Acetonitrile

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1983

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1984

Report date:

1984

Reference 2

Reference Type:

other company data

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: US EPA TSCA 48 FR 50942, November 4, 1983. Docket OPTS 42019A.

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Environmental Protection Agency, Pesticide Programs, Proposed Guidelines for Registering-Pesticides in the U.S.; Hazard Evaluation: Humans and Domestic Animals, Federal Register, Tuesday, August 22, 1978. 43FR37336; for teratogenicity/reproductive effects

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

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 (HPLC grade)
- Physical state: colorless liquid with a sweet etheral odor
- Analytical purity: 99.0%
- Lot/batch No.: 732234
- Storage condition of test material: Room temperature

Specific details on test material used for the study:

- Name of test material (as cited in study report): Acetonitrile (HPLC grade)
- Physical state: colorless liquid with a sweet etheral odor
- Analytical purity: 99.0%
- Lot/batch No.: 732234
- Storage condition of test material: Room temperature

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Dutchland Laboratories, Inc. Swampbridge Road, Box 139A, Denver, PA
- Age at study initiation: 5 months old (actual age - 21 weeks)
- Weight at study initiation: 2.79 - 5.08 kg
- Housing: Individually assigned to housing based on computer-generated random numbers. Females were housed in vertical order according to dosage and insemination groups in one of eight individual stainless-steel cages (5 square feet of floor area with 16 inch height per individual cage). Because rabbits were scheduled to be terminated prior to natural delivery, nesting materials were not provided.
- Diet: Approxiamtely 180 g Certified Rabbit Chow® # 5322 (Ralston Purina), contained in an individual stainless-steel “J-type” feeder attached to each cage, were given to each rabbit each day.
- Water: Local water which had been passed through a reverse osmosis membrane was available ad libitum from individual glass bottles attached to each cage.
- Acclimation period: females acclimated for approximately four weeks.

ENVIRONMENTAL CONDITIONS
- Temperature: 62°F and 76°F
- Humidity: 35 - 80%
- Air changes: 12 - 15/hour
- Photoperiod: 12 hr light/dark cycle. Each dark cycle began at 1900 hours EST (2000 hours EDT).

IN-LIFE DATES: From: 17 July 1983 To: 19 August 1983

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: deionized water

Details on exposure:

Acetonitrile was administered to female rabbits by stomach tube followed by approximately 5 mL of air to clear the tube.

PREPARATION OF DOSING SOLUTIONS: All dosages were administered to rabbits as aqueous solutions (R.O. water) at a volume of 5 mL/kg/day. The acetonitrile was assumed to contain 100% active ingredient for the purpose of calculating dosages and concentrations.

OTHER: The oral route was selected for use since it has been demonstrated that exposure via gavage and inhalation produce similar effects. The test agent was administered once each day, between 0856 and 1235 hours EDT (0956 and 1335 EST, respectively). Each daily dosage was administered as closely as practical to the same time each day, with all dosages administered to all rabbits approximately 3 to 6.5 hours after the beginning of the light period at 0700 hours EST.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test chemical and test vehicle were analyzed for purity by gas-liquid chromatography prior to initiation of the main study. In addition, homogeneity analyses were performed on several concentrations, including the highest (160 mg/mL) concentration that was used in the preliminary studies and the lowest (0.02 mg/mL) concentration that was considered practical to administer in the main study. These same concentrations were determined to be stable at 24 and 72 hours and at 7 days after preparation. Analysis performed on the first day the test chemical was used demonstrated that the concentrations were correctly prepared and stable during the period of use.

Details on mating procedure:

- Impregnation procedure: Artificial insemination from five untreated proven males. A different buck was used each day of insemination. Twenty female rabbits were inseminated on each of five consecutive days. These female rabbits were intravenously administered 20 USP Units/kg of HCG approximately three hours prior to insemination with an estimated 0.25 mL of semen which had been diluted with normal saline to a concentrations of 6.0 x 10^6 spermatozoa/0.25 mL. Male rabbits were not administered the test agent.
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: Day of artificial insemination referred to as day 0 of presumed gestation.

Duration of treatment / exposure:

Days 6-18 of pregnancy

Frequency of treatment:

Once daily

Duration of test:

Day 29 of gestation

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 2.0, 15.0, 30.0 mg/kg bw/day
Basis:

No. of animals per sex per dose:

4 dose groups of 25 animals each.

Control animals:

yes, concurrent vehicle

Details on study design:

- Sex: female
- Dose selection rationale: Doses selected on the basis of two pilot studies.
- Rationale for animal assignment: Computer-generated (weight-ordered) randomization order was used to assign 100 female rabbits to the four dosage groups. These rabbits were in apparent good health and had body weights ranging from 3.03 to 5.27b kg each.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: several times during pre-study period and on days 0 and 5 of presumed gestation. Observations for physical signs of agent effect, abortion and/or viability were made several times each day during the administration period (days 6 through 18 of presumed gestation) and daily during the post-administration period (days 19 through 29 of presumed gestation).

BODY WEIGHT: Yes
- Time schedule for examinations: More frequent than required. Weights were recorded the day after arrival, once weekly during acclimation period, on days 0 and 5 of presumed gestation and daily during administration and post-administration period.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption was recorded to the nearest 25% of 180 g daily during acclimation. Observations for anorexia during study were recorded.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 29 with carbon dioxide was based on computerized random assignment which rotated throughout dosage groups.
-Organs examined: Lungs were examined, liver weights were recorded, and gross lesions considered appropriate were retained and preserved

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
- Gravid uterus weight: No, this parameter was considered extremely inaccurate due to variation in fluid loss resulting from necropsy procedures. More accurate data, consisting of frequent maternal weights and individual fetal weights, rather than a litter weight, were recorded.
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number of live and dead fetuses: Yes

Fetal examinations:

-Only body weights of live fetuses were used in determining fetal body weight averages for litters.
- Soft tissue examinations: Yes: all fetuses per litter. Only specimens observed to be grossly abnormal at soft tissue examination of fetuses were preserved. Dissected fetal tissue which had been determined to appear grossly normal was not considered necessary to preserve.
- Skeletal examinations: Yes: all fetuses were eviscerated, stained with alizarin red-S(6) and evaluated for skeletal alterations.
-Other: A transverse section was made of each unfixed fetal head, between the parietal and frontal bones, and the head then examined for the presence of internal hydrocephaly. As no viscera had grossly observed abnormalities, none was preserved.

Statistics:

Standard deviations were cited in tables only where appropriate. Maternal physical sign data and the incidence of pregnancy and abortion were analyzed using Fisher’s exact test.

The one-way analysis of variance was used to evaluate average body weights of rabbits assigned to the four dosage groups on days 0 and 5 of presumed gestation. Maternal body weight data for days 0, 6, 9, 12, 15, 19, 24 and 29 and maternal body weight change for days 0 to 6, 6 to 9, 9, to 12, 12 to 15, 15 to 19, 19 to 24, 24 to 29, 19 to 29, 6 to 19, 6 to 29 and 0 to 29 of pregnancy were analyzed using Bartlett’s test of homogeneity of variances and either the one-way analysis of variance or the Mann-Whitney U test. If Bartlett’s test was positive (P≤0.05), then the Mann-Whitney U test was used to analyze the data by testing each group individually against the control dosage group. If Bartlett’s test was negative (P≥0.05), then the one-way analysis of variance was used to analyze the data. If the F-ration was positive (P≤0.05), then Dunnett’s test was used to test each group individually against the control dosage group. The same statistical analyses were used to evaluate maternal absolute liver weight data. Data obtained during Caesarean-sectioning and for malformations and variations were evaluated using Jonckheere’s test with the population of affected fetuses per litter considered to be the basic experimental unit. If Jonckheere’s test was positive (P≤0.05), and if there were fewer than 75% ties in a group, then the Mann-Whitney U test was used to analyze the data by testing each group individually against the control dosage group. If Jonckheere’s test was positive (P≤0.05), and if there were more than 75% ties in a group, the Fisher’s exact test was used with fixed point censoring to evaluate the data.

Indices:

Average number of live fetuses, incidence of resorption, incidence of pregnancy, average number of corpora lutea, average number of implantations, average fetal body weight, sex ratios, average percentage of malformed fetuses per litter.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Five deaths and 2 abortions out of 25 rabbits, and reduced weight gain followed by a 'rebound phenomenon' (weight gain) during the 11 days post-treatment, occurred in the 30 mg/kg group. The 15 mg/kg/day group showed only the 'rebound phenomenon'. Upon necropsy, two of the five high dosage group rabbits which died had a stomach wall that appeared thin in the cardiac region. This finding was considered agent-related, since it was observed only in rabbits whose deaths were attributed to administration of acetonitrile. Necropsy of the middle dosage group rabbit which died revealed a spontaneous umbilical hernia and marked hemorrhage in the herniated portion of the intestine (strangulation). Average maternal body weight change was affected by administration of the middle and high dosages of acetonitrile, as compared with the vehicle. Average maternal body weight gain was decreased for high dosage group rabbits on days 6 to 19 of gestation; the decrease was significant (P 0.05). As compared with control values, the increase in average maternal body weight which was observed for the middle and high dosage group rabbits after agent administration was stopped represented a rebound effect.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

15 mg/kg bw/day (actual dose received)

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
A significant decrease in the average number of live foetuses/litter and a slight (non-significant) increase in resorptions were 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 foetal body weight and sex ration. There were no treatment related gross external, soft tissue or skeletal malformations or developmental variations.

Effect levels (fetuses)

1

Dose descriptor:

NOAEL

Effect level:

15 mg/kg bw/day (actual dose received)

Basis for effect level:

other: fetotoxicity

2

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day (actual dose received)

Basis for effect level:

other: teratogenicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

One 15.0 mg/kg/day dosage group rabbit died on day 23 of gestation. At necropsy, this rabbit had an umbilical hernia which had increased in size during gestation and resulted in intestinal strangulation. This death was not considered agent-related.

Applicant's summary and conclusion

Conclusions:

Administration of aqueous solutions of acetonitrile to pregnant rabbits at dosages of 2.0, 15.0 and 30.0 mg/kg/day on days 6 through 18 of gestation was not a unique hazard to the conceptus. Embryo/fetal lethality was observed only at a maternally lethal dosage. Administration of these dosages to the dams was not demonstrated to adversely affect the surviving fetuses, that is, it did not result in retarded fetal growth or an increased incidence in grossly observed external, soft tissue or skeletal malformations among the fetuses.

Executive summary:

In a guideline (US EPA) and GLP study conducted by Argus Research Labs (1984), oral (gavage) administration of aqueous solutions of Acetonitrile (HPLV grade) to pregnant rabbits at dosages of 2.0, 15.0 and 30.0 mg/kg/day on days 6 through 18 of gestation was not a unique hazard to the conceptus. Embryo/fetal lethality was observed only at a maternally lethal dosage. Administration of these dosages to the dams was not demonstrated to adversely affect the surviving fetuses, that is, it did not result in retarded fetal growth or an increased incidence in grossly observed external, soft tissue or skeletal malformations among the fetuses. Based on these findings, the following NOAELs are established for acetonitrile in the pregnant rabbit:

Maternal toxicity - 15 mg/kg/day

Fetotoxicity - 15 mg/kg/day

Teratogenicity - 30 mg/kg/day

Gastrointestinal symptoms observed in the rabbits that died are considered to be a significant confounder in determining the contribution from systemic chemical toxicity in this study.