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Repeated dose toxicity: inhalation

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sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:

Materials and methods

Test guideline
according to
other: OECD 416 (Two-Generation Reproduction Toxicity Study)
Principles of method if other than guideline:
Rats were exposed to acrylonitrile by inhalation as part of a 2-generation reproductive toxicity study
GLP compliance:
Limit test:

Test material


Test animals

Details on test animals and environmental conditions:
Crl:CD(SD) Sprague-Dawley albino virgin male and female rats, obtained from Charles River Laboratories, NC. Rats were acclimated for 14 days, during which they were observed twice daily for mortality and moribundity. Rats were groups housed by sex for 3 days, then housed individually (except during mating) in suspended wire mesh cages. Following mating females were transferred to plastic maternity cages with nesting material. Basal diet (PMI Nutrition International, Certified Rodent LabDiet 5002) and reverse osmosis treated water were available ad libitum, except during exposure. Animals were maintained on a 12 hour photoperiod, at 71◦F ± 5◦F and 30% to 70% humidity. The F0 generation was approximately 8 weeks old at initiation of exposure, the F1 generation approximately 4 weeks old. Animals in the study were maintained in accordance with the Animal Welfare Act (1966) and the Guide for the Care and Use of Laboratory Animals.

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: No information available
Details on inhalation exposure:
Each group of animals was exposed to acrylonitrile vapour in a 2-cubic-metre stainless steel and glass whole-body inhalation chamber operated under dynamic conditions. Chamber temperature (20-25°C), relative humidity (30-70%), ventilation (12-15 air changes per hour), and negative pressure within the chambers were monitored. Cages were sequentially rotated around the available rack positions within the chamber on a daily basis, to minimise any potential variation due to positioning. The control group was exposed to clean filtered air under identical conditions to those used for the acrylonitrile exposure groups.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Exposure concentrations of the vapour were measured approximately every 35 minutes (i.e. 9 to 10 times) during each daily exposure period via gas chromatography, using sensors placed approximately in the centre of the chamber, within the general breathing zone of the animals.
Duration of treatment / exposure:
Rats were exposed for 6 hours a day, 7 days a week, for 10 weeks prior to mating, during mating, gestation and lactation/.
Frequency of treatment:
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm
Dose / conc.:
5 ppm
Dose / conc.:
15 ppm
Dose / conc.:
45 ppm
Dose / conc.:
90 ppm
No. of animals per sex per dose:
25 rats per sex per concentration
Control animals:
Details on study design:
The study was perfomed as a two-generation reproductive toxicity study. The general toxicity data presented here are from the parental generation.

25 males and 25 females (F0 generation) in each of 5 groups were exposed to acrylonitrile (0, 5, 15, 45 or 90 ppm) 6 hours a day, 7 days a week for 10 weeks. The males were exposed for 10 weeks prior to mating and throughout mating until 1 day prior to euthanasia. The females were exposed for 10 weeks prior to mating and throughout mating, gestation, and lactation until 1 day prior to euthanasia. Exposure of the dams was suspended for 5 days following parturition (lactation days (LDs) 0 to 4) to avoid confounding nesting and nursing behaviour and neonatal survival. Exposure of the dams resumed on LD5, they were removed from the litters for 6 hours exposure at about the same time each day.

Animals found to be in good general health were allocated to groups based on body weight stratification and randomised in a block design by a computer generated program.
Positive control:
Not required for this study type


Observations and examinations performed and frequency:
Detailed physical examinations were recorded weekly for all parental animals (F0). All animals were observed twice daily for appearance, behaviour, moribundity, mortality and pharmacotoxic signs prior to exposure and within 1 hour after exposure. Females were also observed twice daily during the period of expected parturition for dystocia or other difficulties.

Individual body weights were recorded weekly throughout the study and prior to scheduled necropsy. Individual female bodyw eights were recorded weekly until evidence of copulation was observed and on GDs 0, 4, 7, 11, 14 and 20, and on LDs 1, 4, 7, 14, 21 and 28.

Parental food consumption was determined on the same days as the body weight measurements, except during the mating period when measurement of food consumption was suspended due to cohabitation.

Plasma and red blood cell (RBC) cholinesterase determinations were conducted on 10 rats/sex of the F0 parental generation from the control and 90 ppm groups, and from 10 rats/sex of the F1 parental generation from the control, 5, 15 and 45 ppm groups. Blood samples were collected from the tail vein following the daily 6 hour exposure 2 days prior to scheduled euthanasia. EDTA was used as the anticoagulant.
Sacrifice and pathology:
Surviving F0 adults were euthanised and necropsied following completion of weaning of their offspring (F1 and F2 pups respectively). Selected F0 and F1 parental tissues and organs were fixed by immersion in 10% neutral-buffered formalin for possible histopathological examination. Microscopic evaluations were performed on the following tissues for 10 randomly selected parental animals per sex (with confirmed sire or pregnancy) from the control and high-exposure groups: adrenal glands, prostate, brain, pituitary, seminal vesicles, right epididymis (caput, corpus and cauda), right testis, vagina, cervix, coagulating gland, uterus, oviducts, and ovaries (one section from each ovary was examined). Nasal cavities, lungs and gross lesions from all control, 5, 15 and 45 ppm groups were examined microscopically. Periodic acid-Schiff (PAS) and haematoxylin staining were used for the right tests and epididymis and haematoxylin-eosin staining was used for all other tissues. Organs weighed from all parental animals included adrenals, brain, total and cauda epididymis (weighed separately), kidneys, liver, lungs (prior to inflation with 10% neutral-buffered formalin), ovaries, pituitary, prostate, seminal vesicles with coagulating glands and accessory fluids, spleen, testes (weighed separately), thryroid, and uterus with oviducts and cervix.
Other examinations:
Plasma and red blood cell activities were assessed using a modified Ellman method.
All statistical analyses were conducted using two-tailed tests unless otherwise specified, comparing each exposure group to the control group. Data obtained from nongravid animals were excluded from analyses following the mating period. Parental body weight and food consumption data, absolute and relative organ weights, and RBC and plasma cholinesterase data were subjected to a one-way ANOVA among all groups. If the ANOVA was significant, Dunnett's test was used for the pairwise comparisons to the control group. Histopathologic findings in protocol-specified tissues were evaluated using a two-tailed Fisher's Exact test. Significance was accepted at the 5% and 1% level.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
There were no treatment-related mortalities at any exposure level evaluated. Spontaneous deaths occurred in parental animals of F0 and F1 generations; 1 F0 female each in the 5 and 45ppm groups were found dead. There were no signs of toxicity although there was evidence of dystocia in the 45 ppm female and the 5ppm female failed to initiate parturition before death.

There were no effects on bodyweights, weight gains, or food consumption at exposure levels of 5 and 15 ppm. Bodyweight gains for the 45 and 90 ppm males were statistically reduced relative to controls during the first 3 weeks of exposure, resulting in persistant and generally statistically significant body weight depression (up to 11.8%). Food consumption was also decreased for these males, generally in parallel with the bodyweight effects. Decreased food consumption and body weight gains were also noted for the females exposed to 45 and 90 ppm during the first 2 weeks of treatment and throughout gestation, resulting in decreased bodyweights (generally statistically significant) for 45 ppm females at study week 2 (-4.5%) and 90 ppm females throughout the 10 week premating period and gestation (7.5-9.1%). Body weights in the 90 ppm females were also depressed during lactation (5.8-11.5%) but did not achieve statistical significance, and were not accompanied by food consumption deficits.

Clincal findings consistent with the irritant properties of acrylonitrile (clear/red material around the nose, eyes and mouth and on the forelimbs) were observed for the rats exposed to 90 ppm throughout the exposure period within 1 hour following completion of daily exposure, but did not generally persist to the following day. Wet, cool tails were also noted for these animals, to a greater extent in the males, within 1 hour following exposure.

Increased absolute and/or relative (to final body weight) liver weights were noted for the 90 ppm group F0 males and females.

RBC cholinesterase activity was unaffected in males and females at exposure levels of 90 ppm. Plasma cholinesterase activity in the females exposed to 90 ppm was 40% lower than controls and was also lower than the mean value in the performing laboratory’s historical control database for approximately age-matched animals. However, there were no corresponding clinically observed functional deficits or inhibition of RBC cholinesterase activity in these females, and no effects on plasma or RBC cholinesterase activity were noted for the males. The reason for this could not be conclusively determined, but was not considered to be of toxicological significance in the absence of corresponding changes in RBC cholinesterase levels or associated clinical observations.

There were no treatment-related findings at histopathological examination.

Effect levels

open allclose all
Dose descriptor:
Effect level:
5 ppm
Based on:
test mat.
Basis for effect level:
other: Histopathology of the nasal cavity (local irritant effects) at this (lowest) dose level
Dose descriptor:
Effect level:
< 5 ppm
Based on:
test mat.
Basis for effect level:
other: Histopathology of the nasal cavity (local irritant effects) at all dose levels

Target system / organ toxicity

Key result
Critical effects observed:
Lowest effective dose / conc.:
5 ppm
respiratory system: upper respiratory tract
nasal cavity
Treatment related:
Dose response relationship:
Relevant for humans:

Any other information on results incl. tables

The analyses of the chamber atmospheres indicated that the mean analytical values of acrylonitrile ± SD for the 5, 15, 45 and 90 ppm groups were; 5.0±0.30, 15.1±0.69, 45.3±1.51, and 89.4±3.58 ppm, respectively. No test chemical was detected in the control atmospheres.

Applicant's summary and conclusion

Sub-chronic inhalation exposure of rats to acrylonitrile vapour resulted in reduced weight gain and food consumption and clinical signs consistent with respiratory irritation. Histopathological investigation revealed findings in the nasal cavity consistent with local irritation in all exposure groups. A NOAEC cannot be determined for this study.
Executive summary:

A two-generation reproductive toxicity study was conducted in Sprague-Dawley rats; the data presented here relate to the repeated dose inhalation toxicity effects on the parental animals (F0 generation). Twenty-five rats/sex/group were exposed to vapour atmospheres of acrylonitrile via whole-body inhalation at concentrations of 0, 5, 15, 45 and 90 ppm, 6 hours daily, on 7 days a week for 10 weeks. Males were exposed for 10 weeks prior to mating and throughout mating until 1 day prior to termination. Females were exposed for 10 weeks prior to mating and throughout mating, gestation, and lactation until 1 day prior to termination. Exposure of the dams was suspended for 5 days following parturition (lactation days 0 -4) to avoid confounding nesting and nursing behaviour and neonatal survival. Exposure of the dams resumed on Day 5; rats were removed from the litters for 6 hours exposure at about the same time each day. There were no exposure-related mortalities. Bodyweight gain was significantly reduced at 45 and 90 ppm. Food consumption was also reduced at these dose levels, but the difference was only significant at 90 ppm. Clinical signs indicative of the irritant properties of acrylonitrile were observed in rats exposed to 90 ppm throughout the exposure period and within 1 hour of cessation of exposure; the irritant effects of the test material did not generally persist to the following day. Acrylonitrile-related microscopic alterations were limited to morphologically similar nasal lesions in the F0 males and females at 45 ppm, F1 males at 5, 15, and 45 ppm, and the F1 females at 15 and 45 ppm. Four levels of the nasal cavity were examined microscopically for the 5, 15, and 45 ppm groups. Lesions showed a clear exposure-response relationship in incidence and included respiratory/transitional epithelial hyperplasia, sub-acute inflammation, squamous metaplasia, and/or degeneration of the olfactory epithelium. The majority of the lesions were present in the most rostral section (level I) of the nasal tissues examined and are consistent with site-of-contact irritation resulting from exposure to irritant chemicals as reported in the literature by a number of authors. All of the nasal lesions noted in this study are common findings in the nasal epithelium of the rat following sub-chronic to chronic inhalation exposure with an irritating compound and represent the effects of local irritation, rather than a systemic effect. No other treatment-related histopathological findings were noted at any exposure level. Based on the incidence of local irritant effects in the nasal cavity at all exposure levels, a NOAEC cannot be determined for this study. A LOAEC of 5 ppm is determined.