Acetonitrile

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

chronic toxicity: inhalation

Remarks:

combined repeated dose and carcinogenicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

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.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: US National Toxicology Program

Principles of method if other than guideline:

Comparable to OECD 453 - inhalation exposure

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Acetonitrile

EC Number:

200-835-2

EC Name:

Acetonitrile

Cas Number:

75-05-8

Molecular formula:

C2H3N

IUPAC Name:

acetonitrile

Details on test material:

- Name of test material (as cited in study report): Acetonitrile
- Analytical Purity: at least 99%
- Lot/batch No.: Lot 2485
- Storage condition of test material: To ensure stability, the bulk chemical was stored at approximately 22° C in the original containers.

Specific details on test material used for the study:

- Name of test material (as cited in study report): Acetonitrile
- Analytical Purity: at least 99%
- Lot/batch No.: Lot 2485
- Storage condition of test material: To ensure stability, the bulk chemical was stored at approximately 22° C in the original containers.

Test animals

Species:

mouse

Strain:

B6C3F1

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.

Administration / exposure

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Details on inhalation 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

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 50, 100, 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:

- Post-exposure period: No
- 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

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

1

Dose descriptor:

NOAEC

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Forestomach hyperplasia. Interpretation is confounded by likely ingestion from preening behaviour and mucociliary clearance.

Remarks on result:

not determinable

Remarks:

no NOAEC identified

2

Dose descriptor:

NOAEC

Effect level:

200 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: all other endpoints

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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 vapor 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 vapor 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. 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.  Hyperplasia seen in the forestomach is considered an adverse finding, but is likely not a result of inhalation. Grooming of contaminated fur or mucocillary clearance followed by ingestion likely played a central role in this finding, although a role for inhalation cannot be ruled out and represents an area of uncertainty. Because of this uncertainty, a NOAEC for this endpoint cannot be identified. The NOAEC for other endpoints in this study is 200 ppm.