Acetonitrile

Carcinogenicity

Description of key information

Reliable animal studies do not indicate that acetonitrile is carcinogenic in rats or mice.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via inhalation route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1988-1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: US National Toxicology Program

GLP compliance:

not specified

Specific details on test material used for the study:

- Name of test material (as cited in study report): Acetonitrile
- Analytical purity: >99% purity.

Species:

rat

Strain:

Fischer 344

Details on species / strain selection:

Widely accepted species/strain, recommended by current guidelines

Sex:

male/female

Details on test animals or test system and environmental conditions:

Tail tattoo and cage position during exposure were used to identify animals.

TEST ANIMALS
- Source: Simonsen Laboratories (Gilroy, CA)
- Age at study initiation: 6 weeks old on the first day of exposure.
- Housing: Individually stainless-steel wire bottom cages which were rotated weekly.
- Diet: NIH-07 diet/pelleted (Zeigler Brothers, Inc., Gardners, PA), ad libitum, except during exposure period.
- Water: ad libitum.
- Acclimation period: 15 days.

IN-LIFE DATES: From: 31 March 1988 To: 4 April 1990

Route of administration:

inhalation

Type of inhalation exposure (if applicable):

whole body

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Liquid acetonitrile was pumped from a reservoir to a vaporizer that consisted of a stainless-steel cylinder heated to approximately 200 degrees F +/- 5 degrees F with a glass fiber wick. Acetonitrile vapor was mixed with charcoal-filtered and HEPA-filtered air. The mixture was drawn into a stainless-steel distribution manifold, diluted to the desired concentration by adjusting the compressed air pressure to the vacuum pumps, and delivered to the exposure chambers once the concentrations in the distribution system had stabilized.
- Method of holding animals in test chamber: stainless-steel wire bottom cages, changed weekly and rotated weekly in the exposure chamber
- Temperature, humidity, pressure in air chamber: 20.9-26.8 degree C, 55.3%-57.8%, Fluorescent light: 12 hours/day
- Method of particle size determination: A Gardner Type CN Small Particle Detector was used prior to study start and again during the study with animals in the chambers to check all chambers for any aerosol inadvertently produced during generation of the atmosphere.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chamber concentrations were monitored with a single on-line HP-5840 gas chromograph equipped with a flame ionization detector and an OD nickel column packed with 80/100 Porapak Q. Each chamber was sampled approximately twice hourly throughout the study. Uniformity of vapor concentration in the inhalation exposure chambers was evaluated prior to the start of the study, and approximately every 90 days thereafter.

Duration of treatment / exposure:

103 weeks

Frequency of treatment:

6 hours/day, 5 days/week

Post exposure period:

No

Remarks:

Doses / Concentrations:
0, 100, 200, or 400ppm
Basis:
nominal conc.

No. of animals per sex per dose:

56 males & 56 females per group. Eight male and eight females were evaluated at 15 months.

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: The doses selected for the 2-year study of acetonitrile were based on reduced survival of 800 ppm males and 1,600.ppm males and females in the 13-week study.

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily for mortality and signs of toxicity or moribundity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Individual observations were recorded every 4 weeks.

BODY WEIGHT: Yes
- Time schedule for examinations: 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.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 15 months into the study.
- Anaesthetic used for blood collection: Yes, 70% CO, 30% air mixture.
- How many animals: 8 males and 8 females

Sacrifice and pathology:

Anesthetization with 70% carbon dioxide followed by exsanguination via the brachial artery.

GROSS PATHOLOGY: Yes. A complete necropsy and microscopic examination were performed on all rats. All organs and tissues were examined for grossly visible lesions.

ORGAN WEIGHTS: Organs weighed at the 15-month interim evaluations were liver, lungs and right kidney.

HISTOPATHOLOGY: Yes. tissues were fixed and preserved in 10% neutral buffered formalin, processed, trimmed, embedded in paraffin, sectioned to a thickness of 5 to 6 pm, and stained with hematoxylin and eosin for microscopic examination. In addition to gross lesions and tissue masses with
regional lymph nodes, tissues examined included: adrenal gland, brain, bone and marrow, clitoral gland, esophagus, gallbladder (mice), heart, kidney, large intestine (colon, cecum, rectum), larynx, liver, lung, lymph nodes (bronchial, mandibular, mediastinal, and mesenteric), mammary gland, nose, ovary, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, seminal vesicle, skin, small intestine (duodenum, jejunum, ileum), spleen, stomach (forestomach and glandular), testis, thymus, thyroid gland, trachea, urinary bladder, and uterus.

Statistics:

The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). Animals found dead of other than natural causes were censored from the survival analyzes; animals dying from natural causes were not censored. Statistical analyses for possible dose-related effects on survival used Cox's (1972) method for testing two groups for equality and Tarone's (1975) life table test to identify dose-related trends. All reported P values for the survival analyses are two sided.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
Survival rates of exposed rats were similar to those of controls. The behavior, general health, and the appearance of exposed rats were similar to that of the controls throughout the study.

BODY WEIGHT AND WEIGHT GAIN
Exposure to acetonitrile by inhalation for 15 months or 2 years had no effect on body weight gain or final mean body weights.

HAEMATOLOGY
At 15 months, minimal anemia was noted in female rats exposed to 400 ppm. The hematologic effects observed were minor and considered of no biological significance.

ORGAN WEIGHTS
Organ weights of exposed rats were similar to those of the controls.

HISTOPATHOLOGY: NON-NEOPLASTIC
The incidences of basophilic, eosinophilic, and mixed cell hepatic foci in 400 ppm males were marginally greater than in the controls. A statistically significant increase in basophilic foci was observed in the 200 and 400 ppm groups, but the foci were not atypical in appearance. Thus it is uncertain if these lesions were preneoplastic.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
The incidences of hepatocellular adenoma (3/48), hepatocellular carcinoma (3/48), and hepatocellular adenoma and carcinoma combined (5/48) were greater in male rats exposed to 400 ppm than in the controls (one carcinoma). The incidences of hepatocellular adenoma and heptacellular carcinoma were within the range of historical controls. However, the incidence of hepatocellular adenoma or carcinoma combined slightly exceeded the range of historical controls (2-8%). There were no exposure-related liver lesions in female rats exposed to acetonitrile.

Relevance of carcinogenic effects / potential:

The survival of male animals exposed to 400 ppm was greater than that in the control group (e.g. 12/56 controls and 21/56 400 ppm animals survived to the end of the study). The first liver neoplasms were noted after about 90 weeks, at this time 31/56 controls were surviving, compared to 37/56 at 400 ppm. Neoplasms in the 400 ppm group were typically noted for the longer survivors (e.g. 5/6 tumours were noted in animals that were 727-733 days on the study). No significant dose-related trend was present after incidences were adjusted for survival using the life table test (US EPA, 1999).

Key result

Dose descriptor:

NOAEC

Effect level:

400 ppm (nominal)

Sex:

male/female

Basis for effect level:

other: highest level tested

The survival of male animals exposed to 400 ppm was greater than that in the control group (e.g. 12/56 controls and 21/56 400 ppm animals survived to the end of the study). The first liver neoplasms were noted after about 90 weeks, at this time 31/56 controls were surviving, compared to 37/56 at 400 ppm. Neoplasms in the 400 ppm group were typically noted for the longer survivors (e.g. 5/6 tumours were noted in animals that were 727-733 days on the study). No significant dose-related trend was present after incidences were adjusted for survival using the life table test (US EPA, 1999).

Conclusions:

These results do not indicate that acetonitrile is carcinogenic to male or female rats.

Executive summary:

In a cancer bioassay conducted by the US National Toxicology Program, inhalation exposure to the maximum tolerated dose of acetonitrile vapour for 2 years did not produce evidence of carcinogenic activity in male or female F344/N rats. NTP concluded equivocal evidence of carcinogenic activity in male F344/N rats based on a marginal increase in the incidence of liver neoplasms at the highest dose (400 ppm). Groups of 56 male and 56 female rats were exposed to 0, 100, 200 or 400 ppm acetonitrile vapour on 6 hours per day, 5 days per week for 2 years. The survival of male animals exposed to 400 ppm was greater than that in the control group (e.g. 12/56 controls and 21/56 400 ppm animals survived to the end of the study). The first liver neoplasms were noted after about 90 weeks, at this time 31/56 controls were surviving, compared to 37/56 at 400 ppm. Neoplasms in the 400 ppm group were typically noted for the longer survivors (e.g. 5/6 tumours were noted in animals that were 727-733 days on the study). No significant dose-related trend was present after incidences were adjusted for survival using the life table test (US EPA, 1999).

 

Two-year survival, mean body weights, organ weights, behaviour, general health, and appearance of male and female rats exposed to acetonitrile were similar to those of the controls. At 15 months, minimal anaemia was noted in female rats exposed to 400 ppm. The hematologic effects observed were minor and considered of no biological significance. The incidence of basophilic hepatic foci in 200 and 400 ppm males were statistically increased, but the foci were not atypical in appearance and are not considered evidence of a hepatotoxic effect. Based on these findings the chronic NOAEC for acetonitrile vapour in rats is considered to be 400 ppm (672 mg/m3).

Reference 2

Endpoint:

carcinogenicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1988-1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: US National Toxicology Program

Principles of method if other than guideline:

US NTP protocol comparable to OECD 453

GLP compliance:

not specified

Specific details on test material used for the study:

- Name of test material (as cited in study report): Acetonitrile
- Analytical purity: >99% purity.

Species:

mouse

Strain:

B6C3F1

Details on species / strain selection:

Widely accepted species/strain, recommended by current guidelines

Sex:

male/female

Details on test animals or test system and environmental conditions:

Toe clip and cage position during exposure were used to identify animals.

TEST ANIMALS
- Source: Simonsen Laboratories (Gilroy, CA)
- Age at study initiation: 6 weeks old on the first day of exposure.
- Housing: Individually in stainless-steel wire bottom cages which were rotated weekly.
- Diet: NIH-07 diet/pelleted (Zeigler Brothers, Inc., Gardners, PA), ad libitum, except during exposure period.
- Water: ad libitum.
- Acclimation period: 15 days.

Route of administration:

inhalation

Type of inhalation exposure (if applicable):

whole body

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Liquid acetonitrile was pumped from a reservoir to a vaporizer that consisted of a stainless-steel cylinder heated to approximately 200 degrees F +/- 5 degrees F with a glass fiber wick. Acetonitrile vapor was mixed with charcoal-filtered and HEPA-filtered air. The mixture was drawn into a stainless-steel distribution manifold, diluted to the desired concentration by adjusting the compressed air pressure to the vacuum pumps, and delivered to the exposure chambers once the concentrations in the distribution system had stabilized.
- Method of holding animals in test chamber: stainless-steel wire bottom cages, changed weekly and rotated weekly in the exposure chamber
- Temperature, humidity, pressure in air chamber: 20.5-26.7 degrees C, 54.0%-54.4%, Fluorescent light: 12 hours/day
- Method of particle size determination: A Gardner Type CN Small Particle Detector was used prior to study start and again during the study with animals in the chambers to check all chambers for any aerosol inadvertently produced during generation of the atmosphere.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chamber concentrations were monitored with a single on-line HP-5840 gas chromograph equipped with a flame ionization detector and an OD nickel column packed with 80/100 Porapak Q. Each chamber was sampled approximately twice hourly throughout the study. Uniformity of vapor concentration in the inhalation exposure chambers was evaluated prior to the start of the study, and approximately every 90 days thereafter.

Duration of treatment / exposure:

103 weeks

Frequency of treatment:

6 hours/day, 5 days/week

Post exposure period:

No

Remarks:

Doses / Concentrations:
0, 50, 100, or 200 ppm
Basis:
nominal conc.

No. of animals per sex per dose:

60 males & 60 females per group. Ten male and ten females were evaluated at 15 months.

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: The doses selected for the 2-year study of acetonitrile were based on reduced survival and gross and histopathologic lesions in 400, 800, and 1,600 ppm groups of male and female mice in the 13-week study.

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily for mortality and signs of toxicity or moribundity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Individual observations recorded every 4 weeks.

BODY WEIGHT: Yes
- Time schedule for examinations: 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.

Sacrifice and pathology:

Anesthetization with 70% carbon dioxide followed by exsanguination via the brachial artery.

GROSS PATHOLOGY: Yes. A complete necropsy and microscopic examination were performed on all rats. All organs and tissues were examined for grossly visible lesions.

ORGAN WEIGHTS: Organs weighed at the 15-month interim evaluations were liver, lungs and right kidney.

HISTOPATHOLOGY: Yes. tissues were fixed and preserved in 10% neutral buffered formalin, processed, trimmed, embedded in paraffin, sectioned to a thickness of 5 to 6 pm, and stained with hematoxylin and eosin for microscopic examination. In addition to gross lesions and tissue masses with
regional lymph nodes, tissues examined included: adrenal gland, brain, bone and marrow, clitoral gland, esophagus, gallbladder (mice), heart, kidney, large intestine (colon, cecum, rectum), larynx, liver, lung, lymph nodes (bronchial, mandibular, mediastinal, and mesenteric), mammary gland, nose, ovary, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, seminal vesicle, skin, small intestine (duodenum, jejunum, ileum), spleen, stomach (forestomach and glandular), testis, thymus, thyroid gland, trachea, urinary bladder, and uterus.

Statistics:

The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). Animals found dead of other than natural causes were censored from the survival analyzes; animals dying from natural causes were not censored. Statistical analyses for possible dose-related effects on survival used Cox's (1972) method for testing two groups for equality and Tarone's (1975) life table test to identify dose-related trends. All reported P values for the survival analyses are two sided.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
Two-year survival of exposed male and female mice was similar to that of the controls, except that the survival of male mice in the 200 ppm group was significantly greater than that of the controls. No clinical observations in any group were clearly related to acetonitrile exposure.

BODY WEIGHT AND WEIGHT GAIN
Mean body weights of exposed groups of male and female mice were similar to those of the controls.

ORGAN WEIGHTS
Mean organ weights of exposed groups of male and female mice were similar to those of the controls.

HISTOPATHOLOGY: NON-NEOPLASTIC
The incidence of squamous hyperplasia of the epithelium of the forestomach was significantly increased at 15 months in 200 ppm females. At 2 years, the increased incidence of this lesion was dose related in the 100 and 200 ppm male groups and in all exposed female groups.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
There were no increases in the incidences of neoplasms that were considered related to acetonitrile exposure in mice.

Key result

Dose descriptor:

NOAEC

Effect level:

200 ppm (nominal)

Sex:

male/female

Basis for effect level:

other: highest level tested

Survival of male mice exposed to 200ppm was significantly greater than that of the control mice. The incidence of squamous cell hyperplasia of the forestomach was increased in both sexes at 200ppm (12/50 males and 19/50 females, compared to 2-3/49 for controls). However, the incidence of squamous cell papilloma was only slightly increased at this exposure level, and was within the range of historical controls.

Conclusions:

There was no evidence that exposure to maximum tolerated doses of acetonitrile caused cancer in either male or female mice.

Executive summary:

In a cancer bioassay conducted by the US National Toxicology Program, inhalation exposure to the maximum tolerated dose of acetonitrile vapour for 2 years produced no evidence of carcinogenic activity in male or female B6C3F1 mice. Groups of 60 male and 60 female mice were exposed to 0, 50, 100 or 200 ppm acetonitrile vapour 6 hours per day, 5 days per week for 2 years. Two-year survival of exposed male and female mice was similar to that of the controls, except that the survival of male mice in the 200 ppm group was significantly greater than that of the controls. Mean body weights and organ weights of exposed groups were similar to those of the controls, and no clinical observations in any group were clearly related to acetonitrile exposure.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

High quality US NTP carcinogenicity studies are available in the rat and mouse

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

No classification of acetonitrile is proposed for carcinogenicity based upon the results of reliable chronic studies in rats and mice.

Additional information

US NTP inhalation carcinogenicity studies are available for acetonitrile in the rat and mouse.

Inhalation exposure to the maximum tolerated dose of acetonitrile vapour for 2 years did not produce evidence of carcinogenic activity in male or female F344/N rats. NTP concluded equivocal evidence of carcinogenic activity in male F344/N rats based on a marginal increase in the incidence of liver neoplasms at the highest dose (400 ppm). Groups of 56 male and 56 female rats were exposed to 0, 100, 200 or 400 ppm acetonitrile vapour on 6 hours per day, 5 days per week for 2 years. The survival of male animals exposed to 400 ppm was greater than that in the control group (e.g. 12/56 controls and 21/56 400 ppm animals survived to the end of the study). The first liver neoplasms were noted after about 90 weeks, at this time 31/56 controls were surviving, compared to 37/56 at 400 ppm. Neoplasms in the 400 ppm group were typically noted for the longer survivors (e.g. 5/6 tumours were noted in animals that were 727-733 days on the study). No significant dose-related trend was present after incidences were adjusted for survival using the life table test (US EPA, 1999). Two-year survival, mean body weights, organ weights, behaviour, general health, and appearance of male and female rats exposed to acetonitrile were similar to those of the controls. At 15 months, minimal anaemia was noted in female rats exposed to 400 ppm. The hematologic effects observed were minor and considered of no biological significance. The incidence of basophilic hepatic foci in 200 and 400 ppm males were statistically increased, but the foci were not atypical in appearance and are not considered evidence of a hepatotoxic effect. Based on these findings the chronic NOAEC for acetonitrile vapour in rats is considered to be 400 ppm (672 mg/m3).

Inhalation exposure to the maximum tolerated dose of acetonitrile vapour for 2 years produced no evidence of carcinogenic activity in male or female B6C3F1 mice. Groups of 60 male and 60 female mice were exposed to 0, 50, 100 or 200 ppm acetonitrile vapour 6 hours per day, 5 days per week for 2 years. Two-year survival of exposed male and female mice was similar to that of the controls, except that the survival of male mice in the 200 ppm group was significantly greater than that of the controls. Mean body weights and organ weights of exposed groups were similar to those of the controls, and no clinical observations in any group were clearly related to acetonitrile exposure.

In summary, the results of the NTP bioassay on acetonitrile do not indicate that acetonitrile was carcinogenic in laboratory rats or mice.

Justification for selection of carcinogenicity via inhalation route endpoint:

A weight of evidence approach is taken to this endpoint.