Acrylonitrile

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Unnamed

Year:

2008

Reference 2

Reference Type:

grey literature

Title:

Unnamed

Year:

2008

Report date:

2008

Reference 3

Reference Type:

publication

Title:

The Effects Of Inhaled Vapors Of Acrylonitrile On Rat Reproduction Over Two Successive Generations.

Author:

Nemec MD, Kirkpatrick DT, Sherman J, Van Miller J & Strother DE

Year:

2006

Bibliographic source:

Toxicol Sci 2006 Mar;90(1-S):251

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Principles of method if other than guideline:

The results of an independent histopatholical re-evaluation of brain slides are also reported.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Acrylonitrile

EC Number:

203-466-5

EC Name:

Acrylonitrile

Cas Number:

107-13-1

Molecular formula:

C3H3N

IUPAC Name:

prop-2-enenitrile

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system 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 (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on 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°C to 25°C), relative humidity (30% to 70%), ventilation (12 to 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.

Details on mating procedure:

Throughout the mating period, each female was housed overnight in the home cage of a specific, non-sibling male (1:1) until evidence of mating was detected. Observation of a copulatory plug in the vagina or the presence of sperm in a vaginal lavage confirmed positive evidence of mating. That day was termed gestation day 0, and the animals were separated.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Exposure concentrations of the vapour were measured approximately every 35 minutes (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 (see study schedule for further information).

Frequency of treatment:

Daily.

Details on study schedule:

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. These animals were randomly bred to produce an F1 generation (avoiding sibling matings). Following weaning on postnatal day (PND) 28, animals selected to be parents from the F1 generation were similarly exposed. Due to excessive toxicity in the 90 ppm group, exposure of the F1 parental animals at 90 ppm was terminated after 16 to 29 exposures. These rats were maintained without exposure for 4 days prior to macroscopic examination. A replication of the breeding procedure was conducted with the remaining 4 groups in F1 (25 rats/sex/group).
The F0 and F1 males were exposed for 10 weeks prior to mating and throughout mating until 1 day prior to euthanasia. The F0 and F1 females were exposed for 10 weeks prior to mating and throughout mating, gestation, and lactation until 1 day prior to euthanasia. Exposure of the F0 and F1 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.
Each dam and litter remained together until weaning on PND 28. Male and female pups were randomly selected (25/sex/group) to compose the F1 generation.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Remarks:

Vapour

Doses / concentrations 2

Dose / conc.:

5 ppm

Remarks:

Vapour

Doses / concentrations 3

Dose / conc.:

15 ppm

Remarks:

Vapour

Doses / concentrations 4

Dose / conc.:

45 ppm

Remarks:

Vapour

Doses / concentrations 5

Dose / conc.:

90 ppm

Remarks:

Vapour

No. of animals per sex per dose:

25 rats per sex per concentration in the F0 and F1 generations.

Control animals:

yes

Details on study design:

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 examined.

Examinations

Parental animals: Observations and examinations:

Detailed physical examinations were recorded weekly for all parental animals (F0 and F1). 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 F0 and F1 bodyweights were recorded weekly throughout the study and prior to scheduled necropsy. Individual F0 and F1 female bodyweights 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.

Oestrous cyclicity (parental animals):

Vaginal lavages were performed daily, and the slides from each F0 and F1 female were evaluated daily beginning 3 weeks prior to pairing and continuing until mating was osberved.
The stage of oestrus was determined on the day of scheduled necropsy for all F0 and F1 females.

Sperm parameters (parental animals):

Sperm samples from the right epididymis were collected from each adult F0 and F1 male and evaluated for the percentage of progressively motile sperm. Motile sperm were evaluated using the Hamilton-Thorne HTM-IVOS computer-assisted sperm analysis system. Sperm morphology was evaluated by light microscopy. The left testis and/or epididymis from all F0 and F1 males in all dose groups were evaluated for homogenisation-resistant spermatid counts (testis and epidydmis) and sperm production rates (testis only).

Litter observations:

To reduce variability among the litters, large litters were reduced to 10 pups/litter (5/sex where possible) on PND 4 using a computer generated random selection procedure.
On the day of parturition (PND 0), pups were sexed and examined for external malformations, and the numbers of stillborn and live pups were recorded. Stillborn and intact offspring dying from PND 0-4 were necropsied using a fresh dissection technique. A detailed necropsy was performed on any pup dying after PND 4 and prior to weaning.
Litters were examined daily for survival and any adverse changes in appearance or behaviour. Each pup was individually weighed and received a detailed physical examination on PNDs 1, 4, 7, 14, 21 and 28. Pups were also individually sexed on PNDs 1, 4, 7, 14, 21 and 28.
Each male pup selected as a parent for the F1 generation was examined for balanopreputial separation beginning on PND 35, and each selected F1 female pup was examined for vaginal perforation beginning on PND 25. These observations continued until all animals attained these criteria. Pup body weights were recorded on the day of acquisition of the landmarks.

Postmortem examinations (parental animals):

Surviving F0 and F1 adults were euthanised and necropsied following completion of weaning of their offspring (F1 and F2 pups respectively). The stage of oestrus was determined on the day of scheduled necropsy for all F0 and F1 females, and 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 F0 and F1 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 from F0 females was examined).
Nasal cavities, lungs and gross lesions from all F0 and F1 animals in the control, 5, 15 and 45 ppm groups were examined microscopically. The lungs and nasal cavities of the 90 ppm animals were not examined histologically because the grossly observable severe irritation indicated that histopathology would not add useful information to the study.
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. Quantitative histopathologic evaluation of 10 sections of the inner third of the ovary (including enumeration of primordial follicles) was conducted on 10 F1 females from the control and 45 ppm groups. An assessment of the presence/absence of growing follicles, antral follicles, and corpora lutea was also performed.
The following tissues were examined for F0 animals in the 5, 15 and 45ppm groups and F1 animals in the 5 and 15 ppm groups that failed to mate or produce offspring, or otherwise exhibited potential reproductive dysfunction: pituitary, cervix, ovaries, oviducts, uterus, vagina, coagulating gland, right epididymis, right testis, prostate, and seminal vesicles.
Organs weighed from all F0 and F1 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.

Postmortem examinations (offspring):

On PND 28, a complete necropsy similar to that performed on parental animals (with emphasis on developmental and reproductive system morphology) was conducted on F1 pups not selected for exposure, and on F2 pups. Brain, spleen, thymus gland, epididymis, ovary, pituitary gland, seminal vesicle, testis and uterus (with oviduct/cervix) weights were also recorded from these pups.

Statistics:

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 mating, fertility, copulation, and conception indices were evaluated by the chi-square test with Yates' correction factor. Parental body weight and food consumption data, oestrous cycle and gestation lengths, precoital intervals, implantation sites, unaccounted-for implantation sites, numbers of pups born, live litter sizes, pup body weights and weight changes, balanopreputial separation and vaginal patency data (day of acquisition and body weight), anogenital distances, absolute and relative organ weights, sperm production rate, sperm numbers, ovarian primordial follicle counts, 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. Sperm motility and morphology and proportional postnatal offspring survival and sex at birth were analysed using Kruskal-Wallis ANOVA followed by the Mann-Whitney U test when appropriate. 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.

Reproductive indices:

Male fertility index (%) = no. males siring a litter/total no. of males used for mating x 100.
Female fertility index (%) = no. females with a confirmed pregnancy/total no. females used for mating x 100.
Male copulation index (%) = no. males siring a litter/no. males with evidence of mating (or females confirmed pregnant) x 100.
Female conception index (%) = no. females with confirmed pregnancy/no. females with evidence of mating (or confirmed pregnancy) x 100.

Offspring viability indices:

Live litter sizes (PND 0).
Pup survival from birth to PND 4 (%). Pup survival from PND 4 to PND 28 (%).

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

: reduced at 45 ppmand 90 ppm

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

: reduced at 45 ppmand 90 ppm

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

: nasal irrtitation

Other effects:

not specified

Description (incidence and severity):

Test substance intake: : not relevant

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

Mortality

There were no acrylonitrile-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 45 ppm groups were found dead. There were no signs of toxicity although there was evidence of dystocia in the 45 ppm female and the 5 ppm female failed to initiate parturition. 1 control group F1 female was found dead and a single 45 ppm F1 male was euthanised in extremis prior to the breeding period; each of these animals were found to have malignant lymphoma.

Bodyweights, food and water consumption

There were no effects on body weights, weight gains, or food consumption at exposure levels of 5 and 15 ppm in the F0 generation. Body weight gains for the 45 and 90 ppm F0 males were statistically reduced relative to controls during the first 3 weeks of exposure, resulting in persistant and generally statistically significant body weight depressions (up to 11.8%) throughout the F0 generation. Food consumption was also decreased for these males, generally in parallel with the body weight effects. Decreased food consumption and body weight gains were also noted for the F0 females exposed to 45 and 90 ppm during the first 2 weeks of treatment and throughout gestation, resulting in decreased body weights (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. Body weight gains in the 45 ppm F1 males were slightly reduced (generally statistically significant) during the first 3 weeks of exposure, but the effects were less pronounced than in the F0 males. Body weights for the 45 ppm F1 males were decreased by up to 9.4% during study weeks 18 to 26. Decreased food consumption in these animals generally paralleled the observed body weight deficits. There were no other effects on F1 female body weights or food consumption.

Clinical signs

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 F0 males and females 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. Marked clinical signs, including sensitivity to touch, vocalisation upon handling, and evidence of local irritation, an approximate 10-15% decrease in food consumption for both sexes, and body weight decrements in excess of 20% for males and approximately 12% for females were noted for F1 animals during the first 3-4 weeks of direct exposure to 90ppm following weaning. As a result of these findings, exposure of the 90ppm F1 weanlings was discontinued following a total of 16-29 exposures, precluding mating and production of F2 offspring at this exposure level. There were no other substance-related clinical findings observed at any F1 exposure level.

Oestrus cyclicity

The regularity and duration of oestrus was not affected by acrylonitrile exposure in either F0 or F1. Mean oestrus cycle lengths in all groups evaluated were similar to controls, with the exception of slightly increased values in the 45 ppm F0 and F1 females. However a similar increase was not observed in the 90 ppm F0 females, and the increase in the 45 ppm F1 females was due to a single female with an atypically long oestrus cycle (16 days). Therefore the increases observed were not attributed to acrylonitrile exposure.

Reproductive parameters

No adverse exposure-related effects were observed on the number of days between pairing and coitus, gestation length, or reproductive performance (fertility, mating, copulation, and conception indices) in either F0 or F1. The process of spermatogenesis (mean testicular and epididymal sperm numbers, sperm production rate, and sperm motility and morphology) was unaffected by acrylonitrile exposure in both generations. A slight statistical decrease in sperm motility (including progressive motility) was noted for the F0 males exposed to 90 ppm when compared to controls. However there were no other andrological changes in this group, nor were there effects on fertility or on reproductive organ weights and histopathology.

Necropsy findings

Increased absolute and/or relative (to final body weight) liver weights occurred for the 90 ppm group F0 males and females and the 45 ppm group F1 males. The histopathological re-evaluation of brain sections of F1 animals exposed to 45 ppm (Garman, 2008) did not reveal any microscopic evidence of cell proiferation or any other findings related to exposure.

Cholinesterase activity

RBC cholinesterase activity was unaffected in males and females at exposure levels of 90 ppm in the F0 generation and 5, 15, and 45 ppm in the F1 generation. Plasma cholinesterase activity in the F0 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 F0 males, F1 males, and F1 females at any exposure level evaluated. Because plasma cholinesterase activity was not evaluated at 90 ppm in the F1 generation (due to early group termination because of excessive systemic toxicity), the relationship of the decreased mean plasma cholinesterase activity in the 90 ppm F0 females to AN exposure could not be conclusively determined, but is not considered to be of toxicological significance in the absence of corresponding changes in RBC cholinesterase levels or associated clinical observations.

Effect levels (P0)

1

Dose descriptor:

NOAEC

Remarks:

reproductive toxicity

Effect level:

90 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No reproductive effects were seen at the highest exposure level

2

Dose descriptor:

NOAEC

Remarks:

parental systemic toxicity

Effect level:

15 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Effects on bodyweights and food consumption

Remarks on result:

other: Generation: all

3

Dose descriptor:

NOAEC

Effect level:

15 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Nasal histopathology at 45 ppm

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Details on results (F1)

Litter parameters

The numbers of F1 and F2 pups born, live litter sizes, sex ratios at birth and postnatal survival were unaffected by parental acrylonitrile exposure. Anogenital differences (absolute and relative to the cube root of pup body weight) on PND 1 were also unaffected by parental acrylonitrile exposure. Slight, generally statistically significant increases in absolute and relative anogenital distance was noted for the F1 males exposed to 45 and 90ppm, although there was no effects on anogenital distance in the 45ppm F2 pups, therefore the slight increases in distance were not considered to be related to acrylonitrile.

F1 pup body weights in the 90ppm group during the last 2 weeks of lactation (PNDs 14-28) were decreased 6.6-12.2% for males and 5.8-10.7% for females, as a result of decreased body weights following the reinitiation of maternal exposure. Slight delays in the occurrence of vaginal patency and balanopreputial separation, and lower body weights on the day of occurrence (relative to control group values) were noted for the F1 males in the 45 and 90 ppm groups, and females in the 90 ppm group. Balanopreputial separation occurred in all F1 male pups by PND 53, and all F1 female pups had vaginal opening by PND 42.

F2 pup weights and weight gains were unaffected by parental exposure to acrylonitrile throughout the postnatal period. Slight but significant body weight decreases were seen relative to controls on PND 28, however the differences did not display an exposure-related pattern, and the mean values were still within the range of the laboratories inhalation reproductive toxicity historical control data.

No internal findings that could be attributed to acrylonitrile exposure were noted at the necropsies of F1 and F2 pups that were found dead or examined at the scheduled PND 28 necropsies. No acrylonitrile related effects on F1 and F2 offspring organ weights were noted in any group.

Effect levels (F1)

1

Dose descriptor:

NOAEC

Remarks:

reproduction

Generation:

F1

Effect level:

45 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects on reproduction were seen at this exposure concentration; the 90- ppm exposure group was terminated early due to excessive toxicity

2

Dose descriptor:

NOAEC

Remarks:

local toxicity

Generation:

F1

Effect level:

< 5 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Histopathology of the nasal epithelium

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

The analyses of chamber atmospheres indicated that the mean analytical values of acrylonitrile ± SD for the 5, 15, 45 and 90ppm groups were; 5.0±0.30, 15.1±0.69, 45.3±1.51, and 89.4±3.58ppm, respectively for the F0 generation, and 5.0±0.25, 15.2±0.59, 45.4±1.57, and 86.5±2.45ppm, respectively for the F1 generation. No test chemical was detected in the control atmospheres.

The sperm motility values in F0 90ppm males were slightly decreased, although the values were similar to those in the F1 generation control group, which illustrates the variability of these data. Sperm motility in the 90 ppm group F0 males was within the range of historical control data for inhalation reproductive toxicity studies conducted in the performing laboratory. Therefore, the slightly decreased sperm motility in the 90ppm F0 males was considered unrelated to acrylonitrile exposure.

Overall, body weight effects in the F0 generation were more pronounced in the males than in the females. Body weight has been shown to affect the time of balanopreputial separation and vaginal patency. Therefore, because the slight delays in acquisition of these sexual developmental landmarks were observed in parallel and/or in the presence of reduced body weights, the delays were not considered to be direct effects of exposure to acrylonitrile.

Reproduction data

	F0 parental and F1 offspring generation Acrylonitrile exposure level					F1 parental and F2 offspring generation Acrylonitrile exposure level
	0ppm	5ppm	15ppm	45ppm	90ppm	0ppm	5ppm	15ppm	45ppm
Estrous cycle length (days)	5.0 ± 1.33	5.3 ± 2.11	5.4 ± 1.96	6.9 ± 3.98	5.8 ± 2.51	4.4 ± 0.61	4.3 ± 0.52	4.7 ± 1.00	5.3 ± 2.53
Time to mating (days)	3.5 ± 2.79	2.6 ± 1.64	3.2 ± 2.43	2.8 ± 1.32	3.3 ± 2.96	3.0 ± 1.32	2.8 ± 1.42	3.0 ± 2.49	3.6 ± 2.02
Male Mating indices (%)a	92.0	100	100	100	92.0	87.5	96.0	100	87.5
Female Mating indices (%)a	92.0	100	100	100	92.0	87.5	96.0	100	88.0
Gestational length (days)	22.3 ± 0.59	22.0 ± 0.49	22.0 ± 0.31∗	22.0 ± 0.36∗	22.0 ± 0.21	22.2 ± 0.49	21.9 ± 0.34	22.0 ± 0.35	22.3 ± 0.44
Gestation wt gainb	113 ± 27.0	128 ± 16.8∗	130 ± 17.8∗	122 ± 22.7	105 ± 14.8	125 ± 15.8	131 ± 11.6	129 ± 14.8	132 ± 14.3
LDs1-4 Lactation weight gain	22 ± 7.8	20 ± 9.9	14 ± 9.5	19 ± 13.5	15 ± 9.8	16 ± 9.2	12 ± 11.2	16 ± 9.4	16 ± 9.9
LDs1-28 Lactation weight gain	−2 ± 12.3	−7 ± 18.2	−5 ± 16.1	5 ± 13.3	9 ± 15.6	−4 ± 16.0	1 ± 22.2	0 ± 13.6	−4 ± 13.9
Male fertility indices (%)c	84.0	92.0	88.0	96.0	88.0	83.3	92.0	100	83.3
Female fertility indices (%)d	84.0	92.0	88.0	96.0	88.0	83.3	92.0	100	80.0
Male copulation index (%)e	91.3	92.0	88.0	96.0	95.7	95.2	95.8	100	95.2
Female conception index (%)f	91.3	92.0	88.0	96.0	95.7	95.2	95.8	100	90.9
No. ovarian primordial follicles	-	-	-	-	-	65.6 ± 37.47	NP	-	76.9 ± 29.38
No. implantation sites/damg	14.2 ± 33.4	15.2 ± 2.32	15.1 ± 2.24	14.4 ± 3.12	14.4 ± 1.37	14.0 ± 2.70	15.3 ± 1.72	15.2 ± 1.85	15.7 ± 2.41
No. pups born per dam	13.4 ± 3.25	14.6 ± 2.42	14.5 ± 2.39	14.0 ± 3.57	13.6 ± 2.11	13.3 ± 2.71	14.4 ± 1.65	14.4 ± 2.25
No. sites unaccounted for	0.7 ± 0.87	0.5 ± 0.86	0.7 ± 1.32	0.7 ± 1.21	0.8 ± 1.19	0.7 ± 1.17	0.9 ± 1.29	0.9 ± 1.54	0.8 ± 0.92
No. of litters produced	21	23	22	24	22	20	23	25	20
Sex ratio at birth (% males/litter)	52.6 ± 12.15	50.7 ± 11.28	50.9 ± 12.11	46.6 ± 15.15	48.6 ± 12.66	51.8 ± 10.33	50.3 ± 14.15	51.2 ± 14.72	51.6 ± 13.46
Live litter size (PND 0)	13.0 ± 3.14	14.3 ± 2.46	14.4 ± 2.44	13.7 ± 3.57	13.3 ± 2.05	13.0 ± 2.78	14.0 ± 1.64	13.8 ± 2.18	14.9 ± 2.29
Pup survival PND 4 (%)h	95.2 ± 8.14	96.1 ± 6.06	98.1 ± 3.83	95.5 ± 7.20	93.8 ± 11.94	96.4 ± 6.69	94.7 ± 6.91	95.0 ± 6.79	94.8 ± 11.59
Pup survival PND 28 (%)i	99.5 ± 2.29	99.5 ± 2.13	98.2 ± 5.88	98.7 ± 4.58	98.2 ±5.01	99.0 ± 3.08	98.3 ± 3.88	100 ± 0.00	100 ± 0.00

Note. Number of animals or litters evaluated per endpoint ranged from 18 to 25 per group, except for 10 F1 females/group (control and 45 ppm) evaluated for ovarian primordial

follicle counts.

∗Statistically significant at p < .05.

a Mating index = no. of males/females with evidence of mating (or confirmed pregnancy)/total no. of males/females used for mating × 100.

b Weight gain over the entire gestation period (GDs 0–20)

c Male fertility index = no. of males siring a litter/total no. of males used for mating × 100.

d Female fertility index = no. of females with confirmed pregnancy/total no. of females used for mating × 100.

e Male copulation index = no. of males siring a litter/no. of males with evidence of mating (or females confirmed pregnant) × 100.

f Female conception index = no. of females with confirmed pregnancy/no. of females with evidence of mating (or confirmed pregnancy) × 100.

g The uterus was examined to determine the number of former implantation sites (the attachment site of the placenta to the uterus); no attempt was made to differentiate between

early and late resorptions for sites that were unable to be accounted for.

h Offspring survival from birth to PND 4 (preselection).

i Offspring survival from PND 4 (postselection) to PND 28.

NP, ovarian follicle counts were also assessed for two F1 females with no evidence of mating in the 5 ppm group (data not presented).

Mean Anogenital Distance (mm) in male pups

Parameter	0 ppm	5 ppm	15 ppm	45 ppm	90 ppm
F1
Mean ± SD	3.45±0.378	3.54±0.317	3.49±0.343	3.67*±0.205	3.66±0.202
Relative^a	1.76±0.168	1.81±0.158	1.80±0.170	1.88*±0.119	1.90*±0.094
F2
Mean ± SD	4.66±0.305	4.59±0.411	4.66±0.422	4.49±0.325	NA
Relative^a	2.40±0.148	2.37±0.197	2.38±0.198	2.32±0.156	NA

* statistically significant (p≤0.05)

Applicant's summary and conclusion

Conclusions:

The results of this study do not indicate concern for reproductive toxicity as a result of acrylonitrile exposure. The critical effect of expousre in this study was identified as nasal irritation.

Executive summary:

The effect of acrylonitrile exposure on reproduction was investigated in succesive generations of Sprague-Dawley rats. Inhalation exposure was chosen as this is the most likely form of human exposure.

Twenty five rats/sex/group were exposed to vapour atmospheres of acrylonitrile via whole-body inhalation at concentrations of 0, 5, 15, 45 (two offspring generations) and 90 ppm (one offspring generation), 6 h daily, 1 litter/generation, through F2 weanlings on postnatal day 28. After approximately 3 weeks of direct exposure following weaning, exposure of the F1 animals at 90 ppm was terminated due to excessive systemic toxicity in the males.

There were no exposure-related mortalities in adult animals, no functional effects on reproduction or effects on reproductive organs, and no evidence of cumulative toxicity or of enhanced toxicity in pregnant and lactating dams or in developing animals. Adult systemic toxicity was limited to body weight and/or food consumption deficits in both sexes and generations (greater in males) at 45 and 90 ppm and increased liver weights in the 90 ppm F0 males and females and 45 ppm F1 males. Neonatal toxicity was expressed by F1 offspring weight decrements at 90 ppm. Clinical signs of local irritation during and immediately following exposure were observed at 90 ppm. Microscopic lesions of the rostral nasal epithelium, representing local site-of-contact irritation, were observed in some animals at 5 to 45 ppm. An independent histopathological re-evaluation of brain sections of F1 animals exposed to 45 ppm (Garman, 2008) did not reveal any microscopic evidence of cell proliferation or any other findings related to exposure.

The no-observed-adverse-effect level (NOAEL) for reproductive toxicity over two generations and neonatal toxicity of acrylonitrile administered to rats via whole-body inhalation was 45 ppm. The NOAEL for reproduction was 90 ppm for the first generation. The NOAEL for parental systemic toxicity was 15 ppm; a NOAEL for local effects was not identified.