000- 740,000 126,000 Total Air - 22,000-95,000 135,000-866,000

W .Water - 166 .6 500 Soil - 3.35 10 ' emission data provided by industry 2 calculated from emission data (3 x 256 .6 kg/day per site) 3 .1 .0 .2. Degradation 3 .1 .0.2.1 . Abiotic degradation a) Air The maximumof absorption for acetonitrile in the IJV range is lower than 160 nm, therefore direct photolysis of acetonitrile in the atmosphere is not expected to be an important fate process (Silverstein and Bassler, 1967; Howard, 1991) . -15-Acetonitrile is reactive tooxidising materials and compounds in the atmosphere,

being the reaction with hydroxyl radicals quoted as one of the main mechanisms for its removal from the environment (Lobert et al ., 1990)

. The rate constant for the reaction of acetonitrile with hydroxyl radicals has been determined by several authors to range from 1 .9 - 4 .94 x 10-'° cm3. molec-' . sec-' for the temperature range 20- 274C . In a moderately polluted atmosphere, with a mean concentration of 10' hydroxyl radicals/cm3, the calculated half-life was about 20 days (Atkinson, 1985; Guesten et al ., 1981 ; Gusten et al ., 1984; Harris et al . 1981 ; Wallington et al ., 1988, Lyman et al ., 1982) . The Arrhenius activation energy, as determined by Harris et al (1981), was 1500 cal . mole-' . In conditions of an average tropospheric OH radical concentration, [OH]= 5 x 105cm-3, Kl6pffer et al .(1988) obtained a half-life about 10 times longer, t 2>160days. Acetonitrile reaction with chlorine radicals is not thought to be significant in relation to hydroxyl radical reaction (Arijs et al ., 1983) . The reaction of acetonitrile with ozone is slow, with a reported reaction rate constant of s1 .5 x 10-'9 cm3 . molec' . sec'' (Harris et al ., 1981), .which will yield a half-life of z54 days, considering an average ozone abundance of 1 x 10'2 molec%m3 (Munshi et al ., 1989) . 

### A reaction rate constant between acetonitrile and single oxygen of 2.4 x 10-'e cm 3. molec'' . sec'' has been reported (Graedel, 1978), which yields an atmospheric half-life of >5000 years. An overall half-life for acetonitrile in the troposphere of about 42 days has been estimated, considering both the OH radical and ozone reaction rate constants (USEPA, 1987) . 

### b) Water The hydrolysis of acetonitrile is unimportant for the aquatic fate of this compound at the normal pH range of natural waters (Ellington et al ., 1988) . The half-life for acetonitrile in water has been estimated to be >150,000 years based on a hydrolysis rate constant at ph 7 and 25°C of 5.8 x 10-3 M-' hour-' (Howard, 1991) . 3 .1 .0.2.2. Biodegradation Ludzack et al . (1958 and 1959) studied the biodegradation of acetonitrile in Ohio river water under aerobic conditions, by measuring C02 nroduction. An initial assay with a dose of 10 mg/I acetonitrile needed about 21 days for reaching 75%of theoretical oxidation, while repeated dose studies showed the acclimatization of microorganisms and the oxidation rate was 5 times quicker in the second serial feeding. The effect of temperature on biodegradation showed that the rate of assimilation observed at 5°C was about a fifth of that at 20°C . The study with aged sewage showed that degradation in the sewage was slower than in the river water. Sasaki (1978), using the Japanese MITI (Ministry of International Trade and Industry) test, reported acetonitrile as readily biodegradable, being degradation higher than 98% after 28 days and higher than 30% in 2 weeks. The available biodegradation test results for acetonitrile are summarised in Table 3.6. -16-Although there are not biodegradation studies following OECDguidelines all the

available information suggests that acetonitrile can be considered as readily biodegradable. 

### Table 3 .6. Biodegradation test results for acetonitrile ype yp TT e Source of Method Result Detection Ref

microorganisms ic aerobic [ activated sludge other 17% 02demand 1 1 day ro jc

aerobic river water other 100% 02 demand 1 4day aerobic other other 98% OZ demand 1 28day aerobic activated APHAe >58% 02 demand 2



### sludge,adapted 5day BOD/ThOD aerobic other Directiveb 30% OZ demand 3 14day 

### BOD/ThOD anaerobic - other half-life - 4 4-16weeks e American Public Health Association 1980 ° Directive 84/449/EEC.C7 1 Ludzack and Ettinger 1960 2 Babeu and Vaishnav 1987 3 Envirofate 1994 4 Howard 1991 Several authors have isolated a number of genera and individual strains of bacte- :a able to growon acetonitrile under aerobic conditions. Mimura et al (1969) isolated the bacterium Corynebacterium nitrilophius from activated sludge and reported the ability of this microorganismto assimilate acetonitrile. Asano et al (1980) identified Arthrobacter sp. J-1 as being capable of growth on acetonitrile . Goud et al (1985) isolated bacteria froma sewage treatment plant at a petrochemical industry and found that Azobacter and Pseudomonas isolates were able to growwell on acetonitrile. Various bacteria isolates fromsoil capable of degrading acetonitrile have also been reported (Nawaz et al ., 1989 ; Li et al ., 1991 ; Martinkova et al ., 1992; Chapatwala et al ., 1992) . -17-

### 3

.1 .0.3. Distribution Acetonitrile is a volatile liquid with a vapour pressure of 9,864 Pa at 20°C (Verschueren, 1983) and it is expected a rapid volatilisation of this compound to the atmosphere when released to the environment . The Henry's Law

constant of 2.07-2.6 Pa. m3. mole-' (Snider and Dawson, 1985; Hine and Mookerjee, 1975) indicates that volatilisation of acetonitrile from surface waters and moist soils is likely to be significant (Howard , 1993) . Chen et al (1981) reported a decrease to 5% of the original acetonitrile level in river water after 72h. 

### The removal of acetonitrile from the atmosphere by precipitation has been quoted in several reports as one of the main fate processes for this compound, as important as the photo-oxidation reaction with hydroxyl radicals (Hamm et al ., 1989

; Hammand Wameck, 1990) . A residence time of about 3 years has been estimated for acetonitrile in the troposphere due to wet deposition (Hamm et al ., 1984

; Arijs and Brasseuur, 1986) . An overall residence time foracetonitrile near 1 .5years was calculated considering the two principal removal processes, precipitation and reaction with hydroxyl radicals (Hamm and Wameck, 1990 ; Arijs and Brasseur, 1986) . Nevertheless, a shorter residence time was calculated based on the tropospheric mass balance of this compound (Hammand Wameck, 1990) . They estimated a mass content (G) in the troposphere of 370-570 x 103 tonnes acetonitrile and a global source strength (Q) of 600-1,100 x 103 tonnes acetonitrile / year, which would yield an overall residence time, calculated as G/Q, of 0 .23-0 .95 years. The dissolution in the ocean was proposed as an effective process for acetonitrile removal, that would take up 659'0 of the total acetonitrile emission, and the combined residence time for the process (precipitation, hydroxyl radical reaction and ocean dissolution) was estimated to be of 0 .45 years, which is in agreement with the mass balance estimate. The adsorption coefficient, Koc, can be estimated for acetonitrile using the octanol-water partition coefficient, log Pow -0.34 (Leo et al ., 1971), and FCo, values of 0 .3-16 1/kg were obtained using regression equations reported in Lyman et al (1982) . These Koc values indicate a lowpotential for adsorption and then leaching of acetonitrile to groundwater may be important when spilled on soil (Howard, 1993) . 3 .1 .0.4. Accumulation No experimental data on bioaccumulation are available. A bioconcentration factor for fish of 0

.3 1/kg was calculated using the log Po,, = -0.34value and regression equations defined in Lyman et al (1982) . This bioconcentration factor is in agreement with other calculated values which are in the range of 0.3-0.4 (EPA; 1991) and indicates a lack of bioaccumulation potential in the environment. 3 .1 .1 . Aquatic compartment (incl . sediment) 3 .1 .1 .1 . Calculation of Predicted Environmental Concentrations -18-a) PEC local Only emission data to the aquatic compartment from the use of acetonitrile have been provided by industry. An aqueous discharge of 4 .70 tonnes/year, corresponding to 15.7 kg/day, has been reported . 

### Therefore, the PEC,,, for water has been calculated using the equations given in the Technical Guidance Document, assuming that the release occurs to a waste water treatment plant. PEC, .w(.,e,) = Ceff / (1 + KP(. .P~ . C.P) . D [g/I] where: Ceff = concentration of the chemical in the WWTP effluent [g/I] 

### KP(,ap) = Kw

x f,,,$,,p = FC= x 0

.1 = 16 x 0

.1 = 1 .6 (1/kg) CW = concentration of suspended matter in the river = 15 mg/I = 15 x 10-6 kg/I D = dilution factor = 10 The WWTP effluent concentration is calculated using the following equation : Ceff=W

.(100-P)/100 .Q where : 

### W = emission rate (kg/day) Q= volume of waste water (m3/day) = 2000 M3 /day P = percentage removal in the WWTP (%) = 91% Acetonitrile is readily biodegradable under aerobic conditions and using the tables in Appendix II of TGD based on the SIMPLETREAT model, log PoW = -0.34 and log H = 0

.41, the removal of acetonitrile in the WWTP is 91% . The fate of acetonitrile in the WWTP is 91%degraded and 9% dissolved in water . The release of acetonitrile to waste water during production was estimated in 50 kg/day, Table 3.5. Ceff = 50 x 9 / 100 . 2000 = 2.25 x 10-3 g/I PECIocw(Wate, - 2.25 . 10-3 / (1 + 1 .6 . 15 . 10-6) . 10 = 2.25 . 10-3 / 10.00024 = 0 .225 . 10-~ g/I = 225 Ng/1 Acetonitrile is used in various processes in the chemical industry. The highest percentage is used in the pharmaceutical industry, assuming that this volume (50% x 5,000 tonnes) is used in one site, the release of acetonitrile to water would be 58.3 kg/day, Table 3

.5. -19-Ceff = 58.3 . 9 / 100 . 2000 = 2 .62 . 10-3 g/I PEC,oca,(we,er) = 2.62 . 10-3/ 10.00024 = 2.62 . 10"3 g/I = 262 E1g/I 

### This value will be used as- the worst case PECbca,( eter)~ Other important percentage (15°/a) is used in butadiene production . Assuming that this volume is used in one site, the release of acetonitrile to water will be 17.5 kg/day, Table 3

.5. 

### Ceff = 17.5 . 9 / 100 . 2000 = 0.788 .10-3 g/I PEC, .w( er) = 0

.788 . 10-3 /10

.00024 = 0

.0787 . 10-3 g/I = 78.7jjg/I 

### Table 3.7 shows the PEC,=,,( a,er) calculated for the aquatic environment . Table 3 .7. Local PECs for the aquatic environment Process Release to waste Ceff (ug/I) PECbC,,

water (kg/day) (Ng/I) Production of 50 2,250 225 acetonitrile Pharmaceutical 58.3 2,620 262 industry Butadiene 17 .5 788 78.7 

### production The PECbcw(s,d) can be calculated using the equations given in the TGD and the values for PECwcai(water)~ PECIO~(sed) = lew+ Kp(sed) ~ es ~ Psoud / Psed l . PECIocW(water) g/kg where ew =volume fraction of pore water of moistened sediment = 0.8 I/I es = volume fraction of solids of moistened sediment = 0 .2 I/I Psolw = density of dry weight solid phase = 2.5 kg/I Psed =density of moistened sediment = 1 .3 kg/I K,(sed) = sediment / water partition coefficient Kplsedl = foc X Koc + foc(sed) = 0.05 -20-Kp(s,d) = 0 .05 x 16 = 0

.8 I/kg Using this equation and the various PECs local (water) calculated above, the PECs local (sed) have been calculated and are shown in Table 3 .8. 

### Table 3.8

. Local PECs for sediment Process PEC,,~ ,,,e,Fcg/I PECWIWSea Ng/kg Production of acetonitrile 225 249 .2 

### Pharmaceutical industry 262 290 .2 B utadiene 78.7 87.2 

### b) PEC regional and PEC continental The calculation of predicted regional and continental environmental concentrations of acetonitrile has been done using the Mackays model Level I . A summary of the results are shown in Table 3.9. 

### Table 3.9

. Summary of regional and continental modelling Regional Continental Amount released 2,314,251 21,096,224 moles/day Concentration in air (Ug/m3) 2 .3 0 .36 Concentration in surface 1 .03 0 .16 

### water (Ng/I) Concentration in sediment 7.7 1 .19 

### (ng/kg) Concentration in soil 3 .9 0 .59 

### (ng/kg) Molecular weight = 41 .05, vapour pressure = 9,864 Pa at 20°C, water solubility = 74,000 g/m3 at 25°C and log Ko,,, = -0.34

### 3.1 .1 .2. Measured levels

Table 3.10

. Levels of acetonitrile in water and sediment Country Location Sample Concentration USA Rock Sprigs, Wyoming Retort water', shale 42 Ng/I oil processing Hanna, Wyoming Process water`, 620jrgll coal gasifica6on plant Gillette, Wyoming Process water, 1,246 jrg/I coal gasification plant Colorado Waste water", shale 23-43 mg/1 oil retorting Coal gasification 365 mg/I condensate` Tucson, Arizona Rainfall' 0 .18pg/I 

# E Japan 47 Prefectures . Sediment' 0 .021 - 0 .54 mg/kg e Izzarl e ., b Benton, 1992 

### c Mohr and King, 1985 ° Snider and Dawson, 1985 ° Office of Heath Studies, 1990 

### No data on measured levels in water or sediment was found for european countries. Acetonitrile concentration found in water and sediment samples in USA and Japan are shown in Table 3.10. Acetonitrile levels were determined in aqueous samples collected fromdifferent operations related with shale oil processing and coal gasification in USA

. Ajapanese environment agency made a study on acetonitrile in fresh and marine surface waters across Japan (Office of Health Studies, 1991)

. In this study, acetonitrile was not detected in surface water (limit of detection 3Mg/I) but was detected in sediment with a frequency of 11 out of 60 samples. In addition, acetonitrile was detected in four rainfall samples collected at a rural site in Tucson, Arizona (Snider and Dawson, 1985) . 3 .1 .1 .3. Comparison of PECs with measured levels The local PECs calculated for surface water ranged from 78.5 to 262 /rg/I at acetonitrile production or use sites and the acetonitrile concentration in NWWfP effluents (Ceff) ranged from 0 .79 to 2 .6 mg/I . These values are in the lower range of -22-those reported in the literature for acetonitrile levels in waste water of various industrial

processes in USA, ranging from0 .04 to 365 mg/I, Table 3.10 . In relation with sediment levels, acetonitrile was found in Japan at concentrations between 21 and 540 pg/kg, which are in agreement with values calculated for local PECsed, which range from87.2 to 290.2 pg/kg. 3 .1 .2. Terrestrial compartment 

### 3.1 .2.1 . Calculation of Predicted Environmental Concentration Direct emissions of acetonitrile to soil from point sources are small and have been estimated in 1 .7 kg/day at production sites and to range from0 .25 to 0 .8 kg/day fromvarious uses of acetonitrile, Table 3 .5. Predicted concentrations of acetonitrile in soil have been calculated using Mackay's model, Level I . The PEC regional obtained for soil was 3 .9 ng/kg and the PECcontinental was 0 .59 ng/kg, Table 3

.9. 

### The PEC for soil porewater has been calculated using the equation in the TGD : PECso;tPorewete, _ (PECsoa

. psoit ) / (ew+ Kp(saq . es ~ Psotin ) where: Kp(soil) 

### = soil / water partition coefficient K%,(, .,) = f., x K

. ; f., = 0

.02 Kv(soiq = 0

.02 . 16 = 0.32 I/kg ew = volume fraction of pore water in soil = 0 .4 I/I es = volume fraction of solids in soil = 0.4 I/I Pso ;, = density of bulk soil = 1 .5 kg/1 Pso,ia = density of solid phase = 2 .5 kg/I This equation and the PECs calculated above have been used to calculate a PEC for the soil porewater. PECregionai soil porewater =3.9 . 1 .5/0.4 + 0.32 . 0.4 . 2.5 = 8.1 ng/I PECcontinental soil porewater= 0.59 . 1 .5/0.4 + 0.32 . 0.4 . 2.5 = 1 .2 ng/I 3 .1 .2.2. Measured levels No information on measured levels in soil was found. -23-

### 3.1 .3 . Atmosphere 3 .1 .3.1 . Calculation of Predicted Environmental Concentrations The major releases of acetonitrile to the atmosphere are from biomass burning and motor exhaust, Table 3 .5. A PEC local for air for acetonitrile emissions fromproduction and various uses can be calculated by using the equation given in the TGD : 

### C . ;, = Emission . CM

W, where: 

### C, ;, = air concentration at 100 mfrom a point source (kg/m3) Emission = emission rate to air (kg/s) C,w,;, = standard concentration in air at source strength of 1 kg/s = 24 . 10-e kg/m3 Using this equation and emissions fromproduction and use given in Table 3.5, various PECs have been calculated and are given in Table 3.11 .

### Table 3.11 . Local PECs calculated for the atmospheric environment Source Emission to air Kg/day PEC local Ng/m3 Production 256 .6 71 .3 Use in Pharmaceutical Industry 8 .3 2 .3 

### Use in Butadiene Production 2.5 0.7

Other uses 5 . 8 1 .6 Mackay's model, Level I, has been used to calculate regional and continental { concentrations. The PEC regional obtained for air was 2 .3 pg/m3 and the PEC continental was 0

.36 Ng/m3, Table 3

.9. 3 .1 .3.2. Measured levels Levels of acetonitrile in air have been determined in the atmosphere over Europe (Table 3

.12) and other parts of the world (Table 3 .13) . Some of these results showthe contribution of biomass burning to the atmospheric levels in rural areas (Becker and lonescu, 1982) and also over the Atlantic Ocean, where the tendency of levels to be higher around the equator (Hamm and Wameck, 1990) can be explained by the high percentage of the world's biomass burning that takes place in the tropics, estimated in more than 80% by Lobert et al . (1990) . 

### 3.1 .3.3. Comparison of PECs with measured levels The local PECs calculated for air ranged from 0 .7 to 71 .3 Ng/m3 at acetonitrile use or production sites. These values are in agreement with those reported in the literature for acetonitrile levels in urban and rural areas in Europe, which ranged from 0 .2 to 58.6 Ng/m3, Table 3.12. The calculated regional PEC is also in agreement with values reported for a mountain area in Switzerland (3.7 to 4 .9 pg/m3) and the estimated continental PEC is similar, although somewhat higher, than values reported for the atmosphere of the Atlantic Ocean near Europe (0.035 to 0 .138 Ng/m3) .

### Table 3.12. Levels of acetonitrile in the European atmosphere Location / Sample Concentration (ng/m3) Reference Rural area, Germany 5,210 - 10,420 Becker and lonescu, 1982 

### Rural area, Germany before agric. burning during agric. burning 6,720 58,630 Urban area, Wupertal, Germany 12,430 ± 4,030 Coastal area, Ireland 10,420 Mountain area, Switzerland 3,700 - 4,870 

### Urban area, Mainz, Germany 197-1,453 Hammand Wameck, 1990 North sea 109-328 Deuselbach 197 - 279 FI'ammet al ., 1989 Aircraft flight over Germany 218-260 Aircraft flight over Scandinavia 186-341 

### Table 3.13. Levels of acetonitrile In the atmosphere of other countries Location / Sample Concentration (ng/m3) Reference Rural area, Arizona, USA 94.1 ± 33.6 Snider and Dawson, 1984 Samples collected across 210-42,000 Office of Health Japan Studies, 1991 Atlantic Ocean 30-17°S 122-192 Hammand Warneck, 14°S-Eq 208-302 1990 Eq-160N 170 -257 22-50°N I 35 - 138 I

### 3.2. Effects assessment Hazard identification and Dose (concentration) - response (effect) assessment 3.2.1 . Aquatic compartment (incl . sediment) 3.2.1 .1 . Aquatic organisms The provided information includes a set of data on the acute toxicity of acetonitrile for aquatic organisms. These data have been summarized in Tables 3 .14, 3.15 and 3.16

. It has not been possible to check ail data going back to the original publications. Therefore, unless otherwise stated, references must be considered as indirect references from the IUCLID data base and the additional background documents presented for the assessment . The acute toxicity of acetonitrile on fish has been assessed on different species. The IUCLID data base includes values for Pimephales promelas, Lepomis macrochirus, Leuciscus idus melanotus, Poecilla reticulata and Oryzias latipes . The lowest value is the 48 h TLmfor Oryzias latipes, 730 mg/I, while all 48-96 h LC50 values are in the g/L range. Information on additional fish species can be obtained from other sources. The WHOreport (1993) includes 14 acute LC60 values on seven fish species. Significant values are 24-48 h LC50 values higher than 1 g/L for Oryzias latipes, and two 48 h LC50s, 730 and 880 mg/L on Cyprinus carpio and Ctenopharyngodon idellus respectively. The data set includes flowthrough and static tests as well as different water pH and hardness conditions. Relevant differences on acetonitrile toxicity regarding water quality conditions were not observed . The acute toxicity of acetonitrile on aquatic invertebrates was investigated by Ewell et al . (1986) on six species from different taxonomic groups including Arthropoda, Platyhelminthes, Mollusca and Annelida; all LCSOvalues were reported as > 100 mg/L. Additional data reported by IUCLID on Daphnia pulex, Culex restuans, Hyalella azteca and Palaemonetes kadiakensis are in the g/L range. A reduced number of toxicity results on algae has been found. Toxicity threshold based on the first detectable inhibition of cell multiplication for the green alga Scenedesmus quadricauda and for the blue-green algae Microcystis aeruginosa has been reported as 7300 and 520 mg/L respectively. No ECSOvalues have been found.t

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information provided for the inhibition of algal growth does not include NOEC calculations or enough data for these estimations. Thus, the effect assessment focuses exclusively on acute toxicity data. 

### The lowest L(E)C50 figure is the 48 h LCSO on Cyprinus carpio, 730 mg/L. The TTs for algae are in the same range. Therefore, it seems appropriate in this assessment to consider 730 mg/L as the lowest end of the acute toxicity range for aquatic organisms. This range covers three taxonomic groups, fish, invertebrates and algae, and taking into account the lack of chronic figures, the recommendations of the Technical Guidance Document suggest the application of the factor 1000to the lowest end of the acute toxicity range (730 mg/I), obtaining a PNECfor aquatic organisms of 0.73 mg/I . PNEC,q ,bc ,ro ,,= =1owest end acute toxicity range/1000=0 .73 mg/11 The data set only includes freshwater species. Therefore, in absence of data on saltwater species the above PNEC can be used for both, freshwater and marine environments, while data on marine species are produced. 3 .2.1 .2. Effects on microorganisms The toxicity of acetonitrile on microorganisms (table 3 .17) has been previously reviewed by different authors. Blumand Speece (1991) present data on three types of environmental bacteria that can be relevant when considering the microbial activity of domestic VWfPs. The IC50 values (the concentrations of acetonitrile causing 50% bacterial inhibition compared to controls) were summarized in the internal report by Nielsen and Howe as follows: 1 . Aerobic heterotrophs.- These bacteria predominate in activate sludge systems and in natural aerobic environments where they convert organic material to carbon dioxide and water. The inhibition in oxygen uptake by the bacteria was monitored after 15 hours exposure and used to calculate and IC50 of 7500 mg/L. 2 . Nitrosomas sp.- These bacteria convert ammonia nitrogen to nitrite as the first most sensitive step in the biological oxidation of inorganic nitrogen. The inhibition in ammonia consumption by the bacteria was monitored after 24 hours exposure and used to calculate an IC5 . of 73 mg/L. 3 . Methanogens.- Methanogens form part of the consortiumof bacteria that convert organic matter to carbon dioxide and methane under anaerobic conditions. The inhibition of gas production by the bacteria was monitored after 48 hours exposure and used to calculate an ICso of 28000 mg/L. -31-

### Additional data on Nitrosomas sp, Nocardia rhodochrous, Pseudomona pufida,

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### 3.2.1 .3. Effects assessment for the sediment The available data set includes a single data on the effect of acetonitrile on the sediment organism Hyalella azteca, but for waterborne exposure. The ECSO obtained for this organism is similar to that reported for other aquatic invertebrates suggesting that the first assumption of the partitioning method presented by the Technical Guidance Document can be accepted. In addition, the very lowPow of acetonitriie, indicates that the use of water phase as the single via for uptake is also adequate. Thus, the PNEC may provisionally be calculated using the equilibrium partitioning method with the PNEC for aquatic organisms and the sediment/water partitioning coefficient. 

### PNEC a. = Kp(s n.PNEC,y , . .,ro ,,sms A rapid estimation produces a Kp(sed) value of 0 .8 I/Kg thus, the PNEC value for sediment-dwelling organisms should be 0 .58 mg/Kg. PNEC d=Kp( M

.PNEC,q tjc aq ,,, s= 0 .8 I/Kg x 0,73 mg/L= 0 .58 mg/Kg. 

### 3 .2.2. Terrestrial compartment i The available data set does not include a single data for th~e terrestrial compartment other than those on mammals which would be used for the assessment of secondary poisoning. Taken into account the lack of data, and according to the Technical Guidance Document, the equilibrium partitioning method can be applied as a conservative calculation method to identify a potential risk to the soil compartment . The PNECaquat;c organisms, is used for the risk assessment, thus, no additional effect estimations are required . 

### 3 .2.3. Air compartment There are not data for this assessment . 3 .2.4. Non compartment specific effects relevant to the food chain (secondary poisoning) According to the Technical Guidance Document, the risk characterization for secondary poisoning is required if three specific criteria are fulfilled. These criteria can be summarized as : Indirect exposure likely. Indication of bioaccumulation potential and -34-Mammalian toxicity risk. 

### In the case of acetonitrile, it is toxic for mammalian, but your lowpotential for bioaccumulation indicates that secondary poisonings are of lowconcern. 3 .3 . Risk characterization 

### 3 .3.1 . Aquatic compartment (including sediments) The worst case PEC/PNEC ratios are summarized in tables for water column and for sediments respectively. 

### Table 3.18. :PEC/PNEC ratios for the aquatic compartment : water column WATER PEC PNEC PEC/PNEC LOCAL 262 ~tg/I 730pg/I 0 .36 

### REGIONAL 1 .03 pg/I 730pg/I 0.0014 CONTINENTAL 0.16 pg/I 730 Ng/I 0.00022 Table 3.19

. : PEC/PNEC ratios for the aquatic compartment : sediment SEDIMENT PEC PNEC PEC/PNEC LOCAL 290 Mg/Kg 580 pg/Kg 0.5 

# R 10 

# EGIONAL 0.007 pg/Kg 580 pg/Kg 0.000013 I CO TI CONTINENTAL 0.001 2 pg/Kg 580pg/Kg 0 .000002 In all cases, for both water and sediments, PEC/PNEC ratios are below , therefore, it should be considered that for the aquatic compartment, including sediment, the risk characterization indicates that the present risk of acetonitrile for the aquatic environment is not of concern. The concentration of acetonitrile in the effluent of 1NWTP has been estimated in a range between 0 .78 and 2.62 mg/I . The worst case assumption corresponds to the pharmaceutical industry. The removal acetonitrile inflow concentration should be about 30 mg/I . This value is lower than the estimated PNECm ;croor9anisms of 73 mg/I . Therefore, no significant risk for NWWfP must be expected .

### ResultFor the aquatic compartment, including water, sediments and biological

processes of VWWTP

: 

### ii) There is at present no need for further information and/or testing and no need for risk reduction measures beyond those which are being applied already. The data set only includes freshwater species. Therefore, in absence of data on saltwater species the above result can be used for both, freshwater and marine environments. 

### 3.3.2. Terrestrial compartment The data set does not include a single data on terrestrial organisms. Therefore, only the comparisons of PEC soil/pore water versus PNEC aquatic organisms are possible. These results are summarized in table 3.20. Table 3 .20 . : PEC/PNEC ratios for the terrestrial compartment :

soil porewater SOIL POREWATER PEC PNEC PEC/PNEC LOCAL 

### REGIONAL 0.0081 Ng/I 730 pg/I 0.00001 CO NTINENTAL 0.0012 Ng/I 730 Etg/I 0 .0000016 These ratios are lowenough to consider that the risk for terrestrial organisms at the regional and continental level is of lowconcern. ResultFor the terrestrial environment, at the regional and continental levels: (ii) There is at present no need for further information and/or testing and no need for risk reduction measures beyond those which are being applied already. This assessment does not include the local scale. 

### 3.3 .3. Atmosphere There is not information on the effect of atmospheric exposures on organisms, other than those data considered for the assessment of potential effect on human -36-

### health.

3 .3 .4. Non compartment specific effects relevant for the food chain (secondary poisoning) The lowpotential of acetonitrile for bioaccumulation indicates that secondary poisonings are of low concern. 

### Result (ii) There is at present no need for further information and/or testing and no need for risk reduction measures beyond those which are being applied already.

### 4 . HUMAN HEALTH 4 .1 . Human health toxicity 4 .1 .1 . Exposure assessment 4 .1 .1 .0. General discussion Acetonitrile is a volatile liquid with a high water solubility. It is readily absorbed from the gastrointestinal tract and through the skin and lungs. All three routes of exposure have been reported to lead to systemic effects. 

### There is little information, if any, on acetonitrile absorption in humans. However, human poisoning cases indicate that it is well absorbed by all routes. 4 .1 .1 .1 . Occupational exposure Occupational exposure to acetonitrile occur primarily through inhalation of vapours. Absorption through the skin may occur . Acetonitrile is produced as a by-product of acrylonitrile manufacture. It is a highly polar solvent with a high dielectric constant . It is used: in the petrochemical industry for separating olefin-diolefin mixtures and C4-hydrocarbons. 

### to extract fatty acids from animal and vegetable oils. for extractive distillation, crystallization, as a starting material or intermediate of vitamins, steroids, bactericides and pesticide manufacture. due to its superior solvency with polymers it is used as a solvent for spinning fibers and casting and molding plastics. in the manufacture of photographic film . it is a common laboratory solvent, and is widely used for HPLC and as a solvent for DNA synthesis and peptide sequencing . Synthesis of acetonitrile is carried out in a closed system . Therefore, occupational exposure during production could only occur accidentally. For some industrial processes as well as when it is used in the laboratories, acetonitrile is used in open systems where its high volatility and relatively high odour threshold favor -38-

### worker exposure potential . The different systems of limit values are summarized in the table 4 .1 . : Table 4 .1 . : Different systems of limit values Country OEL-occupational expos.limit (8h. TWA) STEL-short term

exposure limit Skin

notation 

### PPM mg/m3 ppm mg/m3 U.S.A . (ACGIH, OSHA) 40 67 60 101 no Austria 40 70 ----- ----- no Belgium 40 67 60 101 yes 

### Denmark 40 70 60 105 no Finland 40 1 70 60 105 no France 40 70 ----- ----- no Germany 40 70 80 140 no 

### The Netherlands 40 70 ----- ----- no United Kingdom 40 70 60 105 no 4 .1 .1 .1 .1 Manufacture The european production of acetonitrile is limited to Germany and Italy, since the United Kingdomplant was closed for production in 1990 . The number of workers potentially exposed to this compound during its manufacture is estimated to be about 300-400

. The acetonitrile is mainly obtained as a by-product of acrylonitrile manufacture via propylene ammoxidation. The process is carried out in a closed system and involves temperatures of 400 - 500°C . The plants are outdoor, taking advantage of natural ventilation. In this situation, exposure would only be accidental . The likelihood of such exposures increases during maintenance operations, repair of equipment, transferring and quality control sampling of acetonitrile .

-39-The EASE model predicts that inhalation exposure to gas or vapour used in a closed systemwill be in the range of 0 - 0 .1 ppm . The model also predicts that, in these conditions, dermal exposure will be negligible. High exposure may occur during activities which involves breaching the closed systemsuch as sampling, maintenance and filling activities. The estimated exposure, according with the EASE model (non dispersive use with LEV), will be 10-50 ppm . However, these concentrations will be received as brief and intermittent exposures rather than over a full workshift. Assuming that contact was incidental, dermal exposure will be low

. 

### According to an european producer the process is continuous and automated. The filling operations consist on filling railway tank cars with gas exhaust pipe for purifying. The tables 4

.2. and 4.3. showthe measurements made by two european producers: Table 4.2. : Long termoccupational exposure during production Plant Task No. Type of Repres . Comment Resp. location samples sampling 8h. TWA Protec worn? ste, different*

work ---- stationary < 0 .1 ppm position Italy sile2 differenr work stationary 0 .5 ppm position ---- production 2 stationary/personal < 0 .0059 nortnal no Gerrnany production filiny stationary/personal < 0 .0059 filling rail way no operations 2 tank cars * It is assumed that tasks where exposure is expected to be high are included as "different work positions" (such as filling operations, sampling and maintenance) .

### Table 4.3. : Short termoccupational exposure during production Plant location Task No. of Type of RepresentLtive Respiratory samples samples conc. (ppm) protection Germany Take sample stationary/ <0 .0059 no ~ of the product ~ 2 ~ personal I x 20 min In the worst case, the exposure level was below0.5 ppm . This is much lower than the most representative exposure limit value of 40 ppm(8h-TWA) . 4 .1 .1 .1 .2. End uses 

### The actual number of persons exposed or potentially exposed to AN is unknown. It could be a high figure in viewof the wide pattern of uses. Acetonitrile is used as a solvent on chemical industry. According to a producer, acetonitrile is handled in closed systems with the "spent" solvent being continuously distilled and recycled. The main use in this scenario is the extraction of 1,3 - butadiene. 

### The table 4.4. shows measured data fromthree chemical companies : Table 4 .4. : Measured data fromthree chemicals companies company task year mean exposure type of comments level(ppm) sampling sampling, Chemical maintenance, 1994 company cleaning < 0 .1 personal activities company Chemical purifying company ---- 1990 21" ambient acetonitrile by redistillation Chemical process work non dispersive company ---- use,

laboratory 1977-91 10 dilution w ork ventilation * 21 ppmwas the highest value found and it represents an exposure over 5.40 h. It is supposed that an exposure over 8 hours would be less than 21 ppm but we have assumed this level as representative of long-termexposure, covering the worst case situation. -41-In the worst case, the exposure level of 21 ppmis below the current exposure limit (40 ppm, 8 hour TWA) adopted by the majority of member states. No further details were given aboutjob categories but it is assumed that involves activities where there is a need for breaching the closed system . Near 509'0 of the european acetonitrile production is used in pharmaceutical manufacturing processes. In general, acetonitrile is handled in closed reaction and distillation section of the plant during the manufacturing process. 

### Data from HSE shows that acetonitrile was not detected in personal samples taken along 1988 fromdifferent job locations, in a plant manufacturing thiamine. The table 4

.5. shows exposure data fromtwo pharmaceutical companies. No details about the job categories were reported. Table 4 .5. : Exposure data fromtwo pharmaceutical companies company number of Sam- mean concentration type of sampling No. of samples with (PPm)

ples ezposure level range

(PPM) (PPM) <5 5-20 20-40 >40 pharmaceutical 13 2 0-16.8 personal/ambient 12 1 company 1980-1987 pharmaceutical 45 9.42 <5 - 75 personal 40 2 3 company 1992 The meaning of these measurements can only be assessed to a limited extent because the processes and the activities involved are not described and information on the duration, frequency of exposure and pattern of control is not available. Data from Germany collected in different companies (plastic and pharmaceutical) and research laboratories showthat 95% of results are below7.3 ppm . The higher exposures (between 4 .76 - 7.3 ppm) correspond to research laboratories where acetonitrile is used as mobile phase in chromatography. By analysing all measured exposure data we conclude that an exposure level of 20 ppmis a reasonable worst case situation, and more, taking into account that it has been measured in a sampling time lower than 8 hours (21 ppmx 5.40h and 16.8 ppmx 3 h.) . Values higher than 40 ppmhave been reached only in a company and this can be attributed to a particular situation not reflecting the general pattern (even in that company 90% of the samples gave results below5 ppm) .

### For risk characterization purposes, a worst case exposure level of 20 ppmis considered. In general, most acetonitrile uses are in industrial processes (in closed reaction or processing systems), so that exposure is likely in special tasks such as sampling, maintenance and filling activities. Exposure could be estimated by using the same approach as in the previous section. Accordingly, the EASE model predicts that exposure will be in the range of 10 - 50 ppm(non dispersive use with LEV) . These figures are in agreement with the measured exposure data. The situation can be different in laboratories where acetonitrile is used . Acetonitrile is fed by tube into the HPLC and the waste solvent is collected upon exiting the instrument, so that the likelihood of an exposure is very low . However, acetonitrile is usually transferred or filtered before entering the instrument and the likelihood of an exposure increases. 

### In this situation, dilution ventilation has been chosen as pattern of control in research laboratories as a worst case situation. The EASE model predicts that exposure will be in the range of 100-200 ppm . On the other hand, these activities take only a fewminutes not a whole shift. Therefore, the range quoted is likely to be an over estimate. Data from Germany showthat exposures around 7 .3 ppm could be obtained in research laboratories where acetonitrile is used in chromatography. Since acetonitrile is absorbed by skin, potential exposure will occur via skin contact . Exposure is likely to occur during activities such as sampling, maintenance, cleaning or laboratory work. Assuming a non dispersive pattern of use and incidental skin contact, the EASE model predicts that exposure to the hands and forearms will be low, in the range of 0- 0 .1 mg/cm2/day. Gloves are required in the industrial processes and in the laboratories for special tasks reducing the expected exposure to a minimum . 4 .1 .1 .2. Consumer exposure No quantitative data have been obtained for the evaluation of consumer exposure, neither from the chemical industry, nor through literature reviewed. Most of the acetonitrile produced is used in the synthesis of other substances within the chemical industry and does not reach consumers. Looking at the accidental intoxication cases to acetonitrile we could conclude that acetonitrile could be present in nail remover . However, acetonitrile is not on the EC Cosmetics Inventory (96/335/EC) and is not approved for use in cosmetics in Europe .Therefore it is not of concern for consumers. -43-4 .1 .1 .3. Indirect exposure via the environment The indirect exposure is assessed by estimating the total daily intake of a substance based on the predicted environmental concentrations for (surface) water, groundwater, soil and air. The concentration of a substance in food is correlated to its concentration in water, soil and air and is also dependent on its bioaccumulation behaviour. 

### Release of acetonitrile at environment can occur fromproduction and uses as well as from other sources: biomass burning and automobile exhaust . The USES model has been used (Appendix A) . Some values are reproduced in table 4

.6.

### Table 4.6. : Estimated human daily intake of acetonitrile Source Local Regional Air 4 .45 x 10-5 0 .00039 

### (mg/Kg/day) Drinking water 7.58 x 10-6 1 .9 x 10-5 

### (mg/Kg/day) Fish 1 .25 x 10-8 3.15 x 10-8 

### (mg/Kg/day) Stemof plant 4 .41 x 10-' 3.83 x 10'e 

### (mg/Kg/day) Root of plant 1 .5 x 10-' 1 .02 x 10-e 

### (mg/Kg/day) Meat 6 .02 x 10-'3 5.36 x 10-12 

### (mg/Kg/day) Milk 6 .00 x 10-13 5.34 x 10-12 

### (mg/Kg/day) Total human dose 5.27 x 10-5 0.00042 (mg/Kg/day)4 .1 .2. Effects assessment 

### 4.1 .2.1 . Toxicokinetics, metabolismand distribution 4 .1 .2.1 .1 . Studies in animals 

### In vfvo studies Absorption There are not available quantitative analytical data on the pulmonary absorption of acetonitrile. However, it is known that acetonitrile is easily absorbed fromthe lungs of animals exposed to acetonitrile vapour, (IPCS, 1993) The studies conducted by Pozzani et al ., (1959) in order to knowthe skin penetration of acetonitrile in rabbits demonstrated effective skin absorption of acetonitrile. Although there is evidence that acetonitrile is easily absorbed from the gastrointestinal tract, no quantitative data are available. (IPCS, 1993) . 

### Distribution Haguenoer et al ., (1975) studied the tissue distribution of acetonitrile and its metabolites in Wistar rats after intraperitoneal administration of 2,340 or 1,500 mg/kg of the compound . Acetonitrile was found in heart, lungs, liver, spleen, kidneys, stomach, intestines, skin, muscle, brain and testes. Free and conjugated hydrogen cyanide were also detected in all studied organs. Whole body autoradiography with 2-'4C-acetonitrile in mice demonstrated heavy location of acetonitrile and its metabolites in the liver, kidney, gastrointestinal tract, gallbladder and urinary bladder 5 minutes after dosing. At 24 and 48 hours radioactivity was still retained in the liver and gastrointestinal tract. Delayed accumulation and retention of 2-'°C-acetonitrile was demonstrated in the male reproductive organs and, to a lesser extent, in the brain. (Ahmed et al ., 1992) . There are no indications that repeated administrations of acetonitrile result on its accumulation in animal tissues 

### Biotransformation The toxicity of acetonitrile has been attributed to its metabolite cyanide (Willhite and Smith, 1981) which has been hypothesized to result from a cytochrome P-450- dependent oxidation of the nitrile at the alpha carbon, and subsequent degradation of the intermediate cyanohydrin, (Ohkawa et al ., 1972)

. -46-The results of human and animal studies indicate that cyanide formed in vivo is subsequently conjugate with thiosulphate to form thiocyanate, which is eliminated in urine. This conjugation is catalysed by the widely distributed enzyme rhodanese ("thiosulphate cyanide sulfur transferase: EC 2 .8.1 .1) (Pozzani et al ., 1959 ; Silver et al ., 1982; Willhite, 1983) . The release of cyanide from acetonitrile and its subsequent metabolism to thiocyanate have been studied under a number of experimental conditions and in several animal species. Experimental studies with rats, monkeys and dogs have documented cyanide in the blood and thiocyanate in the urine following exposure to acetonitrile via inhalation or injection, (Pozzani et al ., 1959) . In a study with mate CD-1 mice treated with various nitriles including acetonitrile, WiIlhite and Smith (1981) showed that tissue concentrations of cyanide taken at death or sacrifice 2 .5 h after injection of an ip LDso dose of certain nitriles were elevated in all cases, but considerable variation was noted from nitrile to nitrile. The livers consistently had higher concentrations of cyanide than the brains . Even within groups of animals given the same nitrile, there was considerable variation in the cyanide concentrations. Thehigher concentrations correlate with the more severe signs of intoxication. This large variation may be due to individual differences in the rate of cyanide liberation in vivo, to differences in the rate of cyanide`, biotransformation to thiocyanate or both. wllhite (1983) studied the in vivo biotransformation of acetonitrile in the golden hamsters. Non-pregnant female hamsters were given an oral dose or an intraperitoneal injection of 100, 200, 300 or 400 mg/kg body weight AN . The animals were killed 21/2 hours after dosing and the blood, brain, kidneys and liver level of cyanide and thiocyanate were determined. Elevated levels of cyanide and thiocyanate were detected in all studied tissues after both oral and intraperitoneal dosing. The liver t and kidneys contained greater concentrations of cyanide and thiocyanate than the

brain, and whole blood, liver and kidney thiocyanate concentrations were up to 10 times those in the brain. An increase in acetonitrile dose lead to in increase in metabolite concentrations. The pattern of biotransformation was similar regardless of the route of administration . After a single intraperitoneal administration of acetonitrile (2,340 mg/kg) in rats, all animals died in 3 to 12 . h and acetonitrile was found to be distributed in various organs. The free cyanide varied from 170pg/kg in the liver, to 3 .5 mg/kg in the spleen . Concentrations of combined cyanide in the liver, spleen, stomach and skin were 3 .6, 13.2, 17 .6 and 10.5 mg/kg tissue respectively, (Haguenoer et al ., 1975) . Thiosulphate is an effective antidote of cyanide poisoning. Willhite and Smith have reported significant reduction in mortality following thiosulphate prophylaxis of mice exposed to letal concentrations of acetonitrile. This indicates that cyanide played -47-an important role in toxicity; significant reductions in both mortality and the in situ cyanide concentrations were observed following thiosulphate prophylaxis of acetonitrile-injected mice. Pretreatment with a hepatotoxic dose of CCI, (a well-known modifier of the hepatic microsomal oxygenase system) provided significant protection against death fromip injection or inhalation of acetonitrile and resulted on a significant reduction in the in situ cyanide concentrations following treatment with acetonitrile, (Willhite, 1981 ; Willhite & Smith, 1981) . This indicate that there is little, if any, acute toxicity due to the parent nitrile itself in the absence of normal hepatic function. The metabolite of acetonitrile was more acutely toxic than the compound, (Willhite, 1983) . Ahmed and Farooqui (1982) measured cyanide levels one hour after administration of LD50 doses of several satured and unsatured nitriles to male Sprague-Dawley rats. Animals in groups of 6 were fasted overnight and a LD50 2460 mg/Kg of acetonitrile was administered orally. Control animal received equivalent volume of 0.9% NaCl . Following the treatment the animals were observed for 1 h and then killed by decapitation. At the time of killing blood was collected by completely draining the decapitated animals into heparinized tubes. Liver, kidney, brain . and gastric content were collected, frozen immediately in liquid nitrogen and stored at - 304C until the time of analysis. The conversion of acetonitrile to cyanide proceeds at slower rate than that of other nitriles. In fact, one hour after acetonitrile administration, the blood level of cyanide was much lower than those after acute doses of other nitriles. Peak concentrations of blood cyanide were found 7.5 h afteracetonitrile dosing and were comparable to those of other nitriles measured one hour after dosing. Brain cyanide concentration one hour after acetonitrile dosing was also lower than those after exposure to potassiumcyanide (KCN) or other nitriles. Urinary excretion of thiocyanate after exposure to various nitriles indicated that for acetonitrile the percentage of the dose excreted was lower than for other nitriles even though the absolute given amount of acetonitrile, based on its oral LDso, was much higher. These data indicate that the toxicity of acetonitrile is lower than those of cyanide and other nitriles as we can see comparing the oral LDso value of acetonitrile (2460 mg/kg body weight) with the oral LDso of the other nitriles (i .e . 40 mg/kg for propionitrile or 90 mg/kg for acrylonitrile or 10 mg/kg for potassiumcyanide 1 h. after oral LD50) (Ahmed & Farooqui (1982) . This is most probably due to the slower transformation of acetonitrile to cyanide and consequently, to the more efficient detoxification via thiocyanate excretion. Ohkawa et al ., (1972), have suggested that aliphatic nitriles may be activated by hepatic microsomal enzyme oxidation of the carbon alpha to the cyanide group. The resulting cyanohydrins may be unstable in vivo (as was acetone cyanohydrin) . Spontaneous decomposition of the intermediate cyanohydrin could liberate free cyanide. Several lines of evidence implicate cytochrome P450dependant enzyme system in the metabolism of nitriles to CN- Freeman & Hayes,(1985), in a study with rats pretreated with acetone (an inducer of cytochrome P-450), found that acetone -48- 

# 53stimulate acetonitrile metabolism when the two compound are administered simultaneously. They suggested that acetone potentiates acetonitrile toxicity via a biphasic effect on acetonitrile metabolism, consistent on, an initial inhibition followed by a stimulation of cyanide generation upon acetone elimination. Upon further characterization of this inhibition, acetone was found to fit a competitive model of inhibition, (Freeman $ Hayes, 1987) . 

### Elimination Pozzani et al ., (1959) first reported the presence of cyanide in the blood and thiocyanate in the urine following exposure to acetonitrile via inhalation or injection. The studies were conducted in rats, monkeys and dogs under different experimental conditions : 

### Fifteen male and fifteen female rats were exposed to acetonitrile vapour

(166, 330 and 655 ppm) seven hours per day, five days per week, for a total of 90 days. Some thiocyanate was excreted by the rats inhaling 330 and 166 ppmacetonitrile vapour, although the amount of thiocyanate excreted was not proportional to the concentration of acetonitrile inhaled. Thiocyanate was not completely eliminated between daily exposures, but was almost completely excreted during the 2.5-day rest period over weekends. Repeated inhalation studieswere also performedwith monkeys and dogs exposed to 350 ppmof acetonitrile in air, in the same manner as rats. The concentrations of thiocyanate in the urine of three dogs increased from69 to 252 mg/1 after the 5days inhalation period and from60 to 114 mg/I in the monkeys. Unlike the rats, dogs and monkeys continued to eliminate thiocyanate beyond the 2.5-day rest period over the weekend. In order to knowhow

much of the formed thiocyanate would be excreted three monkeys were injected intravenously with acetonitrile (0.1 ml/kg) . After four to eight weeks of this injection, they were injected with sodium thiocyanate (1 .55 ml/kg of a 10%solution in saline) . The percentages of the dose excreted as thiocyanate were 12% after acetonitrile and 55% after sodiumthiocyanate injection. It appears that more than 12 % of the injected acetonitrile was converted into thiocyanate. Haguenoer et al ., (1975) studied the elimination of acetonitrile and its metabolites in rats after a single dose of 600 mg/kg body weight of acetonitrile. It seems that the acetonitrile is almost completely excreted within four days . However, small amounts are found in the urine (2 .98 - 3.32% of the administered dose) . This is possible because there is an important elimination of acetonitrile via the lungs and because a part of acetonitrile is biotransformed to cyanide. Small quantities of free hydrocyanic acid (equivalent to 0 .03-0.04% of the administered acetonitrile) and bound hydrocyanic acid (equivalent to 2.01-2.60% of -49-

### the administered AN) were eliminated within 11 days of administration. Silver et al ., (1982) reported that the 24 h urinary thiocyanate excretion following po or ip administration of acetonitrile (30.8 mg/kg) in rats, was 11 .8 ± 2 .5 and 4

.4 ± 0 .5% of the administered dose, respectively. Compared with the excretion of other nitriles, the lower excretion of SCN- after administration of acetonitrile could be explained by either its greater water solubility or its relatively high volatility which could result in greater elimination of the parent compound . In fact, a high ratio of unmetabolized nitrile relative to SCN- was reported in the urine of rats dosed with acetonitrile by Haguenoer et al ., (1972) . This is also consistent with the hypothetic slowtransformation of acetonitrile to cyanide. In vitro studies 

### The production of cyanide and thiocyanate from acetonitrile have also been demonstrated in a variety of "in vitro" studies, (Ohkawa et al ., 1972) . Ohkawa et al ., (1972), found that the amount of hydrogen cyanide released in mouse liver microsomal preparations was increased greatly by the addition of NADPH. Freeman and Hayes (1987) found that microsomal metabolismof acetonitrile to Cyanide was oxygen and NADPH-dependent, inactivated by heat and antagonized by NADH . They studied in vitro the effects of known inhibitors of cytochrome P-450- dependent metabolic pathways, and found that carbon monoxide, metyrapone and SKF 525-A all inhibited the metabolismof acetonitrile to cyanide. Dimethyl sulfoxide and ethanol were also competitive inhibitors of acetonitrile metabolism and aniline HCI appeared to be a mixed inhibitor. Tanii and Hashimoto (1986) also reported that the metabolism of acetonitrile was NADPH dependent and enhanced by pretreatment with ethanol . Cobaltic protoporphyrin 9-chloride, which has been demonstrated to deplete hepatic cytochrome P-450content (Drummond &Kappas, 1982), markedly decreases the acetonitrile metabolism in isolated hepatocytes prepared from rats pretreated subcutaneously with cobalt-heme (90 Nmol/kg) 48 h. before killing, (Freeman &Hayes, 1987) . It is known that acetone as well as ethanol induces hepatic cytochrome P450 LM3a (LMeb) in rabbits. This isozyme is apparently similar to cytochrome P450j which has been isolated fromthe hepatic microsomes of isoniazid-treated rats by Ryan et al ., (1985) . Since the metabolismof acetonitrile is increased in animals pretreated with acetone, Freeman & Hayes (1987), suggested that acetonitrile is likely metabolized by this cytochrome P-450isozyme, cytochrome P-450j (LM3a, Web) . Agents (acetone, ethanol, dimethyl sulfoxide and aniline) that are substrates for, or inhibitors of this isozyme inhibited the acetonitrile metabolism . Therefore, acetoneand acetonitrile seem substrates for the same cytochrome P-450 isozyme. -50-



# 55. Dahl and Waruszewski (1989), studied the metabolismof acetonitrile to cyanide in male Fischer-344 rats nasal and liver tissues and found that the maximum cyanide production rates in nanomols cyanide/mg protein/minute by nasal maxilloturbinate and ethmoturbinate microsomes and liver microsomes for 1 millimolar initial substrate concentrations were 0, 0 .9 and 0

.098 respectively. The authors conclude that detoxication of inhaled acetonitrile may occur to a substantial extent in the nasal cavity. 

### 4 .1 .2.1 .2 . Studies in humans Acetonitrile is well absorbed by all routes. Studies on smokers showed that 91 ±49'0 of the acetonitrile inhaled in cigarette smoke was retained, (Dalhamn et al .,1968a) . Asignificant portion of acetonitrile could have been retained in the mouth, (Dalhamn et al ., 1968b) . There are no studies of oral or dermal absorption in humans; however, human poisoning cases indicate that acetonitrile is well absorbed by both routes. Amdur (1959) reported the presence of cyanide in the blood, urine and tissues, and thiocyanate in serum of workers accidentally exposed to acetonitrile vapour . No blood cyanide was found in three volunteers inhaling 40, 80 and 160 ppmof acetonitrile vapour for 4h, (Pozzani et al ., 1959) . There is no specific human study describing acetonitrile biotransformation and elimination. However, accidental poisoning cases indicate that acetonitrile is biotransformed to cyanide and thiocyanate, which are then excreted fromurine, (IPCS, 1993) . In case of suicidal oral acetonitrile ingestion, elimination half-lives of 32 h for acetonitrile and 15 h for cyanide were calculated during the hospitalization of the patient prior to death (Michaelis et al ., 1991) . 

### 4.1 .2.1 .3. Summary of toxicokinetics Acetonitrile is well absorbed from the lungs, gastrointestinal tract and through the skin, although there are not available quantitative data. Acetonitrile has a widespread distribution . It has been found in heart, lungs, liver, spleen, kidneys, stomach, intestines, skin, muscle, brain and testes. Free and conjugated hydrogen cyanide were also detected in all the studied organs, (Haguenoer et al ., 1975) .

There are no indications that repeated administrations of acetonitrile result on its accumulation in animal tissues. -51-The acetonitrile is metabolized to cyanide via cytochrome P-450 . Firstly, a cyanohydrin intermediate is formed and spontaneously decomposes liberating free cyanide. Several studies have indicated that cyanide formed in vivo is subsequently conjugate with thiosulphate to formthiocyanate, which is eliminated in urine, (Willhite, 1981 ; Willhite and Smith, 1981 ; Pozzani, 1959; Haguenoer et al ., 1975) ; Silver et al ., 1982; Ohkawa et al ., 1972) . The cyanide is responsible for the acetonitrile toxicity. The conversion of acetonitrile to cyanide proceeds at a slower rate than that of other nitriles, (Ahmed & Farooqui, 1982) . This explains the lower toxicity of acetonitrile comparing with other nitriles. 

### The microsomal metabolismof acetonitrile to cyanide was found to be oxygen and NADPH dependent, inactivated by heat and antagonized by NADH. Since the metabolism of acetonitrile is increased in animals pretreated with acetone, Freeman 4 and Hayes (1987) suggested that acetonitrile is likely metabolized by the cytochrome

P450j (LM3a, LMeb) . Elimination of acetonitrile occurs primarily through urinary excretion of the unchanged compound and free and bound hydrogen cyanide. Pulmonary clearance of unchanged acetonitrile via exhalation is also an important pathway of elimination, especially at high exposure levels, (Haguenoer et al ., 1975) . 4 .1 .2.2. Acute toxicity 

### 4 .1 .2.2.1 . Studies in animals Oral A large number of LDso values for male and female Carworth Farms-Wistar or Nelson albino rats, in various assays over a five-year interval, ranged from 1 .7 to 8 .5 ml/Kg (1327-6762 mg/Kg using a density value of 0 .79g/ml) by gastric intubation. The males appeared to be two to four times as susceptibles as females to undiluted acetonitrile by this route, but this difference was not important when the animals were fasted overnight before intubation .ln many instances delayed deaths occurred at or, above the LDso dosage level . However, the highest dosage levels caused rapid deaths. Acetonitrile diluted in corn oil, water or 1% aqueous Tergitol 7 is apparently better absorbed fromthe intestine and in most assays is somewhat more toxic for both male and female rats than is the undiluted compound, (Pozzani et al ., 1959) . In the same study Pozzani, obtained a LDSO0 .177 ml/Kg for male guinea pigs by gastric route (fasted) ; the acetonitrile was undiluted. Significant differences in LDso values were found between 14-day-old and adult rats, but not between young adults (80-160g body weight) and older adults (300- 470 gr body wight) (Kimura et al ., 1971) . Male Sprague-Dawley rats in groups of 6 -52-were used for studies in the young and older adult rats, groups of 6-12 rats of both sexes were used for the newborn and 14-day old rat toxicity studies. The acetonitrile was given orally via straight needle in undiluted formin nonfasted rats, and the animals were observed for a week following medication. LDso value of 0 .2 ml/Kg was obtained using wealing (14-day) rats and LDso of 3 .9 and 4

.4 ml/Kg using young and older adults respectively. Tanii and Hashimoto (1984) studied the mechanismof acute toxicity of nitriles in mice, using four animals per dose level and four different doses. The LD50 was 269 mg/Kg, using male mouse strain ddY by oral route and the observation period was 7 days. 

### Inhalation Pozzani et al ., (1959) investigated the mammalian toxicity of acetonitrile. Groups of 1-3 male dogs were exposed to 2000, 8000, 16000 and 32000 ppmacetonitrile during 4 hour . There were no deaths in either of the lower two groups (1 and 2 dogs exposed) . All those exposed in the upper two groups died (1 and 3 exposed) . Exposure for 8 hours (12 male and 12 female Nelson rats at 6concentrations, 32000,16000, 8000, 4000, 2000 and 1000 ppm) generated LC, values of 7551 ppm and 12435 ppm for males and females respectively. This apparent difference in sensitivity between males and females is not evident in the 4 hour tests (LCso 16000 ppm) . Prostration and convulsive seizures often preceded death. Marked to moderate pulmonary haemorrhage and congestion was evident both in survivors and in those which died. A LC502828 ppm . was stableished for rabbit exposed to 3 concentration of acetonitrile during 4hour; and LC50 5655 ppm was stableished for guinea pig, when groups of 6 (males and female) were exposed to 3concentrations of acetonitrile during 4 hour .Dogs appeared to be most resistant to the vapour . The rabbit and guinea pig are somewhat more sensitive. There was agreater difference in individual susceptibility to the vapour in rats than in the other three species tested . Willhite, (1981) exposed male CD-1 mice for 60 min. to concentrations of acetonitrile ranging from 500 to 5000 ppmand were maintained for the following 14 d.The LCso value for acetonitrile was 2,693 ppm . Exposure to 5000 ppm killed all mice within 2 hours. Toxic effects included: intense dyspnea, gasping, tremors, convulsions and corneal opacity after 30-300 minutes. Gross autops, of mice which died showed only particularly red livers in some cases. Groups of 6 or 12 pregnant Syrian golden hamsters were exposed on day 8 of pregnancy to 1800, 3800, 5000, 8000 ppmacetonitrile during 1 hour (Willhite, 1983) . Sacrifice occurred on day 14. There were no overt signs of toxicity in those exposed to the lowest concentration. One of 6 animals died in the 3800 and 5000 ppm groups and 3 of 12 died in the highest concentration group. Overt effects included: Eye irritation, dyspnea, tremors, hypersalivation, ataxia, hypothermia, respiratory difficulty and coma. Histopathological examination of liver, kidneys and lung of affected animals -53-did not reveal any abnormalities. Onefemale monkey exposed to 2510 ppm acetonitrile vapour, appeared normal after the first inhalation day (7 h) but, showed poor coordination followed by prostration and laboured breathing during the second inhalation day . Death occurred a fewhours later. Engorgement of the dural capillaries and pleural effusion was found. Tissues were not examined microscopically because some autolysis occurred before autopsy (Pozzani et al ., 1959) . 

### Dermal Acetonitrile has and LD50 by rabbit skin penetration of 1 .25 (0.84 to 1 .85) ml/Kg of the undiluted compound applied under polyethylene sheeting . It is of interest to note that the toxicity of acetonitrile by skin penetration is increased when application is made as a 75% (v/v) aqueous solution. The LD50 is 0

.50 (0.37 to 0

.67) ml/Kg of the 759'o aqueous solution (Pozzani, et al ., 1959) . Neat acetonitrile was applied in covered contact to the clipped skin apparently of 6rabbits (Smyth and Carpenter, 1948) . Contactwas maintained for 4days, followed by observation for 10 days or until death (Smyth and Carpenter, 1944) . The LDso was given as 5ml/Kg; a density of 0 .79 g/ml has been used . 

### Other routes Female rats (Wistar or Nelson albino) were given acetonitrile undiluted by i .p. route. Two extreme LD50 values of 7.96 and 0 .85 ml/Kg were found. Adensity value of 0 .79 g/ml has been used to convert these to 6288 and 672 mg/Kg respectively. When diluted in saline the LD50 range was 3073-4440 mg/Kg. It is unclear why the range for neat acetonitrile should be so great (Pozzani et al ., 1959) . Different values for the LD50 by i .p. route on mice (NMRI-SPF or CD-1 strains) have been obtained by several authors using acetonitrile in water or saline : 175 mg/Kg (Willhite and Smith, 1981) ; 198 mg/Kg (Pozzani et al ., 1959) ; 400 mg/Kg( Zeller et al ., 1969) and 521 mg/Kg (Yoshikawa 1968) . In the study of Willhite and Smith, five or six doses between 50-862 mg/Kg of acetonitrile in water, were administered i .p. to groups of nine or ten CD-1 male mice. The animals were observed for the following 7 days and the mean time to death was 423 ± 503min. Intoxication syndrome was produced within 1 to 5 hr, included: intense dyspnea, gasping, ataxia, corneal opacity, hypothermia and convulsions. Cuny et al, 1932, reported a. LD50 of 3950 mg/Kg for acetonitrile when it was injected subcutaneously in to rats (cited in Pozzani et al ., 1959) . There is only a reference with respect to route of administration intravenous in the IUCLID (Pozzani et al ., 1959) . An unstated number of male or female Wistar or -54- 

# syalbino rats, treated with neat acetonitrile, had LDso of 1 .68 ml/Kg (1327 mg/Kg using a density value of 0

.79 g/ml for conversion) in both cases. 4 .1 .2.2.2. Studies in humans 4 .1 .2.2.2.1 . Inhalation In a human volunteer study, Pozzani et al ., (1959) studied the acute inhalation toxicity of acetonitrile in three men between the age of 31 and 47 inhaled 40 ppm for 4 hours. Only the youngest subject experienced a slight tightness in the chest during the evening after inhalation and was a slight increase in the urinary thiocyanate levels. There was no detectable blood cyanide in any of the subjects . The two older subjects inhaled 80 ppmacetonitrile vapour for 4 h one week after the 40 ppmtrial, with no symptoms. No blood cyanide was detected in any of the samples taken after the inhalation period. Nine days later, these two subjects inhaled 160 ppm for 4 hours. One subject reported a slight transitory flushing of the face 2h after inhalation, and a slight feeling of bronchial tightness about 5 h later. The blood cyanide and urinary thiocyanate levels of both subjects, did not change significantly. Some cases of severe intoxications for exposure to high concentrations of acetonitrile vapor have been reported: Grabois (1955) reported on 16 workers at a Chemical plant accidentally ~ poisoned with acetonitrile vapour during the brush painting of the inside walls of a storage tank. Acomprehensive discussion of this incident has been given by Amdur (1959) . The paint contained 30-409'o acetonitrile and the thinner contained 90-95% acetonitrile. Because of the viscosity of the paint, the tank was heated to 25oC and thinned on the second day before application. Ventilation of the tank was stopped. One died after two days exposure, two were seriously ill and the remaining 13 workers were also affected . Symptoms included weakness, nausea and vomiting. Convulsive seizure and coma were reported prior to death. Post-mortemexamination revealed cerebral, thyroid, liver, splenic and renal congestion, and a "peach pit" odour of all tissues. The blood and urine cyanide concentrations were 7960 and 2150 mg/litre, respectively. There was a trace of cyanide in the gastric fluid. Spleen, kidney and lung concentrations of cyanide were 3180, 2050 and 1280 mg/Kg tissue, respectively. No cyanide was detected in the liver. The worker who died had worked inside the tank for about 12 hours. Of the two other seriously affected workers, one had worked inside the tank near three hours and the other had spent 12 hours painting around the ports fromthe outside of the tank but inside the last hour . Two other men, less severely affected, had painted inside the tank for no more than 2

.5 hours each on the 2nd day. Following the incident, the paint ingredients were not heated, adequate ventilation was provided and the concentration of organic cyanide was kept below 17 ppm . There were no further incidents . Dequidt et al ., (1974) reported a heart failure and death occurred by acute -55-acetonitrile poisoning in a 19-year-old male photographic laboratory worker . After handling acetonitrile for 2 days, he poured an unknown amount of acetonitrile and boiling water on the floor to clean it. Four hours after work he complained of epigastric pain and nausea and vomited repeatedly. Next day he became comatose and had convulsions. Large amounts of cyanide, thiocyanate and acetonitrile were found in the blood and urine. He died 6 days after the poisoning. 

### Dermal Acase of paediatric accidental exposure to an acetonitrile-containing cosmetic were reported by Caravati & Litovitz (1988) . 

### The exposure occurred both via the skin and by inhalation. Approximately 30 ml of a nail remover containing 98-10096 acetonitrile spilled on a 2-year-old (12Kg) previously healthy boy and his bed (amount of contact to the skin was not specified) . No symptoms were noted immediately after the exposure. Eight hours later, the boywas moaning, poorly responsive, and had vomited. He became lethargic and pale . Whole-blood cyanide levels were: 6mg/litre 12 h after the exposure, 60-70 pmol/litre from 24 to 48 h and 15 Nmol/litre after 60 h. The patient was discharged 3 days later in good condition. 

### Oral Caravati and Litoviz (1988) reported on a 16 month-old (11 .8 Kg) boy who ingested 15-30 ml of a nail remover (1-2g acetonitrile/Kg body weight) . The child vomited spontaneously about 20 min after the ingestion. About 12 h after the ingestion he was found dead . Postmortemexamination showed moderately severe pulmonary oedema, a blood cyanide level of 119 mg/Kg and brain cyanide level of 0 .2 mg/Kg. A case of acute acetonitrile intoxication in a 26-year-old man has been reported by Jaeger et al ., (1977) . The man ingested 40 g of acetonitrile in a suicide attempt . After a 3 h latent period, he suffered fromvomiting, convulsions, coma, acute respiratory insufficiency, severe metabolic acidosis and two cardiac arrests. Recovery occurred over the following three months after treatment . A 22 year old female ingested an unspecified amount of acetonitrile and acetone. She died about 30 hours later. Postmortemexamination revealed pulmonary oedema and haemorrhagic gastritis (Boggild et al ., 1990) . Turchen et al ., (1991) reported the case of 39 year old woman, who was found vomiting and confused 7 h after ingesting 59 ml of nail polish remover containing 99% acetonitrile . After 12 h ingestion, she developed severe metabolic acidosis, seizures and shallowrespiration. Eight hours after ingestion she had a whole blood cyanide level of 3130 Ng/litre. At 65 h the serumcyanide level was 10 mg/I and thiocyanate 120 -56-_~ C'r-/,Ot4 ;Vti~ (~ 

## 10A's- P_



# . mg/I, whereas at 77 h were 12 mg/I and 30 mg/I, respectively. She responded to the treatment with sodiumnitrile and sodiumthiosulphate. On the fifth hospital day the cyanide level was 360pg/I . and thiocyanate level 30 mg/I . The patient was discharged on day six. A case of acute acetonitrile poisoning of a 3-year-old (17.2 Kg) child was reported by Geller et al ., (1991) . The child ingested 15-30 ml of a nail tip and glue remover containing acetonitrile . The amount of acetonitrile ingested was estimated to be 0 .8-1 .7 g/Kg. Gastric lavage was performed. Three hours and 45 min. after ingestion, the cyanide blood level was 1 .24 mg/l and thiocyanate 11 mg/I . Thirteen hours after the ingestion the child vomited, was confused and developed seizures. The child was treated and discharged fromhospital 42 h after the ingestion. Kurt et al, (1991) reported a case of a 15.8 Kg 2-year-old girl who ingested 5- 10 ml (0.25-0

.5 g/Kg) of a nail glue containing 84%acetonitrile. The following morning she was moaning, restless and vomiting. Toxic clonic seizures also appeared about 14h after ingestion, and became comatose with hyperpnoea and tachycardia and with marked hypoxia and acidosis. She was treated and discharged from hospital after 2 days. Vomiting occurred within 6 hours of a 23 month-old child ingesting 60 ml of a product containing at least 98%acetonitrile. He became unresponsive 24h after the ingestion. He was treated and discharged from hospital on the third day (Losek et al ., 1991) . . . - , Michaelis et al ., (1991) reported a case of suicidal oral acetonitrile ingestion in a previously healthy 30-year-old- man. He ingested --about-5 mi .-(64 mg/Kg) . of acetonitrile (98 %) and, 30 min, -later, about 1 ml of ammonium :,and . vomited once. : . Gastric lavage was performed 5 .5 h after ingestion. Peak serumacetonitrile and blood cyanide levels were 99.2 and 15.0 mg/I . Half-lives were calculated for acetonitrile and cyanide and found to be 32 and 15h, respectively. 

### Two fatal cases of a couple who ingested acetonitrile by mistake was reported by Jones et al ., (1992) . They were found dead with traces of vomit . Acetonitrile levels were 0.8 g/I in blood, 1 .0 g/I in urine and 1 .3 g/I in stomach contents. Blood inorganic cyanide levels were 4.5 mg/I (male) and 2.4 mg/I (female) . 4 .1 .2.2.3. In vitro studies Knox et al ., (1986) studied cytotoxicity of acetonitrile in BCL-DL cells. The method used was dybinding and the harvest time 72 h, the results were IC20>24 mM, IC50>24mMand ICso>24mM . Clothier and Hulme (1987) using Mouse 3T3-4 cells, FRAME Kenacid blue method and harvest time after 72 h obtained IC50 = 562 mM Dierichx (1989) obtained IC50 = 494 mMusing human hepatoma Hep G2, 24 h harvest time and cellular -57- 

# 62protection content as method. In like manner, the IC50 values in mouse neuroblastome cells and in rat glioma cells, were 17.8 and > 20 mM, respectively. 

### 4.1 .2.2.4. Summary of single exposure studies Different animal species and individuals of the same species varied widely in susceptibility to acetonitrile in single-dose toxicity studies by various routes. The range of LDso values acetonitrile in mammals is between 165 and 6762 mg/Kg body weight. Mouse and Guinea-pig seemto be the most sensitive species. These studies were performed without GLP information. Astudy showed that acetonitrile was more toxic to immature rats (14-day-old) than to older rats given oral doses of 160-3500 mg/Kg. Other study reported that using male or female wstar or Nelson albino rats, the males where more susceptible than the females; 8

.53 ml/Kg for females and 1 .68 ml/Kg for males, the route was by gavage. The main symptoms in animals appear to be prostration followed by seizures and convulsions. Acetonitrile has rabbit dermal LDsa of 1 .25 ml/Kg. It is of interest to note that the toxicity by skin penetration is increased when application is made as a aqueous solution . Dermal application of acetonitrile causes systemic toxicity. The 8h inhalation LCSo in male rats is 7500 ppm(13,740 Mg/M3)

. Rabbits and mice were more sensitives than rats, with LC50 of 2828 ppm and 2693 ppm, respectively and time of exposures of 4 hour for rabbits and 60 min for mice. A4-hour exposure of dogs at concentrations up to and including 8000 ppm produced no deaths, but deaths did occur at concentrations of 16,000 and 32,000 ppm . Gross pathology indicated pulmonary haemorrhage and vascular congestion. Animals exposed to acetonitrile via different routes of dosing always showed respiratory symptoms: rapid and irregular respiration, laboured or difficult breathing and intense dyspnea. The determination of blood cyanide and urinary thiocyanate should not be relied upon as evidence of brief inhalation to lowconcentrations of acetonitrile vapour . No blood cyanide was found in human subjects inhaling 40, 80 and 160 ppm vapour, and there was no correlation between thiocyanate excretion and acetonitrile concentration. The variability of subjective responses of humans to 40, 80 and 160 ppm acetonitrile vapour suggests that, even if a concentration of the solvent vapour were selected that would not endanger the health of the majority of workers, it might cause discomfort to some. Symptoms and signs of acute acetonitrile intoxication include chest pain, -58-tightness in the chest, nausea, emesis, tachycardia, hypotension, short and shallow respiration, headache, and seizures . The systemic effects appear to be largely attributable to the conversion of acetonitrile to cyanide. Blood cyanide and thiocyanate levels are elevated during acute intoxication. Two deaths after exposure to acetonitrile vapour in the workplace and one fatal case of a child ingesting and acetonitrile containing cosmetic have been reported . Elevated tissue cyanide concentrations were found in postmortemexamination of these cases. In vitrostudies acetonitrile was weekly cytotoxic in a range of cultured cells. The concentration which reduced cellular protein by 50%were about 0 .7-23 g/I . In the Annex I of Directive 67/548/EEC, acetonitrile is classified as Toxic (T) ; Rphrases 23/24/25: Toxic by inhalation, in contact with skin and if swallowed. 

### 4.1 .2.3. Irritation 4 .1 .2.3.1 . Studies In animals Skin Data sheet IUCLID includes two studies about skin irritation. In one study a cotton pad (2.5 cm

x 2 .5 cm) saturated with acetonitrile is placed on the skin of white rabbits for 15 minutes or 20 hours. After the 15 minutes application the treated area was first washed with undiluted polyethyleneglycol 400and finally washed with 50 % aqueous polyethyleneglycol 400, after 20 hour treatment however, the skin was not washed. The reaction was observed after 1,3 and 8 days as well as after removed of the pad. No reaction was seen at any time, after either exposure period (Zeller H . et al ., 1969) . Smyth and Carpenter (1948) describe the skin irritancy of acetonitrile as similar to that of acetone. The method used in the determination is apparently that of Smyth and Carpenter 1944, in which 0 .01 ml of the neat material was applied to the clipped belly of an albino rabbit . Observations were made after 24 hours. In one standard test (Clayton and Clayton, 1982) acetone is described as slightly irritant to the skin. Eye In studies in 5 rabbits, 1 drop of neat acetonitrile was instilled into the eye. After 24 hours the effects were graded as 5 on a scale 1-10 (Smyth and Carpenter, 1948) . The same authors (1944, 1946) giving details of the test and of data interpretation . The 1946 paper states that a score of >_5 represents severe injury. Zeller, et al ., (1969) tested the acetonitrile, one drop of neat acetonitrile was applied to the conjunctival sac of the rabbit's eye. The reaction was noted after 10 minutes, 1 and 24 hour, 3 and 8 days. An irritation of score of 3, out of a possible maximumof 6 ; strong vascular injection with edema or slight necrosis, was recorded. -59-



# 6yPregnant hamsters exposed to 1800, 3800, 5000 and 8000 ppm acetonitrile via inhalation for 60 min. caused irritation to the eyes and nose in 4 of 12 in the top concentration group and irritation (without to specify) to all exposed to 5000. (Willhite, 1983) . Corneal opacity has been observed after either inhalation or intraperitoneal administration of acetonitrile in male mice (Willhite, 1981 ; Willhite and Smith 1981) . 

### 4.1 .2.3.2. Studies in humans Humans accidentally inhaling 500ppm for brief periods, reported some irritation of nose and throat (IPCS 1993) . 4 .1 .2.3.3 . Summary of irritation The informationfrom

studies in animals indicate that acetonitrile is slightly irritant to the skin and one drop of neat acetonitrile applied to the conjunctival see produced strong vascular injection with edema or slight necrosis. When the exposition is via inhalation, acetonitrile cause irritation to the eyes and nose in concentrations of 8000 

### ppm . Although these studies were performed without GLP information and without according to OCDE guidelines, all available data indicates that liquid acetonitrile is a eye irritant and the risk phrase R36 (Irritating to eyes) should be included in the proposal of classification. 4.1 .2.4. Corrosivity The studies in animals and humans in 4 .1 .2.3. indicate that acetonitrile is not corrosive to the skin or eyes. 4 .1 .2.5. Sensitization No data is available on sensitization of acetonitrile in experimental animals. 4 .1 .2.6. Repeated dose toxicity 4 .1 .2.6.1 . Studies in animals Inhalation 

### Studies in rat The National Toxicology Program (1994), has well conducted a subchronic inhalation study, according to OCDE guideline No 413, on rats exposed to acetonitrile for 13 weeks. Groups of 10 male and 10 female F344/N rats were exposed to 0, 100, 200, -60- 

# 65

### 400, 800 or 1600 ppm (equivalent to 0, 168, 335, 670, 1,340 or 2,681 mg/m3) acetonitrile by inhalation for 6 hours per day, 5 days per week for 13 weeks. Animals were approximately 6 weeks old on the first day of exposure. Six male and three female rats exposed to concentrations of 1,600 ppm and one male exposed to 800 ppm died during the study

; all but one of these deaths occurred during the first 2 weeks of the study. At exposure concentrations up to and including 800 ppm, the final mean body weights and body weight gains were generally similar to controls. At the 1600 ppm concentration, body weight gains were lower than controls; the final mean body weights were 81 %of the control value in males and 91 % of the control value in females. Hypoactivity and ruffled fur were observed in 800 ppm males and 1,600 ppm males and females during the first week of the study. Additional clinical findings in 1,600 ppmmales that died during week 1 were ataxia, abnormal posture, and clonic convulsions. An anaemia evidenced by decreases in red blood cell count, haemoglobin concentration, and haematocrit, occurred in the 1,600 ppmmales and females and in 800 ppm

female rats. The anaemia was characterized as nonresponsive, normocytic, and normochromic because the reticulocyte counts, mean erythrocyte volume, and mean erythrocyte haemoglobin concentrations in these groups were similar to control values. In the 1,600 ppm female rats, decreases in triiodothyronine (T3) concentration occurred in the absence of alterations in thyroxine (T4) and thyroidstimulating hormone (TSH) concentrations. 

### Minor, sporadic changes in other parameters were considered unrelated to treatment . Gross and histopathologic changes were restricted to the 800 ppmmale and 1,600 ppmmale and female rats that died during the study; these included lung lesions consisting of congestion, edema, and haemorrhage in alveoli . Aspectrumof lesions that included brain haemorrhage; cellular depletion of the bone marrow

; thymic atrophy; lymphoid depletion of the spleen (females) and depletion of corpora lutea in the ovary were typically noted primarily in animals dying early. 

### In this study a NOAEL: 400 ppmand a LOAEL: 800 ppmwere established . In a 92-day unpublished study, reported as an abstract, acetonitrile was administered by inhalation to Fischer-344 rats at concentrations of 0, 25, 50, 100, 200 and 400 ppm

., 6 hours/day, 5 days/weed. All groups were composed of 10 animals of each sex. One male in the 400 ppmgroup died during the study. There was slight cytoplasmic vacuolization of hepatocytes in females at 400 ppm . Marginal decreases in mean leucocyte counts were reported in males at 100 and 200 ppmand in both males and females at 400 ppm (Coate W .B., 1983) . In a published study of Pozzani et al ., 1959a, rats Wistar were exposed to 0, 166, 330, and 655 ppm acetonitrile vapour, 7h/day, 5 days/week, for a total of 90 days. All groups were composed of 15 animals of each sex, however, there were two sets -61-

### of controls groups (60 animals in all) . The body weight gains and the organ weights did not differ significantly from the controls. Furthermore, the fewdeaths that did occur were attributable to extraneous infection, and their incidence was unrelated to concentration. The tissues of the rats that survived the inhalation of acetonitrile for 90 days were examined microscopically. Of the 28 rats inhaling 166 ppmvapour, 1 had histiocyte clumps in the alveoli, and another had atelectasis. Only three cases of tissue abnormality were observed in the 26 rats inhaling 330 ppmof the vapour . These involved lung changes such as bronchitis, pneumonia, atelectasis, and histiocyte clumps in the alveoli . A small lung granuloma was found in 1 of the 29 rats which served as controls for these two inhalation levels. After the inhalation of 655 ppm acetonitrile vapour, 10 out of 27 animals showed alveolar capillary congestion and/or focal oedema in the lug, often accompanied by bronchial inflammation, desquamation and hypersecretion. Tubular cloudy swelling of the kidneys in eight rats and swelling ( of the livers of seven rats were observed .

Statistical analysis showed that these lung, kidney and liver effects occurred to asignificantly greater degree than in the control animals with P-values of 0 .001, 0 .005 and 0 .04 respectively. No lesions were found in the adrenals, pancreate, spleens, testes and tracheas examined. Focal cerebral haemorrhage was observed in one of the five brains examined. The significance of the effects in the lower groups is unclear. 

### Some thiocyanate was excreted by the rats inhaling 330 and 166 ppm acetonitrile vapour, although the amount of thiocyanate excreted was not proportional to the concentration of acetonitrile inhaled. The urinary thiocyanate apparently was not excreted entirely during the 17-hour rest period between inhalation periods but was virtually all excreted during the 2.5 day rest period over the week end.The haematocrit and haemoglobin values of the five female rats inhaling 655 ppmacetonitrile did not differ significantly from the controls. A NOAEL: <166 ppmand a LOAEL: 166 ppmwere established. Roloff et al ., (1985, abstract) exposed groups of male and female rats (strain unspecificied) to acetonitrile vapour: 0, 618, 1847, 6239 ppm(0, 1038, 3104, 10485 mg/m3) for 1 month (6h/day, 5days/week) . Acitation of this summary states that more of 40 tissues were examined per animal (Johannsen and Levinskas, 1986) . Afull report of this study has apparently not yet been published. Death and reduced body weight gains were observed at the highest exposure level . Respiratory and/or ocular irritation were noted in animals exposed to 1847 and 6239 ppm . Mild anaemia was seen in animals exposed to acetonitrile ; it is not clear at which concentration. A NOAEL of 618 ppmwas established. 

### Studies in mice The National Toxicology Program (1994), has well conducted a subchronic inhalation study, according to OECD Guideline No 413, on mice exposed to acetonitrile for 13 weeks .Ten mice (B6C3F1) of each sex were exposed to acetonitrile vapour at 0, 100, 200, 400, 800 and 1600 ppm, 6h/day, 5 days/week. All groups were composed of 10 mice of each sex. -62-All male and female mice in the 1,600 ppm groups died by week 3of the study. Six additional animals, one female receiving 400 ppmand one male and four females receiving 800 ppm

also died before the end of the study. The final mean body weights and body weight gains of all female exposure groups that survived were similar to controls. The final mean body weight of all exposed male groups were slightly lower than that of the controls. Hypoactivity and a hunched, rigid posture were observed in 800 and 1,600 ppmmice during the first week of the study. Absolute liver weights were significantly greater than those of the controls in males exposed to concentrations of 200 ppmand above and in 800 ppmfemales; relative liver weights were significantly, greater in all exposed male groups and in females exposed to 400 ppmor higher. 

### Microscopic lesions were observed in the forestomach, liver, and adrenal gland. Grossly, focal or multifocal pale, dark brown or black lesions were consistently observed in the mucosa of the anterior forestomach of male and female mice exposed to concentrations of 200 ppm (7), 400 ppm (8), 800 ppm(8) and 1600 ppm (5), in females and in males the incidence was 400 ppm(5), 800 ppm(6) and 1600 ppm(1) . Microscopically, these lesions corresponded to focal or multifocal squamous epithelial hyperplasia. The average severity of these lesions was similar between exposure groups, with the exception of female mice exposed to 200 ppmin which the lesions were less prominent . Focal ulcers associated with areas of epithelial hyperplasia occurred in one female exposed to 200 ppm and one male and five females exposed to 1600 ppm . A high incidence of hepatocellular cytoplasmic vacuolation occurred in the male and female mice exposed to 400 ppm and 800 ppm

. Vacuolation appeared to be a slight distension of preexisting cytoplasmic clear spaces and is considered to represent increased glycogen storage. The absence of such changes in hepatocytes of 1,600 ppmmice that died may be indicative of increased utilization of hepatocyte glycogen stores .Additional alterations that occurred only in mice that died during the study, including lymphoid depletion and lymphocytolysis in the thymus, spleen, and bone marrow, and pulmonary congestion, were considered to be nonspecific changes typically observed in moribund animals. In this study a NOEL females: 100 ppm and 200 ppm for males were established. An unpublished study, acetonitrile was administered by inhalation to B6C3F1 mice at concentration of 0, 25, 50, 100, 200 and 400 ppmduring 92-day (Coate, W .B.,1983) . All group were composed of 10 animals of each sex. One male in each of the 50, 200 and 400 ppmgroups died. There was an increase in body weight gain in all males exposed to 50, 100, 200 and 400 ppm, and in females of the 200 and 400 -63-ppm groups ; body weight gain was decreased by comparison with controls in the 25, 50 and 100 ppm

female groups. The statistically significant effects in the mice included decreased blood urea nitrogen levels, red blood cell cholinesterase counts and haematocrits in females exposed to 200 or 400 ppm of acetonitrile. Hepatic vacuolization and hypertrophy were observed at all dose levels evaluated including controls, but the intensity of the hepatic lesions appeared greater in male and female mice exposed to 200 or 400 ppmof acetonitrile . Elevated relative liver weights were observed in males at 400 ppm and in females at 100 and 200 ppm . Adose related decrease in leucocyte count and serum Ig G levels occurred, but did not appear to indicate specific T- or B- lymphocyte dysfunction, and the biological significance of these findings is unclear. 

### A NOAEL 100 ppmand LOAEL 200 ppmwere established. Immuquest Labs-Inc. (1984) exposed groups of unspecified numbers of female B6C3F1 mice to acetonitrile at 0, 100, 200 or 400 ppm, 6 hours/day, 5 days/week for 90 days. There were no effects on physical appearance, body weights or gross appearance at necropsy. Mice in the 200and 400 ppmgroups had atrophy of thymus, which was noted upon histopathological examination. Slight vacuolization of hepatocytes accompanied by hydropic degeneration was also observed at 400 ppm . There were no treatment-related effects on selected clinical 'chemistries, but mice at 200 and 400 ppm had dose-related decreases in haematoc~its, blood haemoglobin concentration, erythrocyte and leucocyte counts. Changes in haematological parameters were not significant at 100 ppm . No significant effects on organ weights were observed, but the investigations noted a trend toward depressed thymus weights at 200 and 400 ppm . After 14 days, Immuquest Labs, evaluated immune function in mice exposed by the same protocol . Asignificant dose-related depression of serumconcentration of Ig G was observed in all exposed mice, but no effects were observed on the Cunningham plaque-forming response to sheep erythrocytes, the lymphocyte blastogenesis test, delayed hypersensitivity response or susceptibility to challenge with PyB6 tumor cells. 

### Study in dog and monkeys Three male dogs inhaled 350ppm acetonitrile (7h/day, 5days/week) for 91 days (Pozzani, et al ., 1959) . A statistically significant drop in the mean body weight was observed on the third and fifth inhalation days. The haematocrit and haemoglobin values of the three dogs were depressed the fifth week of inhalation, but with the exception of one dog. There was a return to preinhalation values toward the end of the 91-day inhalation period.The depressions in the those values were not accompanied by any significant deviation in the erythrocyte counts. No significant gross pathologic change was seen in the dogs. Histological examination of the tissues revealed some focal emphysema and proliferation of alveolar septa LOAEL: 350 ppm . Two studies have been carry out with Rhesus monkeys. In an inhalation study -64-99 days (7 h/day, 5 days/week) four Rhesus monkeys: one female was exposed to 2510 ppm, two females to 660 ppmand one male to 330 ppm(Pozzani, et al, 1959) . None of the four monkeys experienced an appreciable weight loss during their respective inhalation periods. The monkey at the highest concentration 2,510 ppm, appeared normal after the first inhalation day but showed poor coordination followed by prostration and laboured breathing during the second inhalation day. Death occurred a fewhours later. In the two monkeys exposed to 600 ppm there was also incoordination fromthe second week. One monkey died on day 23 and the other on day 51 .The monkey which inhaled 330 ppm

exhibited overextension reflexes and considerable excitability toward the end of its 99-day inhalation period and was sacrificed then. These three animals on gross examination showed focal dural or subdural haemorrhage in the parietal and/or occipital tissues immediately adjacent to the superior sagittal sinus. Engorgement of the dural capillaries and pleural effusion were found in the female which died at the 2,510 ppmlevel . Tissues from Monkey, which inhaled 2,510 ppm, were not examined microscopically because some autolysis occurred before autopsy . Examination of the tissues frommonkeys which inhaled 660 ppm, revealed focal areas of emphysema and atelectasis with occasional proliferation of alveolar septa. Focal cloudy swelling of the proximal and convoluted tubules of the kidneys was seen. The cause of death was not explained by the lesions observed in the tissues examined microscopically

. The monkey, which inhaled 330 ppm vapours, had chronic pneumonitis as evidenced by diffuse proliferation of alveolar septa, monocytic infiltration, and pleural adhesions.The cause of death of the other two was not explained by the lesions observed in the tissues examined microscopically In another inhalation study (Pozzani, et al, 1959) three male Rhesus monkeys were exposed to 350 ppmacetonitrile (7 h/day, 5 days/week) for 91 days, and at the end of the study the animals were sacrificed. At autopsy, slight to moderate haemorrhage of the superior and inferior sagittal sinuses were found in the brains of all three monkeys. Small discrete caseous nodules were seen in the lungs of two monkeys, as well as a pale liver in one of these two. Microscopic examination of tissues showed focal emphysema and diffuse proliferation of alveolar septa. Acute bronchitis was observed in one animal, and lung tissues from all three monkeys invariably contained focal accumulations of pigment-bearing macrophages. 

### Cloudy swelling of the convoluted kidney tubules, primarily proximal, was noted in two of the three monkeys . NOAEL < 330 ppm, LOAEL = 330 ppm . Oral Data regarding repeated dose toxicity of acetonitrile could not be located in the available literature . In the absence of oral data, inhalation data may be utilized . The available inhalation studies have been reviewed above. In this studies, mice appear to be more sensitive than rats to the effects of acetonitrile . Although monkeys and dogs may be more sensitive than mice, these species were only examined in preliminary evaluations utilizing few animals at a single exposure level . Considering all of the evidence. The NOAEL established to mice in a well- conducted 13-week -65-

### inhalation study was 100 ppm . The mg/Kg/day dose was calculated as follows: 168 mg/m3 (100 ppm) x 0 .039 M3 /24h (mouse ventilatory volume, U.S.E.P.A. 1980) x 6h/24h x 5 days/7 days: 0 .03 Kg = 39 mg/Kg/day. An absorption efficiency of 50%is utilized to estimate an oral NOAEL resulting in an equivalent oral estimate of 19.5 mg/Kg/day. 

### Other routes Two studies are included in data sheet IUCLID about subcutaneous administration. Two species, rat and rabbits were tested during 28 days, rats were exposed to 0

.02, 0 .04, 0 .08 ml/animal and rabbits of 4-month-old daily subcutaneous injections of 0

.1 ml acetonitrile. Two groups of four male rabbits developed pronounced (more than twice normal size) thyroid hyperplasia whereas one group of four females showed no effect . (Marine et al ., 1932a) . A further study (Marine et al ., 1932b) suggested that young rabbits were more susceptible than adults and that the effect varied with the strain . 

### 4 .1 .2.6 .2. Studies In humans No data are available in humans. 4.1 .2.6.3. Summary of repeated exposure studies There is no information available on the effects of repeated exposure to acetonitrile in humans. Clinical findings observed on rats and mice exposed to acetonitrile for 13 weeks were hypoactivity and ruffled fur during the first week at highest concentrations (800 and 1600 ppm) . Females rats exposed to 1600 ppmhad significantly greater absolute and relative heart, kidney and liver weights than those of the controls. In mice this findings appears at 200 ppm . In rats clinical pathology findings included anaemia, evidenced by decreases in red blood cell count, haemoglobin concentration and haematocrit and lesions consisting of congestion, edema and haemorrhage in alveoli at highest concentrations (800 and 1600 ppm) . Therefore the NOAEL in mice is 100 ppmand the NOAEL in rats is 400 ppm . This is consistent with the results of other subchronic inhalation studies. In other study was observed a depressed haematocrit, haemoglobin, red blood cells and white blood cells in mice exposed 6 .5 h/day, 5 days/week, for 13 weeks at 200 and 400 ppm

. Female rats exposed at 400 ppmhad slightly more pronounced cytoplasmic vacuolization of the hepatocytes. Dogs and monkeys exposed exposed 7 h/day, 3 days/week for 91 days at a -66- 

# 7Imean acetonitrile vapour concentration of 350 ppmshowed minor variations in body weight, haematocrit, and haemoglobin. Both, monkeys and dogs had pulmonary abnormalities. Autopsy of monkeys noted cerebral haemorrhage and cloudy swelling of the kidney tubules was noted in two of the three exposed monkeys. In summary monkeys were more sensitive than dogs to the same dose of inhaled acetonitrile (350 ppm) . The NOAEL established to rat was 400 ppmand to mice 100-200 ppm . in a well-conducted 13-week inhalation study, according to OECD Guideline No 413. An oral NOAEL of 19.5 mg/Kg/day is the result frominhalation exposure of mice to 100 ppm, 6 hours/day, 5 days/week. 

### 4 .1 .2.7. Mutagenicity 4 .1 .2.7.1 . In vitro studies 

### Several test methods for investigating the mutagenicity and genotoxicity of acetonitrile are available in vitro. Bacterial studies Florin et al ., (1980) in a screening of tobacco smoke constituents for mutagenicity using the Ames test, acetonitrile was assayed qualitatively using strains TA98, TA100, TA1535 and TA1537 with and without S-9 fromaroclor-induced rats, at 3 l .cmol/plate and the results were negative. Schlegelmilch et al ., (1988) tested acetonitrile in the Ames test (Salmonella/microsome assay) performed with strains TA98 and TA100 . The compound was tested with and without metabolic activation, using S9 preparations fromphenobarbitone-pretreated and autoinduced rats. Autoinduction was performed by chronic (7days) application of a dose equivalent to a 5 % oral LD5o value of acetonitrile . The concentrations used were 0 .27, 1 .35, 2

.71, 13.6, 27 .1, 136, 271 y 1350 mM with both strains in triplicate plates. Acetonitrile not found to be mutagenic in the Ames/Salmonella assay with or without metabolic activation . The concentration 1350 mM results to be toxic with strain TA98 in the presence of S9-mix (phenobarbitone) . Mortelmans et al ., (1986) tested acetonitrile in the Salmonella preincubation assay, strains TA1535, TA1537, TA97, TA98 and TA100within a dose range up to 10 mg/plate and in the presence and absence of the metabolic activation systems prepared from Sprague-Dawley rats or Syriam hamster liver pretreated with Aroclor 1254. This study employed a 20 minute preincubation period at 37oC. -67-All tests were repeated using either the same or different S9 concentrations (10% and 30%) . Each trial consisted of triplicate plates of concurrent positive and negative controls and five doses of acetonitrile 100, 333, 1000, 3333 and 10,000 Mg/plate. In the absence of toxicity 10,000 pg/plate was selected as the high dose. All assays were repeated. 

### The test was performed in two different laboratories. Under the conditions of the study, acetonitrile did not cause a positive response, with or without metabolic activation, in any of the tested strains . Yeast studies Acetonitrile was tested for their genotoxic potential in an assay using Saccharomyces cerevisiae (strain D7), with and without metabolic activation (S9 preparations fromphenobarbitone-pretreated andautoinduced rats) . Tenconcentration were used: 360, 405, 450, 495, 540, 585, 630, 675, 720 and 768 mM) in triplicate plates. No reversion from isoleucin auxotrophy to prototrophy exceeding the spontaneous rate could be detected in strain D7 after various doses of acetonitrile. There was no effect on mutation rate by the presence of a metabolic activation system . Aweak recombinogenic effect was detectable, when phenobarbitone-induced S9 was added to the incubation mixtures, the number of trp` convertants on selective plates increased up to twice the spontaneous number although the corresponding doses were very high (675 and 720mM) . No convertogenic effect could be demonstrated when autoinduced S9 or no metabolic activation systemwere added to the incubation mixtures (Schlegelmilch, et al ., 1988) . Acetonitrile induced chromosome loss Saccharomyces cerevisae strain D61 M, with or without a cold interruption phase and in the absence of metabolic activation (Whittaker et al ., 1989) . The study was carried out in two laboratories with similar results. The following additions are made to 2ml of D61 M culture: 1, 3, 5, 10, 20, 30, 50, 75 and 100 NI, to yield final concentrations of 0 .05-4.76% (v/v) respectively. Positive and negative controls are also included. Cultures are incubated according of the two following protocols: 1) Standard 16 h incubation at 30oC (tubes are shaken at 300rpmat a 45o angle) . 2) Cold interruption (tubes are incubated at 30oC for 4h, transferred to an ice/water bath for 16h, vortex mixed, warmed for 2 min. in a 30oC water bath and further incubated at 30o C for 4h) . Zimmerman et al ., 1985, obtained similar results using 2.91% and 4.76% -68-acetonitrile. All treatment were started by pipetting the acetonitrile into a growing cell culture at a titer between 3 and 10 million cells per ml 4h of incubation at 28oC were followed by a storage of about 17h in ice. Cells were plated aftet 4-5h of additional incubation at 28oC

. Acetonitrile strongly induced aneuploid but not recombination or point mutation. Groschel-Stewart et al ., 1985 demonstrated that theaneuploid-inducing activity of aprotic polar compounds in yeast is associated with their ability to block assembly in vitro of porcine brain tubulin. In the case of acetonitrile, this block could be achieved at concentrations much lower than those necessary to induce aneuploid in yeast. 

### Mammalian cells studies A well-conducted gene mutation assay (HGPRT locus) in cultured Chinese hamster ovary (CHO) cells, was carried out by Bioassay Systems Corporation (1984) . This assay was conducted both in the absence and presence ofAroclor-1254-induced rat liver metabolic activation. The optimal dose levels for the mutation assay were selected following a preliminary toxicity test based on colony forming efficiency. Approximately 40% of the cells survived 15.4 mg/ml while less than 0.03% survived at 77.1 mg/ml . Based on those results concentrations ranging from 0 .1 to 30 mg/ml were selected for the first nonactivated mutagenesis assays and eight concentrations ranging from 1-25 mg/ml were tested in the second nonactivated mutagenesis assay . The exposure time was 16 hours. When the assay was carried out in the presence of 0 .424 mg/ml of S9, CHOcells were exposed during 4 hours at 37oCto solvent ranging from 4-16 mg/ml in the first experiment and eight concentration ranging from 8-20 mg/ml in experiment No. 2 . 

### No significant differences (cr=0.05) between the mutation frequencies of acetonitrile and the negative control were found both in the absence and presence of S9.

Other gene mutation assay was carried out, in this case with L5178Y mouse lymphoma cells and in the presence and absence of metabolic activation system . Acetonitrile was neither mutagenic nor toxic at the maximumtested dose (5mg/ml) (brief abstract Rudd et al ., 1983) . Acetonitrile, was tested in the Chromosome Aberrations test and Sister Chromatid Exchanges test, using chinese hamster ovarycells (Galloway, et al ., 1987) . Acetonitrile was tested both in the presence and absence of Aroclor 1254- induced male Sprague-Dawley rat liver S9 and cofactor mix. Each test consisted of concurrent solvent and positive controls and of at least three doses of acetonitrile. In the absence of toxicity, 5000/jg/ml was selected as the -69-

### high dose. Asingle flask per dose was used, and tests yielding equivocal or positive results were repeated . In the Chromosomal Aberrations test, without S9, cells were incubated with acetonitrile for 12 hours; in the presence of S9, cells were treated with acetonitrile for 2 hour, after which the treatment mediumwas removed and the cells were incubated 12 hours in fresh medium . The concentrations tested were 500, 1600 and 5000Ng/ml both with and without S9 . One hundred first-division metaphase cells were scored at each dose level . Result obtained showed a slight increases in the incidence of chromosome aberrations at 5000 Ng/ml (P<0.05) in the presence of S9 but no induction of aberrations occurred in the absence of S9 . In the SCE test without S9, CHO cells were incubated for 26 hours with acetonitrile at 160, 500, 1600 and 5000 pg/ml doses. Bromodeoxyuridine (BrdU) was added 2h after culture initiation. In the SCE test with S9 cells were incubated with acetonitrile (500, 1600and 5000Ng/ml) and S9for 2h. The mediumwas then removed and replaced with medium containing BrdU and incubation proceeded for an additional 26 hours. Fifty second-division metaphase cells were scored for frequency of SCEs/cell from each dose level . Acetonitrile produced a positive response without S9 at 5000 pg/ml (P<0.01) ; with S9, no increase in SCE was noted. 

### 4.1 .2 .7.2. In vfvo studies Fix and Zeste genetic test systems employing female Drosophila melanogaster were performed by Osgood et al ., (1991 .a,b) . Acetonitrile was found to induce chromosome loss as well as chromosome gain in both assays . Acetonitrile was administered by feeding to either early third instar larvae (LF3), late third instar (LF4) in one experiment, or to young adult (AF) females. The concentrations tested were 2000, 5000, 20000 and 50000 ppm, the solvent system used was water . With Fix acetonitrile was strongly positive only after feeding to third instar larvae (LF3) . With Zeste the chemical induced aneuploid after adult feeding (AF) with no discernable effect after LF3 . Zeste larvae were substantially more sensitive to acetonitrile with 78% mortality at 20000 ppmcompared to 30% for FIX larvae. The magnitude of effect, the increment relative to the concui sent control was approximately 2 fold for FIX and 5 fold for Zeste. (Osgood et al ., 1991 a) . The Drosophila ZESTE system was used to monitor the induction of sex chromosome aneuploid following inhalation exposure of adult females to acetonitrile . Acetonitrile was a highly effective aneuploidogens, inducing both chromosome loss and chromosome gain following short exposure to a concentration of 131 ppm . (Osgood et al ., 1991 b) . Adult females (2-3 days old) were used. Exposures to 0, 10, 30, 50 and 70 min. were carried out. Statistically significant -70- /Jincreases in the frequency of aneuploid offspring were detected following as little as 10-min. exposure. After 30 and 50 min. exposures, the combined frequency of aneuploid offspring was approximately 0 .189'o as compared with a control frequency of 0 .024% a 7-fold increment. Acetonitrife was tested in micronucleus test in NMRI mice (13 weeks old) (Schlegelmich et al ., 1988) . Two dose levels were tested amounting to a 40 and 6096 LDso (340 and 510 mg/Kg) vie i .p. The LDso data were obtained from RTECS. The rat i .p. LDsa value presented in RTECS is 850 mg/Kg. For each dose level and day, four male and four female NMRImice. The animals were killed 24, 48 and 72h after injection of a single dose. Bone marrow was obtained and the number of micronucleated polychromatic erythrocytes was counted by scoring 1000 to 1200 cells per animal from coded slides. 

### When the assay was carried out with autoinduction, the mice received daily a dose of the acetonitrile equivalent to 5%of the oral LD50 value for 7 days. On the eighth day i .p.injection of the high single dose followed. Without autoinduction, the number of micronuclei induced by acetonitrile is higher than under any other condition, when a dose equivalent to a 60% LDso value was administered and the animals were killed 24h after application of the compound. The frequency of micronuclei containing polychromatic erythrocytes under these conditions reached 4 .26 per 1000, compared to 1 .68 per 1000 in negative controls. However, autoinduced mice showed a peak nearly as high as this at 72h after administration of the compound, but no effect at 24 h after application. This study did not include control micronucleus frequencies for comparison to the "induced" levels. Peripheral blood samples were obtained from male and female B6C3F1 mice treated for 13 weeks with acetonitrile (100, 200, 400 and 800 ppm) by inhalation to determine the frequency of micronuclei in 10,000 normochromatic erythrocytes in each of 10 animals per dose group. Results with female mice were negative but in males, a small but significant increase (P<0.006) in micronucleated normochromatic erythrocytes was observed in the 400 ppmgroup (NTP, 1994) . 

### 4 .1 .2.7.3. Studies in humans There is no information availab'e. 4 .1 .2 .7.4. Summary of mutagenicity There are several data available on the genotoxicity of acetonitrile in vitro studies. The information in bacteria indicates that acetonitrile is not mutagenic in Salmonella assay as well as when the assay is carried out employing a 20 minute -71-preincubation period at 37oC . Acetonitrile neither induces mutations in chinese hamster ovary cells or mouse lymphoma L5178Y cells. These tests were conducted with and without S9 metabolic activation enzymes. In cytogenetic tests with chinese hamster ovary cells, acetonitrile induced slight increase in sister chromatid exchanges without S9 and chromosomal aberrations with S9 for both endpoint, the increases were noted at the highest dose tested. In the SCE assay this increases was considered weak evidence of activity (increase at any single dose) . In the chromosomal aberrations test the result was considered equivocal (was absence of a statistically significant increase at any one dose) . 

### Positive results have been reported in assays which measure the induction of aneuploid events. Acetonitrile was found to be a potent inducer of aneuploid, but not point mutations or recombination in a diploid strain of Saccharomyces cerevisae, although relatively high concentrations. Acetonitrile induced sex chromosomal aneuploid (both chromosome loss and chromosome gain) in oocytes of female Drosophila melanogaster fed an aqueous solution of the chemical either as larvae or as adults. In addition, weakly positive results were reported in a bone marrow micronucleus test with acetonitrile administered by intraperitoneal injection to male and female NMR5 mice and a significant increase in micronucleated normochromatic erythrocytes was observed in peripheral blood samples from male mice treated with acetonitrile for 13weeks; the frequency of micronucleated erythrocytes in female mice was not affected by exposure to acetonitrile. In conclusion, acetonitrile did no induce gene mutations in bacteria and showed week clastogenic activity in cultured mammalian cells; in vivo, evidence for chromosomal damage in mice was observed in the formof increased frecuencies of micronucleded erythrocytes, in consequence a possible classification of the substance according to EC criteria is Mutagenic, category 3, with risk phrase R40 (possible risk of irreversible effects) should be included in the proposal of classification . 4 .1 .2.8. Carcinogenicity 4 .1 .2.8.1 Studies in animals The carcinogenicity of acetonitrile has been investiged in rats and mice using the inhalation route. These studies were carried out in compliance with the Good Laboratory Practice (NTP, 1994) . Rats From

the NTPsubchronic rat inhalation study, the following acetonitrile exposure levels were selected for the 2-year inhalation study: 100, 200 and 400 ppm, based on



# 77 -72-

### reduced survival and gross and histopathologic lesions. Groups of 56 male and 56female Fischer 344 rats were exposed to acetonitrile by inhalation at concentrations of 0, 100, 200 or 400 ppm(equivalent to 0, 168, 335 or 670 mg/m3) for 6 hour per day, 5 days per week for 103 weeks. Eight male and eight female rats from each exposure group were evaluated at 15 months. The animals were observed twice daily for mortality and signs of toxicity or moribundity. Individual clinical observations were recorded every 4weeks. The animals were weighed initially, weekly for the first 13 weeks, and at 4-week intervals thereafter. During the final 13 weeks of the study, body weights and clinical findings were recorded every 2 weeks. Blood for haematology determinations was taken fromthe retro-orbital sinus of rats at the 15-month interim evaluation . 

### Necropsies were performed on all animals. Organs weighed at the 15-month interim evaluations were liver, lungs, and right kidney. Complete histopathologic examinations were performed on all animals. In addition to gross lesions and tissue masses with regional lymph nodes, tissues examined included: adrenal gland, brain, bone and marrow, clitoral gland, oesophagus, heart, kidney, large intestine, larynx, liver lung, lymph nodes; mammary gland, nose, ovary, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, seminal vesicle, skin, small intestine spleen, stomach, testis, thymus, thyroid gland, trachea, urinary bladder and uterus. Survival rates of exposed rats were similar to those of the controls . Exposure to acetonitrile by inhalation for 15 months or 2years had no effect on body weight gain or final mean body weights, and the behaviour, general health, and appearance of exposed male and female rats were similar to those of the controls throughout the study. The were no significant treatment-related effects on absolute or relative organ weights. At the 15-month interim evaluation, haematocrit values, haemoglobin concentration, erythrocyte count, mean erythrocyte volume, and mean erythrocyte haemoglobin in 400 ppm female rats were minimally lower than controls . Mean erythrocyte volume and mean erythrocyte haemoglobir~ in 400 ppmmales were also minimally lower than controls; however, the erythrocyte count was slightly greater than that of controls . There was no evidence of significant exposure-related nonneoplastic lesions in male or female rats at 15 months or 2 year . The only questionable finding was the incidences of the hepatocellular adenoma (6%), carcinoma (6%) and adenoma or carcinoma (combined 10%) with a statically significant positive trend in male. The incidences of hepatocellular adenoma were: 0% at 0ppm ; 2%at 100 ppm

; -73-2% at 200 ppm and 6% at 400 ppm

. The incidences of hepatocellular carcinomawere: 2% at 0 ppm

; 0% at 100 ppm

; 0% at 200 ppm and 6% at 400 ppm . And the incidences of hepatocellular adenoma or Carcinoma (combined) were 2%at 0 ppm ; 2% at 200 ppmand 10%at 400 ppm . However pairwise comparisons between exposure groups and controls were not significantly different and the incidence of adenoma and carcinoma (combined) in the 400ppm

group of male was only slightly greater than the historical range for inhalation study controls (range 29'0-8%) . 

### The incidence of basophilic foci was greater in 200 (P<_0.05) and 400 (P<_0.01) ppmmales than in controls and the incidences of eosinophilic and mixed cell foci we marginally greater in 400 ppmmales than in controls. The incidences of liver lesions in exposed female groups were similar to controls. 

### Mice From the NTP subchronic mice inhalation study, the following acetonitrile exposure levels were selected for the 2-year inhalation study: 50, 100 and 200 ppm, based on reduced survival and gross and histopathologic lesions. Groups of 60 male and 60 female mice 136C3F1, were exposed to acetonitrile by inhalation at concentrations of 0, 50, 100 or 200 ppm/equivalent to 0, 84, 168 or 335 mg/m3) for 6 hours per day, 5 days per week for 103 weeks. Ten male and ten female mice from each exposure group were evaluated at 15 months. Clinical examinations, a complete necropsy and microscopic examination were performed on all mice. Survival rates of exposed female mice and male mice exposed to 50 or 100 ppm were similar to those of the controls. Survival of male mice in the 200 ppm group was significantly greater than that of controls. Exposure to acetonitrile by inhalation for up to 2 years had no effect on body weight gain or final mean body weights of male and female mice. There were no significant treatment-related effects on absolute or relative organ weights. There was an increased incidence of alveolar/bronchiolar adenoma in exposed male mice. The incidence was significantly increased in 200 ppmmale (18/50, 36%) and was at the upper limit of the range of historical controls (range 6%-36%, 113/673, 16.8%) . The incidence of alveolar/bronchiolar adenoma or carcinoma (combined) was similarly increased in exposed male mice 21/50 (42%), the range of historical controls was 10%-42% (150/673, 22.3%) . In contrast, the incidences of alveolar/bronchiolar -74-adenoma and adenoma or carcinoma (combined) were inversely related to dose in female groups. The incidences in female controls were at the upper range of historical controls. Proliferative lesions of the alveolar bronchiolar region were present as a continuum, with the distinction between hyperplasia, adenoma and carcinoma based on the size and morphologic characteristics of the lesion. Thus, a time chemicalrelated increased incidence in this neoplasmtype would be expected to manifest as an increase in proliferative lesions of all three stages which was not apparent in the current study

. Although the incidence of alveolar bronchiolar adenomas (18/50, 36%) in the 200 ppmmale group is equal to the highest incidence seen in previous control groups for inhalation studies, the 869'o survival of this group at 2 years was quite high and may have contributed to the apparent effect. For these reasons, the increased incidence of alveolar bronchiolar adenomas in the 200 ppm males was not attributed c to acetonitrile exposure.

The incidence of hepatocellular carcinoma was greater than controls in males exposed to 100 ppm (13/49, 27%) . The incidence of hepatocellular adenoma or carcinoma (combined) was similarly increased in 100 ppmmales (30/49, 61%) ; this increase was significant and exceeded the range for hepatocellular adenoma o carcinoma (combined) of historical controls (range 11%-56% ; 241/673, 35.89'0) . However, the incidence of hepatocellular neoplasms in the 200 ppm groups was less than that in the controls, and there was no indication of an effect of acetonitrile exposure on hepatocellular neoplasmin the females. The lack of evidence for a dose response in hepatocellular neoplasms in males coupled with an absence of confounding factors that would be expected to decrease the neoplasmresponse, such as the markedly lower body weight of 200 ppm males, suggests that this is a sporadic finding unrelated to acetonitrile exposure. Effects on the forestomach were noted in exposed mice, to acetonitrile . At 15 month, the incidence of squamous hyperplasia of the forestomach was greater than controls in males and females exposed to 50 or 200 ppm ; however, the increase was only significant in 200 ppmfemales (P<_0.01) . At 2 years, the increase was dose related in exposed groups; the increases were significant in male mice exposed to 200 ppm(P<_0.05) and female mice exposed to 100 or 200 ppm (P<_0.01) . A fewsquamous cell papillomas were noted in mice exposed to 100 or 200 ppm, and one occurred in a control female. The incidences of this benign tumour were not statistically significant in males or females and were within the ranges of historical controls . Thus, these findings establish an effect of prolonged acetonitrile exposure on the forestomach of mice, but the magnitude of the neoplastic findings is insufficient to attribute to the chemical with any confidence. 4 .1 .2.8.2. Studies in humans -75- 00Off et al ., 1989 researched lymphatic and haematopoietic tissue cancer in a chemical Manufacturing Environment. Exposure Odds Ratios (OR) for non-Hodgkins Lymphoma (52 cases), multiple myeloma (20 cases), non-lymphocytic leukaemia (39 cases) and lymphocytic leukaemia (18 cases) within a cohort of US workers fromtwo chemical manufacturing facilities and a research and development centre between 1940-1978 were examined for twenty-one specific chemicals. For acetonitrile an OR of 5 .2 was estimated for non-Hodgkins lymphoma (based on two cases), and 2 .5 for non-lymphocytic leukaemia (based on one case) . There was a high likelihood of multiple, concurrent exposure to several chemicals in this investigation. There was also a high correlation between individuals exposed to "nitriles" and individual exposed to "antioxidants" . Due the mixed and varied exposures, it is not possible to ascribe any increased tumour incidence solely to acetonitrile exposure. 

### 4 .1 .2.8.3 . Summary of carcinogenicity of acetonitrile Under the conditions of 2-year inhalation studies in rats, the incidences of hepatocellular adenoma (3/48), carcinoma (3/48) and adenoma or carcinoma (combined; 5/48) were greater in male rats exposed to 400 ppm than in the controls. The incidences of adenoma and carcinoma were within the range of historical controls. However, the incidence of adenoma or carcinoma (combined) slightly exceeded the range of historical controls (2%-8%) . In addition, the incidences of basophilic foci, eosinophilic foci, and mixed cell foci were marginally greater than controls in 400 ppm males. The specific relationship between various hepatocellular foci and neoplasia is not clearly understood. However, evidence of chemical-related increased incidences of liver foci does provide support for an acetonitrile-related effect on the liver and suggest that a liver tumour response is biologically plausible. There were no exposure-related liver lesions in female rats. There were no increases in the incidences of lung and liver neoplasms in exposed groups of mice that were considered related to acetonitrile exposure. However, the incidence of squamous hyperplasia of the epitheliumof the forestornach was significantly increased at 15 months in 200 ppm females. At 2 years, the increased incidence was dose related in all exposed male and female groups . These findings establish an effect of prolonged acetonitrile exposure on forestomach of mice, but the magnitude of the neoplastic findings is insufficient to attribute to the chemical with any confidence . 4 .1 .2.9. Toxicity for reproduction 4 .1 .2.9.1 . Studies in animals -76-

### Effects on fertility No studies specifically investigating effects on fertility are available. However, in a study of Morrissey et al ., 1988, evaluation of rodent sperm, vaginal cytology and reproductive organ weight data fromNational Toxicology Program 13-week studies are showed. An analysis was conducted of sperm morphology and vaginal cytology examinations studies carried out at the end of fifty 13-week studies (25 for rats, 25 for mice) over a 3-year period. Reproductive organ weight (testis, epididymis, cauda epididymis) and spermmotility were the most statistically powerful endpoints evaluated, spermhead morphology may also be a sensitive endpoint for detecting reproductive toxicants. ( Ten male and female Fischer 344 rats or B6C3F1 mice in three dose groups

plus the control group were utilized .The three dose groups were selected after Day 70 of the prechronic study. The objective was to select doses which were not causing overt toxic effects (mortality or severe depression of body weight or weight gain) . The doses used were 100, 200 and 400 ppmand the route of administration was by inhalation. Male rats or mice showed no change in (absolute or relative) weight of the right cauda or right testis, and no effect on spermmotility. No data are provided on the effects of acetonitrile on the female reproductive systemrats or mice. Coate W

.B. (1983), in a unpublished study, indicates no adverse effects were seen on spermmotility or morphology or on vaginal cytology in rats or mice exposed during 92 days to 0, 25, 50, 100, 200 and 400 ppmacetonitrile by inhalation. 

### Developmental studies



### J Differents developmental studies were carried out with rats, rabbits and hamsters exposed to acetonitrile by differents routes . 

### Studies in rats The developmental toxicity of acetonitrile was studied in Sprague-Dawley rats after inhalation exposure for 6hr/day, during days 6 to 20 of gestation, at dose levels of 0, 900, 1200, 1500 and 1800 ppm . The number of animals exposed was 20 per dose. Maternal lethality occurred at the highest concentration of acetonitrile (1800 ppm) and resulted in the death of 8 of the 20 dams (40%) . Maternal body weight gain during days 6 to 21 of gestation was significantly reduced at 1800 ppm (p<0.01) . Maternal absolute weight gain was significantly reduced at 1500 ppm (p<0.05) and was approximately 60% of that of the controls at 1500 and 1800 ppm . The incidence of pregnancy was not significantly altered by exposure to acetonitrile . Significant increases in the mean percentage of nonsurviving implants and early -77-embryonic resorptions were observed at 1800 ppm (p<0.01), concomitant with a nonsignificant decrease in the mean number of live fetuses per litter . One litter was completely resorbed at 1800 ppm . Exposure to acetonitrile up to 1800 ppmhad no significant effect on the mean number of implantation sites, fetal sex ratio, or male and female fetal weights. Onefetus fromthe control group exhibited exencephaly, unilateral exophtalmia and open eyelid.There were no significant changes in the incidences of any visceral variations (primarily dilated ureter) or skeletal variations (primaril-extra cervical and/or extra lumbar ribs and reduced ossification of sternebra) across exposure groups, even at maternally toxic doses. For this study the NOAEL for maternal toxicity was 1200 ppm . The NOAF_L for developmental toxicity 1800 ppm(Saillenfait et al ., 1993) . An inhalation developmental toxicity study on acetonitrile was well conducted by Mast et al ., 1994

. Sprague-Dawley rats were exposed to 0, 100, 400 or 1200 ppm acetonitrile, 6 hours/day, 7 days/week. Each of the four treatment groups consisted of 10 non-pregnant females, 10 positively mated females for a distribution study evaluating maternal blood for acetonitrile and cyanide and 33 positively mated females for evaluating developmental toxicity. Rats were exposed for 14 consecutive days (6- 19 days of gestation for pregnant animals) . Body weights were obtained throughout the study period and uterine and fetal body weights were obtained on 20 day of gestation. Llve fetuses were sexed and examined for gross, visceral, skeletal and soft-tissue craniofacial defects. Acetonitrile and cyanide concentrations were determined in the maternal blood of the rats (6/group) on 8 and 18 day gestation. Exposure of rats of these concentrations of acetonitrile resulted in mortality in the 1200 ppmgroup (2/33 pregnant females; 1/10 non-pregnant females) and the 400 ppm group (1/33 pregnant females) . However, there were no treatment-related effects upon body weights or reproduction indices at any exposure level, nor was there a significant increase in the incidence of fetal malformations or variations. The only effect observed in the fetuses was a statically significant increase in the incidence of supernumerary ribs in the offspring at 100 ppmacetonitrile, there was no dose-response relationship and the incidence of this variation at both 400 and 1200 ppm was not different fromthe control group. Determination of acetonitrile and cyanide concentrations in maternal rat blood showed that acetonitrile concentration in the blood increased with exposure concentration for all exposed maternal rats. Detectabie amounts of cyanide in the blood were found only in the rats exposed to 1200 ppmacetonitrile (2mg cyanide/ml of blood) on 8 day gestation, the concentration declined to 0 .8 mg/ml by 18 day gestation while acetonitrile concentrations remained essentially constant over the exposure period . This decrease in the maternal blood cyanide level may have been due to induction of rhodanase, the enzyme thought to be responsible for the detoxification of cyanide (Klaasen et al ., 1986) . In summary, the two highest exposure concentrations were maternally lethal to some rats, however, there was no reduction in body weights, body weight gains, or clinical signs of toxicity in surviving pregnant or non-pregnant rats. The no-observable -78- 

# 3adverse effect-level (NOAEL) for acetonitrile with respect to developmental toxicity in this study was the highest exposure concentrations, 1200 ppm . The maternal NOAEL was 100 ppm

.



## J The developmental toxicity of acetonitrile was examined with an in vivo teratology screen adapted for use in the Long-Evans rat (Smith et al ., 1987) . The screen was extended to an evaluation of growth till postnatal Days 41-42, and weight of several organs at sacrifice. Acetonitrile was dissolved in tricaprylin oil for administration at a volume of 0 .1 ml/100 g body weight and administered by intubation fromdays 7 to 21 of gestation. The doses used for this study and number of rats were 0 (155), 50 (20), 150 (20), 300 (22) and 500 (20) mg/Kg bw/day. 

### There was maternal toxicity in the two top dose groups (300, 500 mg/kg) . At 300 mg/kg, 11 females were clearly moribund and therefore were sacrificed at mdgestation, t while at 500 mg/kg, 16 females died early in the treatment period; two deaths also

occurred in the lowest dose group (50 mg/kg) compared to none of the 155 controls. Maternal weight gain was unaffected amongst those in the 50 and 150 mg/kg bw/day groups and those survivors in the 300 mg/kg bw/day group. In the 500 mg/kg bw/day group there was effect (nonsignificant) on the number of viable litters and on the number of litters resorbed. No foetal abnormalities were reported. The only observations amongst the pups, which achieved statistical significance, were increased pup weight on day 4 (females 500 mg/kg) and increased organ weights on day 41-42; liver (females 300mg/kg), lung (males 300mg/kg) and spleen (males unstated group) . CD-1 rats were administered daily aqueous solutions of acetonitrile by gavage (125, 190and 275 mg/kg) on gestation days 6-19. Although maternal effects occurred at 275 mg/kg bw/day; deaths (2/25), reduced weight gain (2/25), no other maternal I o 1 effects were noted in any treated group. Embryotoxic effects, as shown by increases J in early resorptions and postimplantation losses, were also noted in the high-dose

group, statistically significant when compared with historic but not concurrent controls. There was apparently no increase in the incidence of foetal abnormalities (no data presented) but a slight increase in unossified sternebra in all treated groups compared to concurrent, not historic, controls (IRDC, 1981) . 

### Rabbits

J A study conducted in compliance with FDA GLP was carried out by Argus Research Laboratories, INC (1984) . The purpose of this study was to determine the embryo-fetal toxicity and the teratogenic potential of acetonitrile in the NewZealand White rabbit . Solutions of acetonitrile in reverse osmosis processed deionized water were administered by gavage on days 6through 18 of presumed gestation to artificially inseminated rabbits. Acetonitrile was administered to rabbits (25 rabbits/group) at doses o (vehicle) 2, 15 and 30 mg/Kg/day. -79-



# 51qAnimals given the highest dose showed anorexia and decreased body weight gain, death in 5 out of 25 rabbits and 2abortions occurred at this level (30 mg/Kg/day) . Body weight gain was also reduced in animals receiving 15 mg/Kg/day, but not at the lowest dose level . With respect to the fetuses of the treated animals, a significant decrease (P=0.011) in the average number of live foetuses/litter and a slight (non-significant P>0 .05) increase in resorptions was seen in the 30 mg/Kg group. 

### Acetonitrile did not adversely affect the incidence of pregnancy or the average number of corpora lutea, implantations, average fetal body weight and six ratio. No gross external, soft tissue or skeletal malformations or developmental variations observed for fetuses were attributed to administration of acetonitrile. 

### As compared with control, a significantly increased (P=0.015) incidence of an extra ossification site in the parietal bones was observed for four fetuses in two high dosage group litters. The more valid parameter litter incidence, did not demonstrate statistical significance (P>0.05) . This minor variation is frequently observed in this strain, of rabbit and was considered spontaneous. The maternal NOAEL in this study was 15 mg/Kg bw/d'ay and the NOAEL with respect to developmental toxicity 30 mg/Kg b.w ./day. Hamsters In a study by Willhite (1983), pregnant golden hamsters were exposed to acetonitrile by inhalation, ingestion or ip injection during the early primitive streak stage of embryogenesis. In the study by inhalation, hamsters were exposed at 0, 1800, 3800, 5000 or 8000 ppmacetonitrile during one hour, on the 8th day of gestation. The number of animals exposed were 10, 6, 6, 6 and 12 respectively . Maternal deaths occurred at 3800, 5000 and 8000 ppm(1/6, 1/6 and 3/12 respectively) . Resorptions were significantly increased (P<0.05) in the 5000 ppmgroup. Foetal abnormalities were seen in the 5000 ppm group (6, 11 %

of foetuses) and 8000 ppm

group (29

; 25%) . No abnormalities were seen in either of the other treated groups or in the controls. The most common abnormalities were exencephaly, Encephelocoele and rib fusions. One foetus of the 8000 ppmgroup had ectopia cordis. Average foetal weight was significantly reduced in the 8000 ppmgroup. -80- ~J

### In this study the maternal NOAEL is 1800 ppmand NOAEL to developmental toxicity 3800 ppm

. When, pregnant golden hamsters were exposed to a single gavage or a single intraperitoneal injection of 0, 100, 200, 300 or 400 mg/Kg on the 8th day of gestation, maternal toxicity (deaths) occurred at a single gavage dose of 300 mg/Kg (1/6) and 400 mg/Kg (4/12) . Resorptions were significantly increased in the 200 and 400 mg/Kg groups. There was a significant reduction in body-weight gain associated with intubation of 100-300 mg/Kg acetonitrile, but there was no significant difference in maternal body-weight gain associated with intubation of 400 mg/Kg acetonitrile as compared with intubation of an equivalent volume of water. 

### The incidence of malformations was significantly increased in the 300 and 400 mg/Kg groups, two litters of the 100 mg/Kg group also contained abnormal foetuses compared to none of those of the control group. Malformed foetuses were recovered from dams of the 300 and 400 mg/Kg groups. The skeletal malformations seen included rib fusions and severe axial skeleton dysraphic disorders. The NOAEL with respect to developmental toxicity was 200 mg/Kg/b.w ./day. Exposure of pregnant hamsters to acetonitrile by i .p. injection of 100-400 mg/Kg, failed to result in a significant increase in either the number of malformed fetuses or the number of resorbed conception sites as compared with controls. There was a significant increase in the average fetal body weight associated with i .p. injection of 200-400 mg/Kg acetonitrile when compared to control, the relevance of this observation was not clear. There was no significant change in the average maternal body weight gain throughout days 8-14 of gestation. A lowincidence of abnormalities was seen in the 100 mg/Kg group (2; 3% of foetuses), 200 mg/Kg group (1 ; 2%) and 400 mg/Kg group (4; 3%) . There were no abnormalities in the 300 ppmgroup or in the litters of the 6 control hamsters. Abnormalities occurred to the cranium (craniora chischisis), abdominal wall (gastroschisis) . The statistical and biological significance of these findings is unclear. 

### 4 .1 .2.9.2. Studies in humans Acase-control study of pregnancy outcome among Finnish laboratory workers revealed no association between exposure to acetonitrile and increased risk of spontaneous abortion in mothers, or malformation and birth weight in their children. An odds ratio of 1 .4 (95% Confidence Internal 0 .4-4.7) for spontaneous abortion for exposure to acetonitrile was estimated when 206 cases were compared to 329 controls . No association between solvent exposure and risk of malformation was found in an analysis of 36 cases and 105 controls, or between solvent exposure and birth -81- 

# as

### weight, in an analysis of 500 women (Taskinen et al ., 1994) . 4 .1 .2.9.3. Summary of toxicity for reproduction In relation to fertility, there is no information available in humans and there are no animal studies specifically investigating such effects. However no changes were seen in (absolute or relative) weight of the right cauda or right testis and no effect on sperm motility in rats or mice exposed for 13 weeks with 100, 200 and 400 ppmto acetonitrile. On the other hand, several developmental studies are available in animals. In a well conducted study, rats exposed by inhalation to acetonitrile did not result in significant foetal effects, event at concentrations which were overtly toxic to the dam . In this study a maternal NOAEL: 100 ppmand a NOAEL : 1200 ppm

with respect to developmental toxicity was established. In two studies with pregnant rats that received acetonitrile by gavage no foetal abnormalities were reported. In other study conducted in compliance with FDA GLP, pregnant rabbits administered 2, 15 or 30 mg/Kg acetonitrile orally showed maternal toxicity at 15 and 30 mg/Kg. Embryotoxicity was observed only at the 30 mg/Kg dose level . There were no treatment-related gross external, . soft tissue or skeletal malformations or developmental variations. Exposure of hamsters to 5000 or 8000 ppmacetonitrile on Day 8 of gestation was shown to increase the incidences of resorptions and malformations including exencephaly encephalocele and rib fusions. Malformations identical to those noted following inhalation exposure occurred sporadically in a limited number of offspring after oral or intraperitoneal administration of 100-400 mg/Kg acetonitrile . However, in both experiments, teratogenicity was associated with maternal toxicity. Consequently, acetonitriie is not considered to be toxic to fetuses at doses belowthose causing maternal toxicity. In human, no association between solvent exposure and risk of malformation, birth weight or spontaneous abortion has been observed . 4 .1 .3. Risk characterisation 4 .1 .3.0. General aspects Acetonitrile is well absorbed from the lungs, gastrointestinal tract and through the skin, although there are not available quantitative data. Acetonitrile has a widespread distribution . It has been found in heart, lungs, liver, spleen, kidneys, stomach, intestines, skin, muscle, brain and teste. Free and -82- 

# S)conjugated hydrogen cyanide were also detected in all the studied organs. There are no indications that repeated administrations of acetonitrile result on its accumulation in animal tissues. The acetonitrile is metabolized to cyanide via cytochrome P-450 . Several studies have indicated that cyanide formed in vivo is subsequently conjugate with thiosulphate to form thiocyanate, which is eliminated in urine. The cyanide is responsible for the acetonitrile toxicity. The conversion of acetonitrile to cyanide proceeds at a slower rate than that of other nitriles. Interspecies variations in toxic response are probably related to the relative speed of cyanide formation fromacetonitrile. ( Elimination of acetonitrile occurs primarily through urinary excretion of the

unchanged compound and free and bound hydrogen cyanide. Pulmonary clearance of unchanged acetonitrile via exhalation is also an important pathway of elimination, especially at high exposure levels. The range of oral LD50values acetonitrile in mammals is between 160 and 6762 mg/Kg body weigh. Mouse and Guinea-pig seemto be the most sensitive species. A study showed that acetonitrile was more toxic to immature rats (14-day-old) than to older rats. The main symptoms in animals appear to be ~prostration followed by seizures and convulsions. Acetonitrile has rabbit dermal LDso of 1 .25 ml/Kg. It is of interest to note that the toxicity by skin penetration is increased when application is made as a aqueous solution. The 8 h inhalation LCso in male rats is 7500 ppm . Rabbits and mice were more sensitive with LCso of 2828 and 2693 ppmand time of exposures of 4 h and 60 min. respectively. Animals exposed to acetonitrile via different routes of dosing always showed respiratory symptoms: rapid and irregular respiration, laboured or difficult breathing and intense dyspnoea . Some cases of severe intoxications for exposure to high concentrations of acetonitrile vapour have been reported . The systemic effects appear to be largely attributable to the conversion of acetonitrile to cyanide. Blood cyanide and thiocyanate levels are elevated during acute intoxication. Acetonitrile is slightly irritant to the skin. When acetonitrile is applied to the eye it produces strong vascular injection with edema or slight necrosis. Humans accidentally inhaling 500 ppm for brief periods reported some irritation -83- 

# g8'to nose and throat . Sensitization studies with acetonitrile are not available. There is no information available on the effects of repeated exposure to acetonitrile in humans. Clinical findings observed on rats and mice exposed to acetonitrile for 13 weeks were hypoactivity and ruffled fur during the first week at highest concentrations (800 and 1600 ppm) . Female rats exposed to 1600 ppmhad significantly greater absolute and relative heart, kidney and liver weights than those of the controls. In mice this findings appears at 200 ppm . In rats clinical pathology findings included anaemia, evidenced by decreases in red bloodcell count, haemoglobin concentration and haematocrit and lesions consisting of congestion, edema and haemorrhage in alveoli at highest concentrations (800 and 1600 ppm) . The NOAEL established to rat was 400 ppmand to mice 100 ppm . This is consistent with the results of other subchronic inhalation studies . The dose-response for acetonitrile toxicity is quite steep, with little evidence of adverse effects in animals exposed to concentrations lower than those that resulted in death, or in survivors in exposure groups in which some animals died . Acetonitrile did not induce gene mutations in bacteria and showed weak clastogenic activity in cultured mammalian cells. However, positive results have been reported in assays which measure the induction of aneuploid events, in vitro acetonitrile was found to be a potent inducer of aneuploid en Saccharomyces cerevisae strain D61 Mand in vivo, induced sex chromosomal aneuptody (both chromosome loss and chromosome gain) in Drosophila melanogaster . In addition, weakly positive results were reported in bone marrowmicronucleus test with acetonitrile administered by intraperitoneal injection to male and female NMR5 mice and a significant increase in micronucleated erythrocytes was observed in peripheral blood samples from male mice treated with acetonitrile for 13 weeks. Moreover, further well-validated in vivo study, in order to detect chromosome damage is required . The carcinogenicity of acetonitrile via inhalation has been investiged in rats and mice. In male F344/N rats exposed to 400 ppmacetonitrile there were a marginal increase of neoplasms based on an increased incidence of hepatocellufar adenomas and carcinomas. There was no evidence of carcinogenic activity of acetonitrile in female F344/N rats : exposed to 100, 200 or 400 ppm . There were no chemical-related increases in malignant or benign neoplasms -84-in male or female B6C3F1 mice exposed to 50, 100 or 200 ppm . However, exposure of male and female mice to acetonitrile by inhalation resulted in an increased incidence of squamous hyperplasia of the forestomach. These findings establish an effect of prolonged acetonitrile exposure on the forestomach of mice, but the magnitude of the neoplastic findings are insufficient to attribute to the chemical with any confidence. In relation to fertility, there is no information available in humans and there are no animal studies specifically investigating such effects. However no changes were seen in (absolute or relative) weight of the right cauda or right testis and no effect on sperm motility in rats or mice exposed for 13 weeks with 100, 200 and 400 ppmto acetonitrile. On the other hand, several developmental studies are available in animals. In rats exposed by inhalation to acetonitrile did not result in significantfoetal effects event at concentrations which were overtly toxic to the dam . A maternal NOAEL 100 ppm and a NOAEL 1200 ppm with respect to developmental toxicity was established. Pregnant rabbits administered 2,15 or 30 mg/Kg acetonitrile orally showed maternal toxicity at 15 and 30 mg/Kg. Embryotoxicity was observed only at the 30 mg/Kg dose level . There were no treatment-related gross external, soft tissue or skeletal malformations or developmental variations. Teratogenicity associated with maternal toxicity was observed in two experiments carried out with hamsters exposed by inhalation to 5000 or 8000 ppm acetonitrile or after oral or intraperitoneal administration of 100-400mg/Kg acetonitrile . In summary acetonitrile is not considered to be toxic to fetuses at doses below those causing maternal toxicity. The NOAEL of 100 ppm for maternal toxicity and the NOAEL of 1200 ppmfor developmental toxicity is the same or clearly above of the NOAEL of subchronic toxicity respectively. Therefore, the risk for those aspects will be covered for the risk characterisation of subchronic toxicity . In human, no association between solvent exposure and risk of malformation, birth weight or spontaneous abortion has been observed . In summary, inhalation of acetonitrile produces the characteristical signs and symptoms of cyanide intoxication. Data fromlong and short-termstudies showed that is a wide variation in inter and intraspecies susceptibility to acetonitrile. The results from a well conducted subchronic study shows that mice is one of



# 96 -85-

### the most sensitive species to inhaled acetonitrile being the NOAEL 100 ppm . Monkeys were more sensitive to acetonitrile than dogs to similar dose in a 99- days study conducted by Pozzani . The Pozzani's human study shows that there is a variability of subjective responses of humans to 40,80 and 160 ppm of acetonitrile vapour, suggesting that, even if a concentration that would not endanger the health of the majority of workers it might cause discomfort to some. On the other hand, the dose-response relationship for acetonitrile is quite steep with little evidence of adverse effects in animals exposed to concentrations lower than that resulted in death. 

### 4 .1 .3.1 . Workers Occupational exposure to acetonitrile may occur by inhalation or dermal routes. Since acetonitrile is a volatile substance, the most important route is inhalation . 

### For risk characterization purposes a NOAEL of 100 ppmis considered. The risk assessment for the workplace is based on a NOAEL of 100 ppm . This NOAEL is regarded as meaningful, since it have been obtained in one of the most sensitive species to inhaled acetonitrile and from a good subchronic inhalation study. 4 .1 .3 .1 .1 . Manufacture The acetonitrile is produced within an enclosed system . Data provided by industry indicate that, in the different work positions, including sampling, maintenance and cleaning activities, the 8-hours (TWA) exposure was below 0 .5 ppm . This is 200 times belowthe NOAEL (100 ppm) . Thus, inhalation exposure in the manufacture scenario does not present any grounds for concern in relation to health risk to workers. Dermal exposure is unexpected . Potential contact could only occur in activities that involves breaching the closed system . According with the EASE model, the potential dermal exposure is estimated to be low . The general practice is that where a contact is expected, gloves are required to avoid absorption through the skin and therefore, exposure should be minimal . Contact to the eyes should be rather uncommon regarding the industrial processes. However, in order to avoid the irritant effect of acetonitrile on eye, face shield and/or goggles are expected to be available for employee use in special tasks -86- 

# 916#

### where a contact cannot be excluded. Result : There is at present no need for further information and/or testing for risk reduction measures beyond those which are being applied already. 4 .1 .3.1 .2 . End uses Most acetonitrile uses are in industrial processes involving closed systems. The exposure is expected in special tasks such as sampling, cleaning, maintenance, as the precedent section. 

### In the exposure section a worst case exposure level of 20 ppm has been considered for risk characterization purposes . This is five times belowthe NOAEL in mice of 100 ppm

. This ratio seems lowfor protection of workers, taking into account the inter and intraspecies variability, the steeper dose-response curve and the suspicion of mutagenicity. On the other hand, the exposure level of 20 ppmhas been obtained fromdata provided by industry. These data have limited meaningfulness because the processes, work practices and the activities involved are not described and information on the duration and frequency of exposure is not available. For this reason, more information on these exposure aspects is needed to refine the risk assessment . Result : i) There is a need for further information and/or testing. Regarding the use of acetonitrile in chromatography, the exposure value of 7.3 ppmis 14 times belowthe NOAEL of 100 ppm, Thus, inhalation exposure is not of concern for laboratory workers. Following the EASE model dermal exposure will be low . Accordingly with the safety data sheet, gloves should be worn to prevent absorption through the skin. 4 .1 .3.2. Consumers Since acetonitrile is not present in the consumer's products the conclusion ii) is applied. Result : ii) There is at present no need for further information and/or testing for risk -87-

### reduction measures beyond those which are being applied already. 4.1 .3.3. Man exposed indirectly via the environment For the end-points irritation and subchronic toxicity this is not likely to present a major health problem . Using a NOAEL of 19.5 mg/Kg/day and dose data as reported in 4 .1 .1 .3. we can calculate the safety margin for indirectly exposure as follows: NOAEL for oral subchronic toxicity/total human dose. Model local : 3

.7 x 105 Model regional : 4

.6 x 104 

### Those values mean high margins of safety indicating no concern on human safety after indirect exposure . Result : There is at present no need for further information and/or testing or for risk reduction measures beyond those which are being applied already.

4 .2. Human health (Physico-chemical properties) 4 .2.1 . Exposure assessment 4 .2.1 .1 . Workers Due to the fact that acetonitrile is a volatile, highly flammable liquid, can rapidly lead to a dangerous concentration build up in air. The acetonitrile forms explosive mixtures with air and when is heated to decomposition it emits highly toxic fumes of cyanide. In the industrial setting, these risks are not of concern provided that adequate safety measures are taken. On the other hand information is already provided on the label and in the safety data sheet . -88-



# 434 .2.1 .2. Consumers Acetonitrile has not been detected in consumers products. 

### 4 .2.1 .3. Man exposed indirectly via the environment Not applicable. 4.2.2 . Effect assessment 4 .2.2.1 . Explosivity 

### Acetonitrile forms explosive mixtures with air. The lower explosive limit is 3 .05% in volume and the upper explosive limit 17% in volume. 4.2.2.2. Flammability Acetonitrile is a highly flammable liquid (flash point : 5°Cin open cup and 12.8°C in closed cup) with an auto flammability temperature of 524°C . It is a volatile liquid (vapour pressure 9.4-9.8 KPa at 20°C) . The vapours can forma flammable mixture with air within the range of 4.4%to 16 volume %

. 

### 4 .2.2.3. Oxidizing potential The test method (OECD-EEC) is not applicable to liquid substances. 4.2.3. Risk characterization (physico-chemical properties) Regarding its physico-chemical properties, flammability is aproperty of concern for acetonitrile since it is a volatile liquid which is highly flammable. 

### In the industry setting, the flammability risk is not of concern provided adequate safety measures are taken. Information is provided on the label and in the ,afety data sheet . 

### Concerning use by consumers, acetonitrile is not present in the consumer's products. CONCLUSION There is at present no need for further information or testing or for risk reduction beyond those which are being applied already. -89-5 . (i) 

### CONCLUSIONS There is a need for further information and/or testing. Sensitization No data is available on sensitization of acetonitrile. Therefore, there is need for testing for skin sensitization in accordance with the EU-Annex V-standard. Mutagenicitv Acetonitrile did no induce gene mutations neither in bacteria nor in mammalian cells in culture . Showed week clastogenic activity in cultured mammalian cells. However, was found to be a inducer of aneuploid in a diploid strain of Saccharomyces cerevisae and in Drosophila melanogaster. In addition, positive results, in vivo, were reported in micronucleus test. Additional well-validated, in vivo, study in order to detect chromosome damage is required, before further conclusions can be made with regard to the risk assessment . Workers This conclusion is reached because the highest levels of exposure obtained in the "end uses" scenario have limited meaningfulness since the processes, work practices and the activities involved are not described and information on the duration and frequency of exposure are not available. 

### (ii) There is at present no need for further information and/or testing or for risk reduction measures beyond those being applied already. This conclusion applies to: The environmental risk, with respect to aquatic compartment, including water, sediments and biological processes of WWfP . Terrestrial environment, at the regional and continental levels, as well as non compartment specific effects relevant to the food chain (secondary poisoning) . This conclusion also applies to risk to workers involved in the acetonitrile manufacture, consumers, risk from indirect exposure of man via the environment and from physico-chemical properties.

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## APPENDIX Ap. 107

USES V1 .0 SINGLE SUBSTANCE

-- ._. .----------------- . .---------~---------- Printed on : 0~/03/1997 10 :10 Country : NL Substance : acetonitrile CAS-No : 75-05-8 SUBSTANCE IDENTIFICATION

--- ---~ ---------~-----~----------------- --------- ------- .__ .._-. ..-_._ . __ .. _ ._ ._.. Name : acetonitrile CAS-No : 75-OS-8 EC-notification no . . EINECS no . : 200-835-2 Molecular weight [g .mol-1] : 41 .05 Mol . Formula : C2H3N PARAMETER STATUS Input parameters defaults used . 35 of 59 Model parameters defaults usea~ . 15 of 15 Intermediate results overwritten . 9 of 57

INPUT PARAMETERS

_ ._- . ... .... .. . . .. _ . .. ... _. ..... ... . .._ ..... . . . __ . .- _ ..... ..... ._. .... ._. . ._. TONNAGE ACTUAL DEFAULT

-------------- ._ ..._ .__ ..__ ._ ..-------------------- -~------------- - Tonnage national [tonnes .yr-1] : Tonnage Europe [tonnes .yr-1] : 1 .5e+04 S MAIN CATEGORY Production : Ic Batch production, dedicated equipment Formulation : Ib Dedicated equipment - cleaning iimitea Processing : III Non-dispersive use INDUSTRIAL CATEGORY 0

--------- -------------------------- ---------~-- --- ---- ----~ --- ._ . __ -. .__ . .. .. .. .-- - ----- ---- Ine . cat . : 3 Chemical industr y : chemicals used in s yntnesis USE CATEGORY

- -- --- - - - -- ---. _

.. __ ._.._..-~----. _

.. _ . _ ..-- --._.. _ ._ _

. _ .. ... ..__ . ._. .. .- .._ ._ _ .. Primary : JJ Intermediates EMISSION RELEVANT DURING LIFE-CYCLE STEP ? ACTUAL ------ -... ..--------- ----------- ------- ------------------~----~--~- ---- .. ._.. Emission production Yes Emission formulation No Emission processing

. Yes `nij .ssior orlvate use . No ~=mission recovery . No Bypass STP

. No USES V1 .0 NL 03/03/1997 10 :10 DEFAULT No No No No No No Page : 1 

# idsp. 108

USES V1 .0 SINGLE SUBSTANCE

_.-.-- .._.- ._ ---~---- -- .-_ . ._ .. .--- ------ .. .----------------- .._-- ._ - Printed on : 03/0J/1997 10 :10 Country : NL Substance : acetonitrile CAS-No : 75-05-8 PHYSICO-CHEMICAL PROPERTIES : ACTUAL DEFAULT

-~ -- -- ----- ~------_ . ..------ -- Melt .ing point [oC] : -45 S "''' Vapour pressure [Pa] : 9600 S 0 .001 Octanol-water part . coeff . (lOlog) [-] - -0-34 S Water solubility [mg .l-1] : le+06 S ?? Henry`s law constant [Pa .m3 .mo1-1] : 2 .962 S ?? Air-water part . coeff . [1 .1-1] : 0.00125 E ?? Solids-water part . ccseff .,i,n soil [l .kg-1] : 0.005448 E ?? Solids-water part . coeff . sediment '[l .kg-1] : 0 .005448 E ?? Solids-water part . coeff . susp .mat .[l .kg-1} : 0 .01879 E ?? DEGRADATION ACTUAL DEFAULT

..----~- ----~--___

. .---- .._ .__

.------- ._

.. .-----------------~~--------- .___ . .----- .... ...---.._._.-----.._ ...-~--- .. .- ---~-- DT50 photodeg . in air [d] - 700 S 160 DT50 hydrolysis in water [d] : 676 S le+ob Readily biodegradable . Yes No DT50 bioaeg . in water [d] : 15 S ?? DT50 b

.i,odeg . in .soll [d] : 15 S ?? DT50 bioaeg . in STP [d] : 0.009627 E ?? BIOACCUMULATION : ACTUAL DEFAULT

_ .. ..------- ?? BCF--fish [l .kg-i] : 0-3 S BCF worm [kg .kg-1] : 0.3947 E ?? BCF stem plant [kg .kg-1] : 0.3375

. . E ?? BCF root plant [kg .kg-1] : 2 .889 E ?? BCF air plant [m3 .kg-11 : 0.3242 E ?? BCF meat [d .kg-1] . 1 .148e-08 E ?? BCF milk [d .kg-1] : 3 .631e-09 E ?? ECOTOXICITY : ACTUAL DEFAULT

_ .. _ . . .. . .... .. .. .. . . . . . L(E)C50 for frsrn [mg .l-1] : 630 S ?? L(E)C50 for crustaceans [mg .l-1] : 101 S ?? L(E)C50 for algae ~ [mg .l-1] : 520 S ?? L(E)C50 for other aquatic species [mg .l-1] : ?? D ?? IC50 for micro-org . in STP [m9 .1-1] : 7500 S ?? NOEC for micro-org . in STP [mg .l-1] : ?? D ?? NOEC for fish [mg .l-1] : ?? D ?? NOEC for crustaceans [mg .l-1] : ?? D ?? NOEC for algae [mg .l-1] : ?? D '? ACTUAL DEFAULT

------- ..__._ .------. ._

.._ .._------~--- ._

.. .-----------~------------~-----------~_--~-- . ..._ ._ .._ . . ..__.__ .._ . ...------ ~onsump~ion rate testspec :ies [kg .kg-l .d-1] : 0 .1~5 D 0 .125

-1SE3 V_' .0 NL 0~/0 . :~/ 19y7 10 :10 Nage : 

# jQqp. 109

USES V1 .0 SINGLE SUBSTANCE Printed on : 0 ;/03/1997 10 :10 Country : NL Substance : acetonitrile CAS-No : 75-05-8 TOXICITY DATA FOR MAMMALS : ACTUAL DEFAULT

--~~-------------------------------------------- _-----.. ._----~----- --------- -----_ . _ LD50 [mg .kg-1(bw)] 175 S 1 LC50 inhalatory [mg .m-3] : 4524 S ?? NOEC in food [mg .kg-1] : 195 E ?? Oral NOAEL, non-carcinogens [mg .kg-l .d-1] : 19 .5 S 1 Is this value a LOAEL ? . No No Animal test or human study ? . Animal Animal Subchronic or chronic test, ? . Subchronic Subchronic NOAEL inhalatory [mg .m-3] : 168 S ?? Is this value a LOAEL ? ' . No No Animal test or human study ? . Animal Animal Subchronic or chronic test ? . . Subchronic Subchronic ADI or TDI [mg .kg-l .d-1] : Genotoxic or carcinogenic . Corrosive or sensitizing . Toxic to reproduction . vEL man, oral, carcinogens [mg .kg-l .d-1] : NEL man, inh ., carcinogens [mg .m-3] : ?? D ?? Yes No No No No No le-05 D le-05 ?? D ?? ACTUAL DEFAULT --~-- ---~ ------ ---.._._----__-_ .---- --- - -- -- ------------ -- -------------------------- Consumption rate testspecies [kg .kg-l .d-1] : 0.1 D 0.1 Scenario . No consumer No consumer INTERMEDIATE RESULTS exposure exposure

---- ...------- ._

. . .... ._ .. .----._-.__

. ._ .----~---. ._ ._.__._._ .._.___

..- .___... ..___

._ ..._-_ . .. .__._ .. .__ ..._ .._ ...--- ._ . ..__._ .._._ .._ ._. .__.. . ._ ... .... __._ ... ..---- ... . . . . . ... . . LOCAL EMISSION ~ OVER- CkLCUWRITE LATED _- .-- ~-- ----- -------~- ---~ -------- ---- ---__ ._ .._ ._ . _ .. ...- ._ ---~--------~--- ._ ._ ._. _.....-- -- ---.._

._ _ .. _ ._ _ . ._. . ...- ..----- -- Step with largest emission to water . Processina Release to air [kg .d-1] : 273 .2 U Release to waste water [kg .a-1] : 166 .6 0 Release to surface water [kg .d-1] : 0 Relea~_-e to soil [kg .d-1] . 3 .35 0 Number of emission aays [d] . 1 Distr . factor emission flux to water [-] : 3 .155

uSES Vl .u NL 03/03/1997 10 :10 Pace : 3



# 1iv1P. 110

USES V1 .0 SINGLE SUBSTANCE --~--- ------- ._._..------.. .__ ..--_--------~-- Printed on : 03/03/1997 10 :10 Country : NL Substance : acetonitrile CAS-No : 75-05-8 REGIONAL EMISSION OVER- CALC:UWRITE LATED Release to air [kg .d-1] : 9 .5e+04 0 Release to waste water [kg .d-1] : 166 .6 0 Release to surface water [kg .d-1] : 0 Release to agricultural soil [kg .d-1] : 3 .35 0 Release to industrial soil [kg .d-1] : 0 CONTINENTAL EMISSION ~ ~ OVER- C :AL~" :U- ' WRITE LATED

_ .. __ . ._ .. .- ----~---- -- ------~---- .. _--- --- ----------~--- .__ .-_------ _------------------- --- .__ __ _ .. .._ . .- ------- ~- Release to air ' ~ [kg .d-1 .] 8 .6be+05 1G.: .IJ ne" lease to waste water [kg .d-1] : 500 2466 Release to surface water [kg .d-1] : 0 Release to agricultural soil [kg .d-1] : 10 0 Release to industrial soil [kg .d-1] : 0 LOCAL EMISSION FROM STP OVER- CALCUWRITE LATED Emission STP to air [kg .d-1] : 1 .fJ28 Emission STP " to water [kg .d-1] : 5 .578 Emission STP to susp .matter [kg .d-1] : 1 .62e-05 Emission STP to sludge [kg .d-1] : 0_00__1)8'~S REGIONAL EMISSION FROM STP OVER- CALUUWRITE LA Emission STP to air [kg .c-1] . Emission STP to water . LKg .d-1] . E.mis-_ior STP to susp .matter [kg .d-1] : Emiss :ort SIP to sludge [kg .d-13 0.952 5 .593 1 . Eo2ie-05 0. 00~53c

;,

~:&' .I7INEr~~~H~ EMiSSION FROM STP OVER- CA_C;UWRITE LATEL) Emission S- to ai,- [kg_d-1] . 2,2 Emission STP to water [kg .c~-1] . CRl : .ssion ;:~~FF to susp .matter [kg .d-1] . 4 .87e-05 Emission STP to sludge [kg . d-1~~ : 0 .01152

c ;. v 1 , i:i I',J_ Cizi'C%-'/1 c%G 7 10 :10 Page :



# ilift-- ...---- ._.-------~ -------~--~- -~--=----------- .-_..---- -----

USES V1 .0 SINGLE SUBSTANCE Printed on : OJ/OJ/1997 10 :10 Country : NL Substance : acetonitrile CAS-No : 75-05-8 LOCAL ENVIRONMENT CONCENTRATIONS Cone . influent STP Conc . aeration tank Cone . Cone . Cone . Cone . Conc . Cone . :~onc . Cone . STP

effluent STP sludge STP air (100m from STP) air (100m from source) groundwater agricultural soil surface water (episode) surface water (annual) [Mg .l-1] : [mg .l-1] . [Mg .l-1] : [mg . kg-1] : [mg .m-3] : [ mg m-3] . [Mg . 1-11 : [mg .kg-1] : [mg .l-1] : [mg i-1] . OVER- CALCUWRITE LATED

-- 92 .5L, 3 .128 3 .099 3 . 349 7 .826e-07 0.0002079 2 .206e-06 2 .931e-05 0 .09683 0.0002653 REGIONAL ENVIRONMENT CONCENTRATIONS Cone . Cone . Cone . Cone . Cone . . Cone . air

s.urface water porewater natural soil aaricultural soil inoustr-ial soil CONTINENTAL Cone . Conc . Cone . "ono . Cone . Cone . LOCAL :

. .one . Corl,, : .

l " ~il IL" , _, J t -I ,~ . ENVIRONMENT

air surface water porewater natural soil agricultural soil industrial soil CONCE-NTRATIONS IN air CI'_' I-IKirq w

.. ~ er Frem 04- plants r G, : _ . ~` IJi4~ .r ~S Inc, ~I 11- J. I, OVERWRITE _- [ mg . m-3] [mg 1-1] . [mg .l-1] [mg .kg-1] [Mg .kg-1] [mg .kg-1] CALCULATED ~0 .001861 0 .0006697 0.0006c; 13 0.0003432 0.0002002 0.0003432 CONCENTRATIONS

HUMAN OVER- CALCUWRITE LATED [mg m-31 ---------------------- --- ----- [mg .1-1] : [mg .l-1] : [mg .kg-1] : [mg .kg-1] : [mg .kg-1] : INTAKE MEDIA L Lmy .K~-i~ : [ Mg . kg-1 ~ : [Mg .kn-1 ~mg .kg-1]

. [mg ~u--, - 0 .0008688 0 .0002696 0 .0003109 0 .0001602 '-~ . VUl

_ e-03 U. uCjult ": :.~ uvE .I .- ~ 1~+h .UU

0 . 00012 tG57- 7 .539e-05 7 .729e-05;:) rir,te0 . ,Dr : Country

L J . .. . U3 7 I'v ~

u ~ ~ ~ L7~ 1 i . . . LOCAL : INTAKE BY PREDATORS Cone . earthworms Cone . fish

water LOCAL : HUMAN INTAKE VIA INDIRECT EXPOSURE Intake air Intake drinking Intake fisn Intake stem of plant intake root of plant Intake meat Intake milk Total human dose REGIONAL : CONCENTRATIONS Cone . air Cone . drinking water Cone . fish Co-io . s,Leirr ; ot plart .s

~.,0~-,c . rGOt of piants Cone . meat Cone . miik C;onc . e, : :rtr,worm=_ . ttGJ1 0NAL . t-ftJI-iHN Intake air iNTAKt : i ~_,,

- -------~- ---~ ---~-- ... ...-~--

[mg .kg-1] : [mg kg-1] .

IN

VIA [mg .kg-l .d-1]

[mg .kg-l .d-1] [mg .kg-l .d-1] [mg .kg-l .d-1 ..] [mg .kg-l .d-1] [mg .kg-l .d-1] [mg .kg-l .d-1] [mg .kg-l .d-1] HUMAN INTAKE

f m tg rr,-~,1 - [mg .l-1] : [mg-kg-1 ] : [mg .kg-1] : Lmg .kg-1 ;, : [Mg . kc-1 . Lmg kg-i ; . [mg .kg-1] : INDIRECT EXPOSURE Lm9-<<g- :--aInt at.: e GrInKI .ng water take f is-) Inc&~~:e stem of pl,_=,r,t ~ r~ c a:< e rnot of piar z : - ;ta~~-e meat"

. .r'Lw, .~Ke ir~I K Tn? a 1 human

USES V1 .0 NL dose 7mg .kc- [mg .kg-l .a-1]

GG- 1 ~ ," .g-i .d-1 [Pig . kg-l .d-1] [mg .kg-l .d-1]

0: ./03/19c~7 10 : 10 OVER- CALC:UWRITE LATED

1 .157e-05 7 .959e-05 OVER- CALCUWRITE LATED

_..-- . . .S -_ .. .---------- 4 .45e-05 7 .58e-06 1 .251e-08 4 .417e-07 1 .5e-07 6 .024e-13 6 .001e-13 5 .274e-05 OVER- uWRI TE ED

0 .0018b1 0 .0006913 O .OuU20Uy U . 000b i G8 u .0005783 3 .129e-09 9 .8-4e-lv 7 . 84ye-05 lJVtk- t.AL C,lJwRITE LATED

0 GUUO`~Sc1 . ~~ .75e ..-G= e-08 J . ~. :1Je-l~G 1 .024e-06 5 .363e-12 5 .343e-12 0.0004234

Page : 

# 13p. 113

USES V1 .0 SINGLE SUBSTANCE

_ ._ . . ._ .. .__..... .... .. .-.___ ._... ... ... Printed on : 03/03/1997 10 :10 Country : NL Substance : acetonitrile CAS-No : 75-05-8 NEC ECOSYSTEMS OVER- CALCUWRITt LATEL NEC for micro-organisms [mg .1-i] : NEC aquatic species [mg .!-1] : NEC terrestrial species [mg .kg-1] : NEC predator in food [mg .kg-1] : IF TOXICITY DATA ON TERRESTRIAL ORGANISMS ARE OvE- :- NOT AVAILABLE wri-r I NEC terrestrial species (eq . part. . [mc .~ c ;- : ~ It= T-tE SUBSTANCE IS 750 1 .01 19 .5

? =

c~ c ._ c

:~ ~- , -

. . . . ._ . _ . . . . . . . . : . _- . -ar_or, IC50micro to NL ~7.icro Extrapolation L~:5 ;_1 to wZC Extrapoia7ion 1-5 --US ~o N,- :C SubC :hrOnic Ni :ti, to NEC

_ 1'-' i_ K= J VL 1

a ;- Gf . .~, .uot . 7 -J = L [-] - [- .__._ . .. ._.__ . ._ . 0 .004171 G 46 .iu 4LS .`'8 0 .0031i 0 LOCAL) INTER"fEDIATE DIi-'`, ~

__ . . ... . . . . . _ ...._... ... . . ... . . . .. .. . .._ ._ ... ... ._. . . ._..--------------------------- . ._ . ._.. .... ._ ..__ .__ ..._ ._

. . .... ._. .... t'iaza.rG quGt . aqua~~iC species 0 .09557 U histri .bution factor tor the hazard quot .[-] : 176 .7 176 .7 ?robab :11ty GE, : /NEC > L [-j : 0 .189 U T=R~EOTRIAL ECO_7ti'CT~-_M (LOCAL} INTERMEDIATE LIRE .,7

,:.,-:3ra quot . : .errestriai species -)r.~ d ;-a quot . terr . species (eq . part .) 1-1 . C. 0 001UiJ2

. C

E ; V.1 .0 NL U5/03/ 19' ;7 10' : 10 Dva

: _ .,- OVER-

w~ i` . 1

_ . L ~S I L ~ . .

7r,-- .

age : 

# Ip. 114

-_ _ .. ._ .. .------- .. ...- .-__ .._ ..._. .__ .. .._ ._..__ ._ ..__._._. .--- .... ... . .... . USES V1 .0 SINGLE S'-i& .STANCE

_ ..- .__ . ._ . .. .--~--..._.__._._._

. ._._ .. . ... ._ ._ ._. - .. .... _ ... . ._..._. . .. .._ ..-- Printed on : 03/03/1997 10 :10 Country : NL Substance : acetonitrile CAS-No : 75-05-8 PREDATORS (LOCAL) INTER"iEDIATE DIRECT

-- -- ._ __

. . .... ._ . . .. .- - - -- .... -- - -- ... .. . .... __ ... .- --- ... .._ _. __

...--- --- -- - ~--- - - --. ._ . _

.. ..- - -.__ .._ ._ ._. ..._ ..... ._ ._.._...__ . _

. _ ._ .-- Hazard quot . fi1sh-eating predators [-] : 4 .08ie-06 Hazard quot . worm-eating predators [-] . 5 .932e-07 MAN (LOCAL) INTERMEDIATE DIRECT

---- .. .---------- ._._._. .. . .---.._.__... . .. . ... .._ ..--~-- .- ._ ._. .._._ . .-~----~-- .__ .-_._

.. .... ._._._ . ..... ..._ .._.__

. ._. ._. __._ . ..._.... .__

._ .. ....--- .. . ..._._ ....._

. .--~--- .._ .... ._

. ._.._ .. ... .__. Margin of Safety to, man 0 .1896 ?? AQUATIC ECOSYSTEM (REGIONAL) INTERMEDIATE DIRECT _- ._. __ . .... . . . . ._ ._ _

.. ._ .___... ._ _. . .- .. . ._ _

...-------- ._

. ._.. ._ .__r Hazard quot . aquatic species 0 .0006631 6 .b39e-07 TERF'ESTRIAL ECOSYSTEM (REGIONAL) INTERMEDIATE DIRECT Hazard quot . terrestrial species ~o 00 iya : :ard quet . terr . specles (ec, part . :) [-] : 0 .0006844 4 .47e-07 PREDATORS (REGIONAL) INTERMEDIATE DIRECT

.. . . . _ .. ._ . . .... .._. ... _ .__ .. _ . ... .... ..... _ ... .. . ... . .... ..... _ . ..... ._.. . . .- ..... . .' . .. .. .. . . _

.. .... . .. .. .. ... Hazard quot . fish-eating predators 1 .03e-05 1 .052e-08 Hazard quot . worm-eating predators [-] : 4 .051e-06 2 .645e-09 MAN (REGIONAL) Margin of Safety for man MODEL PARAMETERS 33 .81 ;

_. . ... .... . .. . -------- . .__ . .--- ..._ .__ . ._._ .._ ..___- .._ ._ .- ._-_

._ .._ ..__.._ .... ._

... . ..-- .--- ... . .... ._--._

.. ._..--~--------------------------_ .-._ ._.._.---- JISTRIBUTION PARAMETERS FOR THE LOCAL MODEL : Ai : :TUAL DEFAULT

----------~--------- Size of the local STP [eqj : 1 .2e+04 D 1 .2e+04

. 32 D 32 Dilution factor for STG effluent [-" Distr . facto- for diiution [-j : 148 D 148 Dis--r . factor inTiuent discharge [-] : 41 D 41 Amount of siucioe applieo [l :g .ha-l .yr-1] : 2000 D 2000 DISTRIBUTION PA~'AMETERS FOR THE REGIONAL ACTUAL DEFAULT JYJIGM . Size of the re :~,or~al STP [eq] : le-07 D le+07 INTERMEDIATE DIRECT

UC: .C V1 .0 NL 03/05/1997 10 :10 ' ~~age :p. 115

USES V1 .0 SINGLE SUBSTANCE

-

._ _ .. ._ _ .. .---- - ---_

..-------- ---~--------~- - -~----- Printed on . 03/03/1597 10 :10 Country : NL Substance : acetonitrile CAS-No : 75-05-8 MAN : AC7uhL DEFAULT

.._._ ._ -------- -- --- . .. .... ..... .--~------ _ ._. .-----~-- ..--- Daiiy intake of drinking water [m3 .d-1] 0.002 D 0 .002 Daily intake of fish [kg .d-1] : 0.011 D 0.U11 Daily intake of leaf crops [kg .d-1] : 0.4 D 0.4 Daily intake of root crops [kg .d-1] : 0.124 D 0.124 Daily intake of meat [kg .d-1] : 0.12 D 0.12 Daily intake of dairy products [kg .d-1] : 0.378 D 0 .378 Bioavailability for inhalation [-] : 0.75 D 0.75 Bioavailability for oral exposure [-] : 1 D 1 Bioavailability for dermal exposure [-] : 1 D 1 COUNTRY PARAMETERS EXTRAPOLATION FACTORS . Extrapolation IC50micro to NECmicro Extrapolation LC50 to NEC Extrapolation ,'- . t_C.`,Os to r~~ECtxtrapoiat : o4,v_C ..) . L.er1rJ . at tY'lE: air-wc;ter .Lnter1c .UE [K] . i-lvg . wli~C! sPeeu i6m :~, : .>ovr 5ur-face [m.s-1]

. D :STRiEU' : Ow PARAMETERS FOR THE LOCAL MODEL Distribution factor for flushing factor DISTRIBUTION Pt ;--RAMETtRS FOR THE REGIONAL S YS TtI`i

. ... . __ .. .. . ..... . .. . ... .. .. . .. . .. . . . .. . . . ... . .... . ._ . .. .. ... .. . .. . ... . . . . . . TotaA area of the reg . s\,stem [m2] : Fra_c_ of reo_ionai area that is water --1 : Frac . of t-eg . Gr eG tilCC` ze . I,rYL . soil [-] . 1-r-au . o-~ ;s agr . soil [-] .

c- ; reg . area that is ind . soil [-] : deptn of the reg . water comp . [m] . Sum aisch . st .r . crossing sys .bound [m3 .s-1] : ACT 1C 10V0 1 :_) ()

: .Tu!-;L S ACTUAL

ACi UA ~ 3 .8e+10 0.125 0.415 0.45 0.01

J 2600

.. . .. . ..... ._.. ... _ . _

. .. . .... . _. .... ..... .. . .. .. . .... .... _ .. ..... - ... .._. .... ..._ .. . ... . ... .. . ..... ..... . ._. ... ..... .. _ . ..... .... ... .. ... . ._ USES V10 NL 03/03/1997 10 :10 Pace :_..--------.. ..-_-------------~---------

U,S,,ES V1 .0 SINGLE SUGSTANCE Printed on : G~/05/1997 10 :10 Country : NL Substance . acetonitrile CAS-No : 75-05-8 p. 116

DISTRIBUTION PARAMETERS FOR THE REGIONAL AND ACTUAL THE CONTINENTAL SYSTEM



### ----------- ._.-.-------------------- . .------------- ...---------- -- Atmospheric mixing height [m] : Mixing depth sediment compartment [m] : Mixing depth compartment of nat . soil [m] : . Mixing depth compartment of agr . soil [m] : Mixing depth compartment of ind . soil [m] : Average annual precip~itation [m.s-1] : Frac . rain water infiltrates soil ' [-] = . Frac . rain water that runs off~soil [-] : Dep .velocity aerosol particles [m.s-1] : C .onc . of biota in surfacie water [kg .m-3,] : Settling velocity susp . particles [m.s-1] : _ _ .-_. .... .--- 

### 1000 0 .03 0.05 0.2 0 .05 51e-08 0.4 0 .5 0.001 0.001 89e-05 DISTRIBUTION PARAMETERS FOR THE STP MODEL ACTUAL (IN/OUT) Waste water per inhabitant [m3 .d-1] . Surplus sludge per inn . equiv .[kg .d-l .eq-1] : 0.15 0 .0355 ORGANISMS ACTUAL Daily intake of soil by cattle [kg-d-1] : 0 .41 Daily intake of grass by cattle [kg .d-1] : 16 .9 Daily intake of air by cattle [m_d-i] : 1<< Daily ventilation rate [m3 .d-1] : 20 Average human bodyweight [kg] : 70

_ ..----------- --------------------- - --- USES v'1 .0 NL 03%03,'1997 10 :10 

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