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Key value for chemical safety assessment

Effects on fertility

Description of key information

The reproductive toxicity of acrylonitrile has been investigated in a number of studies including a one-generation drinking water study in the rat, a two-generation inhalation study in the rat and a three-generation drinking water study in the rat. An expert review of all reproductive toxicity studies on acrylonitrile is also provided.

Link to relevant study records

Referenceopen allclose all

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
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
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.
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.
Dose / conc.:
0 ppm
Remarks:
Vapour
Dose / conc.:
5 ppm
Remarks:
Vapour
Dose / conc.:
15 ppm
Remarks:
Vapour
Dose / conc.:
45 ppm
Remarks:
Vapour
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.
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 (%).
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: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
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.
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
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
Dose descriptor:
NOAEC
Effect level:
15 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Nasal histopathology at 45 ppm
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
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.
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
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
Reproductive effects observed:
not specified

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

Relativea

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

Relativea

2.40±0.148

2.37±0.197

2.38±0.198

2.32±0.156

NA

* statistically significant (p≤0.05)

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.

Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Published study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Principles of method if other than guideline:
The study was broadly comparable to OECD 415
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
Male and female Sprague-Dawley rats
Route of administration:
oral: drinking water
Vehicle:
water
Details on exposure:
Rats were exposed to acrylonitrile in the drinking water.
Details on mating procedure:
Two females were mated to one male, but females were not assessed for sperm plugs/presence.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No information available
Duration of treatment / exposure:
Rats were exposed for 60 days pre-mating, and through mating, gestation and lactation.
Frequency of treatment:
Daily in drinking water
Details on study schedule:
Rats were mated and the resulting offpsring raised to weaning. Litters were not culled.
Dose / conc.:
0 ppm
Remarks:
Drinking water
Dose / conc.:
35 ppm
Remarks:
Drinking water
Dose / conc.:
210 ppm
Remarks:
Drinking water
Dose / conc.:
500 ppm
Remarks:
Drinking water
No. of animals per sex per dose:
20/sex/dose
Control animals:
yes
Details on study design:
No further information
Positive control:
Not examined; not required for this study type
Parental animals: Observations and examinations:
Clinical signs, body weight, feed consumption and drinking water consumption. TIme from pairing to parturition.
Oestrous cyclicity (parental animals):
Not examined
Sperm parameters (parental animals):
Not examined
Litter observations:
Pup survival and sex ration. Pup body weights (presented as litter means) were recorded on PND 1, 7, 14 and 21.
Postmortem examinations (parental animals):
Gross necropsy
Postmortem examinations (offspring):
Histopathological evaluation on testes in weanlings and examination for skeletal abnormalities. Pup malformations were characterised on lactation day 21. Pups that died during the study were not necropsied.
Statistics:
No information.
Reproductive indices:
Fertility index: % pregnant / females mated
Offspring viability indices:
No information
Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
: gastric ulceration
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Treatment-related effects on the adults appear primarily be associated with the poor palatability of acrylonitrile in water, which resulted in marked decreases in water consumption (40-60% at the high dose level) associated with body weight and feed consumption decreases. Necropsy findings suggest that some gastric ulceration may have been associated with the high concentration of acrylonitrile, although this finding may have been confounded due to concurrent nematode infestation. There were no obvious effects on reproductive performance in this study.
Dose descriptor:
NOAEL
Remarks:
reproductive
Effect level:
500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects on reproduction were seen in this study.
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
reduced weight gain
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
No adverse effects on pup survival were seen in this study. The body weight gain in the pups in the high dose level was decreased at day 7 compared to control, although not statistically significantly. At the mid dose, the pup body weight gain decrease was only apparent at PND 21. One litter in each of the low-, mid- and high-dose groups had an indication of short or missing tail.
Reproductive effects observed:
not specified

Summary of results

ppm in water

0

35

210

500

Parental toxicity

 

 

 

 

Clincal signs

-

-

-

-

Body weight

-

-

*

*

Feed consumption

-

-

*

Water consumption

-

(slight)

*

*(marked)

Reproductive toxicity

 

 

 

 

Fertility Index (% females pregnant/females mated)

67

75

50

80

Time from pairing to parturition

-

-

-

-

Pup survival

-

-

-

-

Pup sex ratio

-

-

-

-

Pup body weight (litter mean) PND 1 or 7

-

-

-

-

Pup body weight (litter mean) PND 14

-

-

-

*

Pup body weight (litter mean) PND 21

-

-

*

* = statistically significant finding

Conclusions:
Mo adverse effects on reproduction were seen under the conditions of this study
Executive summary:

Sprague-Dawley rats were exposed to acrylonitrile in the drinking water at concentrations of 0, 35, 210 or 500 ppm for 60 days pre-mating and throughout mating, gestation and lactation. Treatment-related effects on the adults appear primarily to be associated with the poor palatability of the test material in water, which resulted in marked decreases in water consumption (40-60% at the high dose level) associated with body weight and feed consumption decreases. Necropsy findings suggest that some gastric ulceration may have been associated with the high concentration of acrylonitrile, although this finding may have been confounded due to concurrent nematode infestation. There were no obvious effects on reproductive performance in this study, although there are some limitations in the study methodology. No adverse effects on pup survival were seen in the study. There was an equivocal increase in pup malformations reported in this study. One litter in each of the low-, mid- and high-dose groups had an indication of short or missing tail. This is a relatively infrequent finding in SD rats in developmental toxicity studies however no dose response was evident so no clear conclusion can be drawn from the available data.

Endpoint:
three-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Three-generation reproduction study in rats, test substance administered in the drinking water
GLP compliance:
not specified
Remarks:
published study
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
Male and female weanling Charles River [CRL:CDBS CD (SD) BR] rats, were obtained from Charles River Laboratories, Massachusetts. The rats were housed individually in plastic cages on hardwood chip bedding and acclimated to the laboratory conditions for 15 days prior to the start of the study. Purina Laboratory Chow (ground) was provided ad libitum. The rats were assigned to treatment groups using a table of random numbers and individually numbered with ear tags.
Route of administration:
oral: drinking water
Vehicle:
unchanged (no vehicle)
Details on exposure:
Acrylonitrile was administered in the drinking water at nominal levels of 0, 100 or 500 ppm for 100 days prior to mating (F0 generation). Exposure continued throughout the mating period and the subsequent gestation and lactation phases in females. Two litterings were produced per generation and exposure was continuous through 3 generations.
Details on mating procedure:
20 F0 females were paired with 10 F0 males for mating over a 6 day period. Any females not bred at the end of the 6 days (as evidenced by the absence of vaginal plugs) were mated to another proven male. After mating females were housed individually. This produced the F1a generation. F1a litters were reduced to 10 pups per litter on day 4, with equal numbers of males and females being retained. Males not mating were not re-mated.
The female F0 rats were remated to produce the F1b generation, with 10 previously unmated F0 females also being mated. Half of these pups were fostered at birth to untreated females, while at weaning (21 days) 1 male and 1 female from each unfostered litter were selected as breeders for the F2 generation. Selected F1b animals were administered acrylonitrile in drinking water for 100 days and then mated with production of an F2a and an F2b litter. Due to high pup mortality in the 500 ppm F1b offspring, some F1a animals were used as replacements. Selected F2b rats were mated to produce F3 litters, with additional animals being used from surviving litters in order to achieve required breeding numbers.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of acrylonitrile concentrations in the drinking water was carried out weekly by gas chromatography.
Duration of treatment / exposure:
100 days prior to mating, throughout the mating period and the subsequent gestation and lactation phases in females.
Frequency of treatment:
Daily
Details on study schedule:
F0 weanlings were exposed to acrylonitrile for 100 days, then mated over a 6 day period. The day the litter was cast was considered Day 0 of lactation. The female F0 rats were remated 2 weeks after removal of the F1a pups to produce the F1b offspring.
Dose / conc.:
0 ppm
Remarks:
Drinking water
Dose / conc.:
100 ppm
Remarks:
Drinking water
Dose / conc.:
500 ppm
Remarks:
Drinking water
No. of animals per sex per dose:
F0 generation: 30 females and 15 males per dose (plus 10 male and 20 female controls). Litters were culled to 10 pups (approximately 5/sex/litter), except in the second littering of the F3 generation, in which the cull was increased, leaving 8 pups (approximately 4 male and 4 female pups per litter).
Control animals:
yes, concurrent no treatment
Details on study design:
Selection of animals for the initial mating of the F0 parents was not random; the lightest and heaviest animals were excluded.
Positive control:
Not examined.
Parental animals: Observations and examinations:
Duration of mating and gestation. F0 parents and F1a and F1b offspring were observed daily, especially with regard to signs of neurotoxicity (evidenced by abnormal gait). Body weights were obtained every 2 weeks. Food consumption was measured weekly. Water consumption was measured twice a week.
Oestrous cyclicity (parental animals):
Not determined.
Sperm parameters (parental animals):
Not determined.
Litter observations:
F1a offspring were examined on Days 0, 4 and 21 of lactation. Body weights were obtained on Day 4 (litter) and on Day 21 (individual). Also, the pup sex ratio, no. live pups per litter, pup survival and growth were recorded.
Postmortem examinations (parental animals):
Evaluation of selected tissues and gross lesions in selected adults.
Postmortem examinations (offspring):
Postmortem was performed on female rats 20 weeks after wearning of the litter. Histopathological examination of uterus, seminal vesicle, prostate and testes for 10/sex 500 ppm and control F3b weanlings.
Statistics:
Student's t-test, Fisher's Exact test, Wilcoxon Rank Sum and the Mantel-haenszel method.
Reproductive indices:
Male fertility index (no. males producing a litter/no. mated). Female fertility index (no. pregnant females/no. mated). Gestation index (no. litters born/no. females pregnant).
Offspring viability indices:
Viability index (no. live pups at 4 days/no. pups born alive). Lactation index (no. pups weaned/no. live pups at 4 days).
Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not specified
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
There was no effect on male or female fertility in the F1, F2 or F3 generation as assessed by the fertility index. Fertility in some groups was occasionally low (e.g. 50-60% in F2 generation for controls and the 100 ppm groups), however slightly higher fertility was consistently recorded in the 500 ppm groups. There was also no indication of an embryotoxic effect, gestation index being similar across all groups and numbers of live pups per litter also being reasonably consistent across the dose groups.

Water consumption was markedly reduced at 500 ppm (approximately 50%), and moderately reduced at 100 ppm in the F0 animals; water consumption was not further evaluated. F0 body weight and feed consumption were also reduced at both dose levels, with effects more marked in males at 100 ppm.

A dose-related tumorigenic effect occurred in female rats held 20 weeks after weaning of the second litter and histopathological examination of these dams showed an increase in astrocytomas and zymbal gland tumours.

There were 5 adult females in the post-reproductive phases at 500 ppm which had gait disturbances reported; three of the affected animals were found to have astrocytomas, and one (based on gross lesions) a likely Zymbal’s gland tumor. These findings could have contributed to the observed gait disturbances.

There was no effect on the duration of gestation. The duration of mating was decreased in both exposed groups in the first mating of the F2 adults; review across generations, however, shows the duration of mating was actually markedly increased for the controls in this generation compared to the controls in the first two generations. This finding is therefore not considered compound-related. The lack of effect on duration of mating suggests that estrous cyclicity in the exposed females was not adversely affected by exposure. There was no obvious effect on male or female fertility (mating) indices, although the poor control fertility, particularly for female rats in the second or third generations could have masked a treatment-related decrease. No decrease was evident in the first generation, however, in which control fertility was high, and in the other generations the high-dose group females generally showed higher fertility than did the controls. There was no effect on the gestation index.
Dose descriptor:
NOAEC
Effect level:
100 ppm (analytical)
Sex:
male/female
Remarks on result:
other: All generations
Clinical signs:
not examined
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Viability Index was reduced in the 500 ppm litters, with statistical significance being attained in the F1a (94%, p < 0.05), F1b (91% p < 0.05) and F3a (95%, p < 0.05) generations. There was a trend towards reduced viability also at 100 ppm, statistically significant in the F1b generation (90%, p < 0.05). Bodyweights of pups at 500 ppm in all 3 generations at 21 days were also reduced (mean control pup weight in grams on day 4 over all generations was 10.5 + 0.55, mean pup weight at 500 ppm was 9.0 + 1, p < 0.05). A marked statistically and biologically significant decrease in the lactation index (No. pups surviving to PND 21/No. pups live at PND 4) was evident in the F1a generation at 500 ppm; this finding was not replicated in subsequent generations/litterings. The study authors hypothesise that the decreased pup survival in the F1a pups may have been due to decreased water intake leading to decreased milk production by the F0 dams during lactation.

Although pup survival rate at 500 ppm was reduced, fostering of pups to untreated mothers lessened their mortality rate indicating that the effect was attributable to maternal toxicity. This was confirmed by findings in the F0 parental generation, in which acrylonitrile at 500 ppm caused reduced bodyweight gain in the first generation parent rats (this parameter was not investigated in F2 and F3 parents).

There is no obvious exposure-related effect on pup sex ratio. The ratio in the 500 ppm F2a pups appeared skewed; however, there was no pattern evident across generations/litterings.

There were no clearly treatment-related effects on pup observations. There appeared to be increased findings of hematomas in the high dose F1a pups, this finding was not replicated in subsequent generations and is considered unlikely to be treatment-related. One 500 ppm pup had an absent tail; this pup was examined skeletally and no other abnormalities were identified. The study authors suggest this may have been an amputation. No conclusions can be drawn regarding exposure-relationship based on the low incidence of this finding. There were no exposure-related findings on histopathological evaluation of the reproductive organs (uterus, seminal vesicle, prostate and testes; evaluated from 10/sex 500 ppm and control F3b weanlings).
Reproductive effects observed:
not specified

Analysis of actual levels of acrylonitrile in the drinking water indicated stability in water, with the exception of the 100 ppm level between weeks 44 and 65. Actual average weekly acrylonitrile concentration in the high-dose group was 522 ppm (SEM 11.6), and in the lower-dose group was 106 ppm (SEM 2.3). This latter value did not include the values determined between weeks 44 and 65, when mean levels ranged between 0 and 100 ppm, with an average of 37 ppm (SEM 6.2). This reduction in measured levels was associated with bacterial contamination of the water, which was controlled by disinfection of water bottles and storage vessels post week 65. The authors calculated that the mg/kg/day intake was approximately 11 ± 5 and 37 ± 10 for males and 20 ± 3 and 40 ± 8 for females for the 100 and 500 ppm concentrations, respectively.

Water consumption data are not available for the F1 and F2 adults, and though it is possible that water consumption was equally reduced compared to the F0 adults, there is no way to confirm this. The results indicate no obvious effect of acrylonitrile on fertility in the rat at an average level of 500 ppm in drinking water (approximately 35 mg/kg/day). The experimental design does however have limitations, in particular the absence of histopathological examination of gonads in the male rats, other than in the F3b offspring in which no abnormality was reported. Sperm parameters were also not investigated. Interpretation of data for parental toxicity and pup growth (and possibly pup survival) is confounded by the severe decrease in water consumption by the high dose group resulting from poor palatability of water containing acrylonitrile at the 500 ppm concentration. It is difficult to assess the biological significance of the decrease in the pup viability index because there was no historical control data on pup survival; the decreased pup survival during lactation post-PND 4 in the F1a generation at 500 ppm is at most equivocally related to exposure.

Evaluation of pup growth in this study is difficult. Reporting was limited to PND 4 (for both sexes combined) and PND 21 (with combined male and female weights presented for the F1a, F2a, and F3a pups, and weights separated by sex for the F1b, F2b and F3b pups). There is wide variation in the mean control weights, e.g., PND 21 mean weights for control male F2b and F3b pups are 53 and 50 grams respectively, while the F1b control pup mean PND 21 weight is 39 grams (S.E. of 2-3 in all groups), which is a 26% difference. The culling procedure was different for the F3b 13 pups (to 4/sex/litter rather than 5), but according to the report the culling procedure for the control F1b pups and F2b pups was the same (to 5/sex/litter). Control PND 21 female mean pup weights also had marked variance across generations. There is no apparent explanation for this marked difference in control PND 21 mean pup weights between generations. There is a marked statistically significant exposure-related decrease in PND 4 and PND 21 mean

pup body weights at 500 ppm. It is very likely due to decreased water intake by the high dose dams compromising milk production, and likely in some part due to decreased water intake or direct toxicity of acrylonitrile to the pups as they initiate self-drinking in the last half of the lactation period. Both factors probably contributed to the pup body weight decreases at 500 ppm. Sporadic statistically significant decreases in PND 21 pup body weight were reported at 100 ppm. This finding is considered to be unlikely to be treatment related, because of the inconsistency across generations/litterings, but as noted previously reaching a conclusive opinion is difficult because of the variation in control pup weights across generations.

Summary of Reproductive Indices

 

F0Parents, F1pups

F1Parents, F2Pups

F2Parents, F3Pups

 

F1a

F1b

F2a

F2b

F3a

F3b

0 ppm

 

 

 

 

 

 

Male fertility

100

100

50

60

60

90

Female fertility

90

80

50

50

70

70

Gestation

100

100

100

100

100

100

Viability

99

100

98

100

100

97

Lactation

92

91

100

100

98

98

100 ppm

 

 

 

 

 

 

Male fertility

80

100

70a

50

90

100

Female fertility

80

85

55a

40

65

75

Gestation

100

100

100a

100a

100

100

Viability

98

90*

94a

96a

99

100

Lactation

93

95

91*a

96

100

98

500 ppm

 

 

 

 

 

 

Male fertility

100

87

80

80

100

100

Female fertility

80

79

70

70

75

85

Gestation

100

100

100

100

100

100

Viability

94*

91*

95

100

95*

94

Viability (fostered

to untreated dams)

n/a

98

n/a

n/a

n/a

n/a

Lactation

66*

88

94*

100

99

92

Lactation (fostered

to untreated dams)

n/a

98

n/a

n/a

n/a

n/a

* = statistically significant finding

a= 100 ppm animals with likely lower than nominal compound exposure (timeframe estimated based on

results of dose formulation analyses presented in the Litton report).

Parental toxicity

Parameter

0 ppm

100 ppm

500 ppm

F0

Preebreed clinical signs (M and F)

-

-

-

Prebreed body weight (M)

-

↓*

↓*

Prebreed body weight (F)

-

↓*

Preebreed feed consumption (M and F)

-

↓*

↓*

Prebreed water consumption (M and F)

-

↓*

↓*

(approx 50%)

F1

Preebreed clinical signsa

-

-

-

F2

Preebreed clinical signsa

-

-

-

* statistically significant (p≤0.05)

aF1 and F2 premating body weight, feed and water consumption not recorded.

Pup body weights (mean±SE/litter)

 

F0 Parents, F1 pups

F1 parents, F2 pups

F2 parents, F3 pups

F1a

F1b

F2a

F2b

F3a

F3b

0 ppm

Pup PND 4 weight

11±0.5

10±0.5

11±0.7

11±0.95

10±0.44

10±0.35

PND 21 weight (M and F)

42±1.4

NA

39±2.5

NA

43±1.03

NA

PND 21 M

NA

39±2.4

NA

53±2.96

NA

50±2.0c

PND 21 F

NA

37±2.3

NA

49±2.77

NA

47±1.17c

100 ppm

Pup PND 4 weight

10±4.3

9±0.5

10±0.3b

10±0.56

9±0.58

10±0.39

PND 21 weight (M and F)

40±2.4

NA

39±0.6b

NA

43±1.78

NA

PND 21 M

NA

36±1.4

NA

46±1.10*

NA

47±0.98c

PND 21 F

NA

33±1.4*

NA

45±1.08

NA

44±0.98c

500 ppm

PND 4 weight

9±0.4*

10±0.4a

9±0.7*

9±0.50

8±0.17*

8±0.41*

PND 4 weight Fostered to untreated dams

NA

11±0.4a

NA

NA

NA

NA

PND 21 weight (M and F)

28±1.9*

NA

30±2.9*

NA

30±1.39*

NA

PND 21 M

NA

34±1.4*a

NA

30±1.43*

NA

32±1.76*c

PND 21 M (fostered to untreated dams)

NA

53±2.2*a

NA

NA

NA

NA

PND 21 F

NA

34±1.2a

NA

29±1.49*

NA

32±1.57*c

PND 21 F (fostered to untreated dams

NA

49±2.2*a

NA

NA

NA

NA

 * statistically significant (p≤0.05). NA, data not available or not calculated.

aData considered unreliable due to cross-fostering procedure

bdata from dose group with significantly lower than nominal compound intake

cinadvertently culled to 4 pups/sex/litter instead of 5 pups/sex/litter

Tumour incidence in female rats after approximately 1 year drinking water exposure

Generation

Dose (ppm)

Total tumours/masses

Astrocytomas

Zymbal gland tumours

F0

0

0/19

0

0

100

3/20

1

0

500

6/24

2

2

F1

0

1/20a

0

0

100

3/19

1

2

500

9/17b

4*

3*

F2

0

0/20

0

0

100

2/20c

1

0

500

7/20d

1

3

* statistically significant (p≤0.05).

a Uterine papilloma

b Includes 1 adenocarcinoma of the leg and 1 mammary adenocarcinoma

c Includes 1 mammary fibroadenoma

d Includes 3 mammary fibroadenomas

Conclusions:
The NOAEL for reproductive and pup toxicity appears to be 100 ppm Sprague-Dawletin drinking water.
Executive summary:

Acrylonitrile was tested for reproductive effects in a three generation drinking water study with two matings per generation. Sprague–Dawley rats were exposed to acrylonitrile in drinking water at 0, 100, or 500 ppm. This corresponds to 0, 11±5 and 37±10 mg/kg, respectively, for males and 0, 20±3 and 40±8 mg/kg per day for the females, respectively. Water consumption was reduced in F0 rats in the 100 and 500 ppm groups. At 500 ppm, acrylonitrile reduced body weight gain and food intake of the first generation parental rats (F0). These parameters were not investigated at subsequent generations. The pup survival (both viability and lactation indices) was reduced at the 500 ppm treatment level in both matings of all three generations. Fostering the 500 ppm pups onto untreated mothers following the second mating lessened mortality, suggesting a maternal effect consistent with decreased water consumption. There was no remarkable change in the reproductive capacity in any of matings in rats at the 100 ppm concentration. In contrast, in all three generations, the body weights of the pups of the 500 ppm treatment level were reduced on Day 21 at both matings. No adverse findings were observed in the tissues of a limited number of third generation weanlings (F3b) upon gross and microscopic evaluation. No effect on the sciatic nerve was evident among the adult female rats held for 20 weeks after weaning of the second litter. There was a dose-related effect of acrylonitrile on gross masses in female rats at each parental generation held 20 weeks after the weaning of the second litter. Histopathological evaluation of these dams showed an increase in astrocytomas and zymbal gland tumors.

Endpoint:
toxicity to reproduction
Remarks:
other: review of literature and proprietary reproductive toxicity studies
Type of information:
other: literature review
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The authors use a weight of evidence approach to review the available animal data on the reproductive toxicity of acrylonitrile.
Qualifier:
no guideline available
Principles of method if other than guideline:
Critical review of the available literature and proprietary reproductive toxicity studies.
GLP compliance:
no
Remarks:
: some individual studies were GLP-compliant
Limit test:
no
Species:
rat
Strain:
other: Sprague-Dawley, Crl:CD®(SD)
Sex:
male/female
Route of administration:
other: drinking water and inhalation
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Duration of treatment / exposure:
In the two-generation inhalation reproductive toxicity study (WIL Research Laboratories, 2005; Nemec et al., 2008), Crl:CD®(SD) rats were exposed to AN via whole-body inhalation (6 h/day, 7 days/wk) at concentrations of 0, 5, 15, 45, and 90 ppm for the F0 generation. Animals were exposed for 10 wk prior to mating, and were bred to produce an F1 generation, with exposure to the parental animals continuous for the males to the day post mating, and for the dams through mating, gestation and lactation, except for the dams during postnatal day (PND)
Frequency of treatment:
6 hours/day; 7 days/week: two-generation inhalation toxicity study.
Remarks:
Doses / Concentrations:
0, 5, 15, 45, and 90 ppm
Basis:
other: atmospheric concentration (inhalation study)
Remarks:
Doses / Concentrations:
0, 35, 210, or 500 ppm
Basis:
nominal in water
Toxicological Research Laboratory, 1975
Remarks:
Doses / Concentrations:
0, 100, or 500 ppm
Basis:
nominal in water
Litton Bionetics, 1980; Friedman and Beliles, 2002
No. of animals per sex per dose:
Various
Control animals:
yes
See discussion below
Dose descriptor:
NOAEL
Effect level:
45 ppm
Sex:
male/female
Basis for effect level:
other: Inhalation study: WIL Research Laboratories, 2005; Nemec et al., 2008
Remarks on result:
other: Generation: all
Dose descriptor:
NOAEL
Effect level:
100 ppm
Sex:
male/female
Basis for effect level:
other: Overall reproductive NOAEL for the drinking water studies
Remarks on result:
other: Generation: all / overall
See discussion below
Reproductive effects observed:
not specified

Two-generation inhalation reproductive toxicity study (WIL Research Laboratories, 2005; Nemec et al., 2008)

Body weight gains and feed consumption were suppressed in the 90- and 45-ppm females during gestation, and body weight gain was suppressed during lactation at the same exposure levels. There were no functional effects on male mating performance or fertility. Sperm motility and progressive sperm motility were statistically significantly decreased in the F0 males at 90 ppm (82* ± 6.4 and 67* ± 9.4 at 90 ppm compared to 87 ± 5.7 and 73 ± 8.2 in the control group for percent motile and progressively motile, respectively). This finding is unlikely to represent an adverse effect of exposure based on the slight nature of the decreases (5.8% and 7%, respectively), the lack of effects on other sperm endpoints (count or morphology), and the percent motile sperm falling within the WIL historical control range. There were no statistically significant effects on sperm motility in the 45-ppm group of F0/F1 males. There were no treatment-related effects on sperm counts or morphology, or treatment-related histopathological findings in male reproductive organs. There were no effects on the conventional measures of female reproductive performance: mating, fertility, and success in rearing pups were uniformly excellent across all groups. There were no dose-related statistically or biologically significant effects on pre-coital interval or gestation length. Mean oestrous cycle lengths in all groups were similar to controls, with the exception of slightly increased values in the 45-ppm F0 and F1 females (6.9 ± 3.98 and 5.3 ± 2.53 days at 45 ppm compared to 5.0 ± 1.33 and 4.4 ± 0.61 days in the control groups for the F0 and F1 females, respectively). This finding is not considered treatment related because the changes were not statistically significant, no dose response was present in the F0 animals (in the F1 animals 45 ppm was the highest dose tested), and the increase in the 45 ppm F1 females was due to a prolonged oestrous cycle in a single female. There were no treatment-related histopathological findings in F0 or F1 female reproductive organs. The mean number of ovarian primordial follicles was unaffected in the 45-ppm group F1 females. The numbers of pups born, live litter sizes, sex ratios at birth and postnatal survival were unaffected by parental acrylonitrile exposure in either generation. There was no evidence of treatment-related malformations. Pup toxicity was present (decreased body weight, statistically significant from PND 14) in the F1 pups at 90 ppm in the presence of maternal toxicity (data not shown). As noted, pups were not directly exposed to acrylonitrile however, exposure to acrylonitrile or its metabolites could have occurred through the milk or nursing activity, as the dams were directly exposed from PND 5–28. F1 male pups showed increases in anogenital distance (AGD), statistically significant at 45 ppm but not at 90 ppm; calculated relative to the cube of body weight, the increases were statistically significant at both 45 and 90 ppm. No increased AGD was seen at 45 ppm in the F2 pups. The AGD mean values (including controls) were quite different for the F1 and F2 male pups, suggesting there may have been inter-observer variability. The increased AGD in the F1 male pups is not considered likely to be treatment related because of the small magnitude of the finding and the lack of replication in the second generation. There was a slight, not statistically significant, delay in the time of balano-preputial separation in the F1 males (46.1 ± 3.77 and 46.9 ± 3.05 days in the 45- and 90-ppm groups, respectively, compared with 44.6 ± 3.11 days in the control) in the presence of marked statistically significant decreased body weights. This finding is unlikely to be biologically significant because of the absence of statistical significance and the correlation with statistically significant decreases in body weight. Vaginal opening was statistically significantly delayed in the 90-ppm F1 females (37.0** ± 2.92 days at 90 ppm compared to 34.3 ± 1.54 days in the control group). The delay may be attributable to the decreased body weight in this group. The time of vaginal opening in the 90-ppm females was within the historical control range for inhalation studies and is not considered to be related to treatment.

One-generation drinking-water reproductive toxicity study (Toxicological Research Laboratory, 1975)

Treatment-related effects on the adults appear primarily to be associated with the poor palatability of acrylonitrile in water, which resulted in marked decreases in water consumption (40–60% at the high dose level) associated with body weight and feed consumption decreases. Necropsy findings suggest some gastric ulceration may have been associated with ingestion of the high concentration of acrylonitrile, but this finding may have been confounded by concurrent nematode infestation. There were no obvious effects on reproductive performance or on pup survival; the decreased water consumption by the dams may have decreased milk production and thus decreased the body weight gain of the pups. Pup body weight gain may also have been impacted late in the lactation period by poor water palatability resulting in reduced water consumption by the pups, or by direct toxicity to the pups. Pup development was not further evaluated in this study.

Three-generation drinking-water reproductive toxicity study (Litton Bionetics, 1980; Friedman and Beliles, 2002)

There were no treatment-related clinical signs in F0 adults during the premating period. Water consumption was markedly reduced at 500 ppm (by approximately 50%), and was moderately reduced at 100 ppm in the F0 animals. F0 body weight and feed consumption were also reduced at both dose levels. There were no indications of male reproductive toxicity. The duration of mating was decreased in both exposed groups in the first mating of the F2 adults; however, the controls for this mating had an increased duration of mating compared to the controls in the first two generations. This finding is therefore not considered compound related. There were also no indications of effects on female reproduction. The lack of increased duration of mating suggests that oestrous cyclicity in the exposed females, which was not directly evaluated, was not adversely affected. There was no effect on the duration of gestation. There was no obvious effect on male mating, fertility, or female fertility indices, although the poor control fertility, particularly in the second or third generations, could have masked a treatment-related decrease. No decreased fertility was evident in the dosed groups in the first generation, however, in which control fertility was high, and in the other generations the high-dose group females generally showed higher fertility than did the controls. Therefore, it is unlikely that a major treatment-related effect on fertility was missed in this study. There was no effect on the gestation index. Slight but statistically significant decreases in the pup viability index were seen in most generations/litters at 500 ppm. It is difficult to assess the biological significance of this finding without historical control data on pup survival; however, the decreases may be related to maternal toxicity. A marked statistically and biologically significant decrease in the lactation index was evident in the F1a generation at 500 ppm (66% compared to 92% in the control group); this finding was not replicated in subsequent generations/litters, in which the lactation indices ranged from 92%-100% (excluding data from the F1b generation 500-ppm pups, half of which were cross-fostered). The study authors hypothesize that the decreased lactation index in the F1a pups may have been due to decreased water intake leading to decreased milk production by the F0 dams during lactation. However, pup survival was not adversely affected in the Toxicological Research Laboratory (1975) drinking-water study (in which the same nominal 500-ppm concentration of acrylonitrile in water also led to marked decreases in maternal water consumption), although pup body weights were decreased in the latter study. It is concluded that the decreased pup survival during lactation post PND 4 in the F1a generation at 500 ppm is at most equivocally related to exposure to acrylonitrile  Although a statistically significant decrease in the lactation index was also seen for the F2a pups at 100 ppm, this finding is not considered exposure related, because it was slight, limited to one generation/littering, and not dose related (no similar decrease in the lactation index was seen at 500 ppm in the F2a pups). There were no exposure-related increased incidences of pups found dead in the study, and no consistently statistically significant effects on number of live-born pups per litter, or obvious exposure-related effects on pup sex ratio. There was a statistically significant exposure-related decrease in PND 4 and PND 21 mean pup body weights at 500 ppm. This is consistent with the pup body weight findings reported in the 1975 Toxicological Research Laboratory one-generation reproductive toxicity study, and is likely at least partly due to decreased water intake by the high-dose dams, compromising milk production. Decreased water intake by the pups or direct toxicity of AN to the pups as they initiated self-drinking in the last half of the lactation period may also have contributed to the body weight decreases. Sporadic statistically significant decreases in PND 21 pup body weight were reported at 100 ppm. The inconsistency across generations/litters, variation in control pup weights across generations, and the incomplete data collection or reporting preclude a conclusion regarding exposure relationship at 100 ppm, but the sporadic nature of the findings makes an exposure relationship unlikely. There were no clearly treatment-related effects on pup observations. There were no treatment-related histopathological findings on adults in the reproductive phase of the study (limited evaluations). There were no exposure-related findings on histopathological evaluation of the reproductive organs, including uterus, seminal vesicle, prostate, and testes, evaluated from 10/sex of the 500-ppm and control F3b weanlings.

Conclusions:
The existing animal data on the reproductive toxicity of acrylonitrile do not raise significant concerns. Decreased pup growth was observed at maternally toxic high-dose concentrations in both drinking-water and inhalation reproductive toxicity studies. The NOAEL for reproductive and pup toxicity is 100 ppm in drinking water; however, confidence in these studies is limited due to study design deficiencies. The NOAEL for reproductive toxicity over two generations and neonatal toxicity of AN administered to rats via wholebody inhalation was 45 ppm (the NOAEL for reproductive toxicity was 90 ppm for the first generation). There is a high level of confidence in the results of the inhalation reproductive toxicity study, which was conducted in compliance with GLP and current standards for evaluating reproductive toxicity.
Executive summary:

The authors critically review the available (published and unpublished) data relating to the reprodcutive toxicity of acrylonitrile.

It is concluded that drinking-water and inhalation reproductive toxicity studies show no clear effects of acrylonitrile exposure on reproductive performance or fertility. Effects at maternally toxic concentrations were limited to decreased pup growth. The drinking-water reproductive NOAEL is considered to be 100 ppm (with moderate confidence due to the limitations of this study). The inhalation exposure reproductive NOAEL is considered to be 45 ppm (with high confidence). The inhalation reproductive toxicity study is considered to provide the most robust data for risk assessment. The authors conclude that acrylonitrile is not expected to be a reproductive toxicant in the absence of significant maternal toxicity.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
35 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
A one-generation and a three-generation drinking water study are available in the rat.
Effect on fertility: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
211 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
A two-generation study inhalation study in the rat is available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The reproductive toxicity of acrylonitrile has been extensively investigated in studies in experimental animals. A number of reports of reproductive effects in exposed humans are also available.

 

Two generation inhalation toxicity study (Nemec et al., 2008)

This study is considered to be key to the assessment of the reproductive toxicity of acrylonitrile as it includes a comprehensive investigation of a number of relevant parameters and uses an appropriate route of exposure. Sprague-Dawley rats (25/sex/group) were exposed (whole body) by inhalation (6 hours/day) to acrylonitrile vapour at concentrations of 0, 5, 15, 45 or 90 ppm. F0 animals were exposed for 10 weeks prior to mating and throughout mating, gestation and lactation of the subsequent F1 litters. Selected F1 offspring were then similarly exposed following weaning and mated to produce F2 litters. In addition to standard reproductive indices, the study included assessment of oestrus cyclicity and sperm parameters. Post mortem investigations of parental animals included detailed histopathological assessment of the reproductive system and associated organs/tissues, detailed histopathological assessment of brain and nasal tissues. Offspring were additionally investigated for developmental ontogeny. F1 animals exposed to 90 ppm acrylonitrile showed excessive toxicity, therefore this exposure level was not investigated further. Mortality was unaffected by exposure. Systemic toxicity in exposed adult rats was limited to reduced weight gain and food consumption and increased liver weights at 45 and 90 ppm.  Local toxicity (nasal irritation) was apparent during and immediately following exposure to 90 ppm; histopathological effects on the nasal tissues consistent with local irritation were also seen in some animals in all exposure groups in the F1 generation, although a NOAEC of 15 ppm for this effect was apparent in the F0 generation. The difference for this effect is attributable to the age at first exposure (8 weeks for F0, 4 weeks for F1) and may be related to differences in nasal morphology, dosimetry There was no evidence of any effect on reproductive parameters, tissues or organs of the reproductive system.  Effects on offspring were limited to bodyweight effects.

 

Three generation drinking water study (Beliles et al., 1980)

Sprague-Dawley rats (20 males, 10 females / group) were exposed to acrylonitrile in the drinking water at concentrations of 0, 100 or 500 ppm for 100 days prior to mating and throughout mating, gestation and lactation of the resulting litter. Two litters were produced per generation and exposure continued over three generations. No effects on fertility were seen in this study. Toxicity (reduced weight gain, food and water consumption) was seen at 500 ppm, with less marked effects also seen at 100 ppm. Offspring toxicity (reduced growth and survival) was also noted at 500 ppm (nominal concentration; actual concentration 552 ppm equivalent to approximately 35 mg/kg bw/d; findings are considered most likely to be secondary to parental toxicity.

 

One generation drinking water study (Schwetz et al., 1975)  

No evidence of an adverse effect on reproduction or fertility was seen in this study following the administration of acrylonitrile in the drinking water at concentrations of up to 500 ppm. High dose effects seen in parental animals (reduced bodyweights, food and water consumption) are considered likely to be secondary to the reduced palatability of the drinking water. Gross necropsy revealed gastric haemorrhage at the highest dose level, however findings may be confounded by the presence of nematode infection. Findings in offspring were limited to bodyweight effects. One litter in each of the treatment groups had an indication of short or absent tail, however in the absence of a clear dose-response relationship this finding is not considered to be treatment-related. 

 

Weight of evidence review (Neal et al., 2009)

The authors of this review considered the available data from published and unpublished animal reproductive toxicity studies, human epidemiology studies, other non-standard investigative studies and relevant endpoints from other toxicology studies and discuss the potential of acrylonitrile to cause reproductive toxicity in exposed humans. The two drinking-water reproductive toxicity studies of acrylonitrile were both pre-GLP studies that evaluated a limited number of reproductive parameters, compared to current study design guidelines. The two generation inhalation reproductive toxicity study reported by Nemec et al. (2008) was conducted in compliance with GLP requirements and with current EPA regulatory testing guidelines. The Nemec et al. (2008) study was therefore considered to provide data of the highest confidence for reproductive toxicity hazard assessment by a relevant route of exposure, and provides NOAELs that are appropriate points of departure for risk assessment. It is noted that there were no obvious compound-related effects on reproductive success in any of the reproductive toxicity studies, even at exposure levels producing toxicity to the parent animals. Both the drinking-water studies and the inhalation study showed decreased pup weight gain at maternally toxic doses, however the severe decrease in maternal water consumption at 500 ppm possibly contributed to the slowed pup growth seen in both drinking-water studies. Lactation may have been affected by maternal dehydration, resulting in a change in quantity or quality of the milk. The decreased weight gain in the pups was seen at a younger age in the drinking-water studies than was the decreased pup weight gain in the inhalation study, supporting a contribution of the drinking-water deficit because of the sole reliance on milk for nutrition in pups early in the lactation period. However, the decreased pup growth in the inhalation study suggests that dehydration may not have been the only mechanism in the drinking-water studies delaying growth at maternally toxic doses. The 3 -generation drinking water study (Friedman & Beliles, 2002) showed a possibly treatment-related adverse effect on pup survival, most notably in the 500-ppm F1a pups. The study authors suggested this finding was due to decreased maternal milk production resulting from the decrease in maternal water consumption. This finding was not replicated in other generations/litters. Additionally, the 1-generation drinking-water study (Schwetz et al., 1975) showed no exposure-related effects on pup viability at the same high exposure level, despite commensurate decreases in maternal water consumption, and the inhalation study, discussed earlier, showed no effect on pup survival at a maternally toxic dose. A dominant lethal study performed with acrylonitrile was negative, demonstrating a lack of male-mediated reproductive toxicity. Data from short-term gavage studies such as Tandon et al. (1988) suggest that some effects on sperm quality might result from high-dose gavage exposure. The majority of these studies are of limited quality. The absence of functional effects on reproductive success in the drinking-water and inhalation reproductive toxicity studies, biologically significant effects on andrology, or male reproductive organ histopathological findings in the inhalation reproductive toxicity study mitigate concerns regarding these endpoints. It is noted that the NTP (2001) chronic study in mice showed an increased incidence of ovarian atrophy in reproductively senescent mice exposed to acrylonitrile; the biological significance of this finding is unclear. There were no similar findings in the inhalation two-generation rat reproductive toxicity study

 

No data were seen in animal studies supporting an increased incidence of stillbirths, pre-term or post-term deliveries or maternal mortality following exposure to acrylonitrile at dose levels producing other evidence of systemic toxicity. There was very weak support in the animal data for increased infant mortality, with pup deaths increased only at the high dose level in a single generation of a three-generation reproductive toxicity study. The pup deaths may have been contributed to by decreased water intake of the dams. No evidence of increased pup mortality was seen in the two-generation inhalation reproductive toxicity study, considered to have the highest confidence level. There is no robust evidence for male-mediated toxicity, with only one equivocal study of poor quality reporting a positive result (Ahmed et al., 1992), and other studies, including a well-conducted dominant lethal study (Working et al., 1987) showing no effects. Effects on male reproductive toxicity (changes in sperm parameters or testicular degeneration) were reported in three studies, one of moderate quality (Tandon et al., 1988) and two of very poor quality. However, several other high- or moderate-quality evaluations showed no effects on the testes or on andrology data, including the Nemec et al. (2008) inhalation reproductive toxicity study, which included the most comprehensive evaluation of these parameters.

 

With regard to human data on the reproductive toxicity of acrylonitrile, Collins et al. (2003) reviewed four epidemiology studies performed on Chinese workers exposed to acrylonitrile and reporting reproductive and developmental effects following maternal and/or paternal exposure. The results of the studies are consistent for an increased risk of stillbirth, birth defects, miscarriage, infertility and low birthweight. However the absence of details on the timing of exposure and on the levels of personal exposure are considered to limit the value of the studies as a clear temporal link and relationship between exposure level and adverse outcome cannot be concluded. While the individual studies have several strengths and are largely consistent with each other in indicating that exposure to acrylonitrile is associated with various adverse reproductive and perinatal outcomes, there are limitations which make a clear causal assessment difficult. In addition, experimental studies provide little support for biological plausibility. It is concluded that additional studies with better designs are required to further investigate the suggested association between acrylonitrile exposure and adverse reproductive outcome. Neal (2009) concluded that, while some Chinese epidemiological studies reported increased rates of birth defects, stillbirths, premature delivery, infertility, and spontaneous abortions; these findings may be artefacts of differential ascertainment, reporting bias, confounding exposures, or other factors. The epidemiological studies are not sufficiently robust, free of potential confounders, or adequately documented for exposure to be an appropriate basis for risk assessment.

Effects on developmental toxicity

Description of key information

Studies of the developmental toxicity of acrylonitrile are available in the rat (gavage and inhalation exposure) and hamster (intraperitoneal exposure), together with some non-standard investigative studies.

An expert review of developmental toxicity and malformations in eight animal studies leads to the conclusion that very high (and maternally toxic) exposures to acrylonitrile results in fetotoxicity, and may result in teratogenicity. Teratogenicity appears to be most likely following oral gavage exposure, which is not a relevant route of exposure to humans. The studies considered to be of highest quality do not show clear evidence of teratogenicity. The Saillenfait et al. (1993) rat inhalation developmental toxicity study (which tested to the highest inhalation concentration) showed decreased fetal body weights at a maternally toxic dose, but no exposure-related malformations. The Nemec et al. (2008) rat inhalation reproductive toxicity study showed only a single high-dose malformation, considered at most equivocally related to treatment. There was no evidence of developmental toxicity in any study in the absence of maternal toxicity.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Proprietary study, guideline comparable and well reported
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
no
Remarks:
: study pre-dates GLP
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
Female pregnant (mated) Sprague-Dawley rats
Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on exposure:
Mated female rats were exposed to 0, 10, 25, or 65 mg/kg bw/d acrylonitrile by gavage on days 6 -15 of gestation
Analytical verification of doses or concentrations:
no
Details on mating procedure:
No information available
Duration of treatment / exposure:
Daily on days 6 through 15 of gestation
Frequency of treatment:
Daily
Duration of test:
10 days
Dose / conc.:
0 mg/kg bw/day
Remarks:
Vehicle control
Dose / conc.:
10 mg/kg bw/day
Dose / conc.:
25 mg/kg bw/day
Dose / conc.:
65 mg/kg bw/day
No. of animals per sex per dose:
29-39 females per group
Control animals:
yes, concurrent vehicle
Details on study design:
Pregnant rats were exposed to 0, 10, 25, or 65 mg/kg/day acrylonitrile by gavage on days 6 through 15 of gestation
Maternal examinations:
Daily clinical examination and periodic determination of bodyweight, food and water consumption.
Ovaries and uterine content:
The numbers and positions of implantation sites, live, dead and resorbed foetuses were recorded.
Fetal examinations:
All foetuses were examined macroscopically for external abnormalities and cleft palate, one-third were then examined for visceral abnormalities under a dissecting stereo-microscope and the heads were examined by the razor-section technique of Wilson. Examinations included sex and body weight. All remaining foetuses were examined for skeletal alterations.
Indices:
Incidence of pregnancy, numbers of implantations per dam, live foetuses per litter, resorptions per litter, foetal body weight and crown-rump length
Historical control data:
No information available.
Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: Clinical signs, reduced weight gain and food consumption at 65 mg/kg bw/d. Local gastric irritation at 25 and 65 mg/kg bw/d.

Details on maternal toxic effects:
Animals receiving 65 mg/kg bw/d acrylonitrile in drinking water showed hyperexcitability and excessive salivation, and body weight was significantly decreased compared with controls between days 6 and 15. Food consumption was decreased in the early stages of the study while water consumption was increased in the later stages. One dam at this dose level died on day 1 of the study. Bodyweight was unaffected by acrylonitrile administration at the lower dose levels.

Treated dams had increased liver weights. Thickening of the glandular forestomach was observed in the majority of animals receiving 65 mg/kg bw/d and in 3/39 animals receiving 25 mg/kg bw/d. Sialodacryadenitis (indicated by the presence of swollen salivary glands) was noted in many animals including controls.

The incidence of pregnancy was significantly decreased in rats at 65 mg/kg bw/d (69% compared with 88% in controls, p <0.05) and implantation sites were detected in 4 apparently non-pregnant dams at this dose level (14%). No effect on the incidence of pregnancy was seen at lower dose levels, and no effect was detected on numbers of implantations per dam, live foetuses per litter or resorptions per litter at any dose level.
Key result
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Key result
Abnormalities:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: Reduced pup weight and skeletal abnormalities

Details on embryotoxic / teratogenic effects:
Foetal body weight was significantly decreased at 65 mg/kg bw/d (7.4% decrease, p < 0.05) and crown rump length was also decreased (1.9% decrease, p < 0.05).
In foetuses examined for skeletal and visceral abnormalities, short tail occurred significantly more often among the litters of dams given 65 mg/kg bw/d than in control litters (in 8/212 foetuses examined at 65 mg/kg bw/d, compared with 1/443 in controls, p < 0.05). Short-tailed foetuses also had missing vertebrae, ranging from lack of one lumbar vertebra to lack of all sacral, lumbar and most thoracic vertebrae, with associated ribs. Additional malformations in these foetuses included short trunk (3/212 foetuses, with 0/443 in controls) imperforate anus (2/212), right-sided aortic arch (1/212), missing kidney (1/212) and anteriorly-placed ovaries (1/212). There were also increases in the incidence of minor skeletal abnormalities in the 65 mg/kg bw/d offspring compared with controls, these included delayed ossification of sternebrae, split sternebrae and delayed ossification of cervical vertebrae. At 25 mg/kg/day no malformation occurred with an incidence statistically that was significantly different to that seen in the controls, although a number of the same malformations seen in the 65 mg/kg bw/d group also occurred at this dose level.
Key result
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
external malformations
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
external: tail
Description (incidence and severity):
hort tail occurred significantly more often among the litters of dams given 65 mg/kg bw/d than in control litters (in 8/212 foetuses examined at 65 mg/kg bw/d, compared with 1/443 in controls, p < 0.05). Short-tailed foetuses also had missing vertebrae, ranging from lack of one lumbar vertebra to lack of all sacral, lumbar and most thoracic vertebrae, with associated ribs. Additional malformations in these foetuses included short trunk (3/212 foetuses, with 0/443 in controls) imperforate anus (2/212), right-sided aortic arch (1/212), missing kidney (1/212) and anteriorly-placed ovaries (1/212). There were also increases in the incidence of minor skeletal abnormalities in the 65 mg/kg bw/d offspring compared with controls, these included delayed ossification of sternebrae, split sternebrae and delayed ossification of cervical vertebrae.
Key result
Developmental effects observed:
not specified
Lowest effective dose / conc.:
65 mg/kg bw/day
Treatment related:
yes

Whilst the study is well-conducted, it may have been compromised by SDA virus infection.

Foetal toxicity

Dose (mg/kg/day)

0

10

25

65

Foetal body weight (g)

5.68±0.28

5.78±0.25

5.80±0.33

5.26*±0.32

Foetal crown-rump length (mm)

44.4±1.0

44.5±1.3

45.0±1.2

43.6*±1.2

* statistically significant (p< 0.05)

Selected developmental anomalies and variants

Dose (mg/kg/day)

 

0

10

25

65

External

Number evaluated (foetuses/litters)

443/38

388/35

312/29

212/17

Missing tail

Fa

0

0

2(0.6)b

4(2)*

L

0

0

2(7)

4(23)

Missing or short tail

F

1(0.2)

0

2(0.5)

8(4.0)*

L

1(3)

0

2(7)

6(35)

Short trunk

F

0

0

0

3(1)*c

L

0

0

0

3(18)

Imperforate anus

F

0

0

0

2(1)c

L

0

0

0

2(12)

Soft tissue

Number evaluated (foetuses/litters)

154/38

135/35

111/29

71/17

Right sided aortic arch

F

0

0

1(1)

1(1)c

L

0

0

1(3)

1(6)

Anteriorly displaced ovaries

F

0

0

1(1)c

1(1)c

L

0

0

1(3)

1(6)

Skeletal

Number evaluated (foetuses/litters)

443/38

388/35

312/29

217/17

Missing vertebra (other than 1 thoracic and 1 lumbar)

F

1(0.2)c

0

2(0.6)c

4(2)*c

L

1(3)

0

2(7)

6(35)

Missing vertebral centra (other than C1 and C2)

F

23(5)

30(8)

31(10)

71(34)*

L

11(29)

16(46)

13(46)

15(88)

Missing ribs (more than 1 pair)

F

0

0

2(1)c

4(2)*c

L

0

0

2(7)

4(24)

* statistically significant (p< 0.05)

a F= foetuses; L= litters

b Number affected (% affected)

c Alteration seen only in foetuses that also showed short or no tail at this dose level

Conclusions:
Some evidence of developmental toxicity was seen in this study at the highest (and maternally toxic) dose level of 65 mg/kg bw/d.
Executive summary:

Mated female Sprague-Dawley rats (29 -39/group) were gavaged with acrylonitrile (in water) at dose levels of 0, 10, 25 or 65 mg/kg bw/d on Days 6 -15 of gestation. Dams were observed for clinical signs, bodyweight, food and water consumption. Dams were killed on Day 21 and the uterine contents examined. All foetuses were examined for external abnormalities, one third for visceral abnormalities and two thirds for skeletal findings.

Mortality, signs of toxicity (hyperactivity and salivation), reduced weight and food consumption and increased water consumption were noted in the high dose group. Necropsy revealed gastric irritation in most animals at 65 mg/kg bw.d and a small number at 25 mg/kg bw/d. Post-implantation loss was increased at the high dose level; reduced foetal weight and length was also apparent in this group. No effects were seeen in the other treated groups. A higher incidence of short tail was seen in foetuses at 65 mg/kg bw/d, with other abnormalities and skeletal variations also increased in this group. No effects were seen in the lower dose groups.

Some evidence of developmental toxiicty was seen in this study at the highest (and maternally toxic) dose level of 65 mg/kg bw/d. The NOAEL for maternal toxicitiy is therefore 10 mg/kg bw/d based on the signs of gastric irritation at 25 mg/kg bw/d; the NOAEL for developmental toxicity is 25 mg/kg bw/d.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Proprietary study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
The effects of maternally inhaled acrylonitrile on embryonic and foetal development were investigated.
GLP compliance:
not specified
Remarks:
: older study, pre-dates GLP
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
Female pregnant Sprague-Dawley rats
Route of administration:
inhalation
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
Pregnant rats were exposed to acrylonitrile by inhalation for 6 hours per day on days 6 through 15 of gestation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Evaluation of acrylonitrile plus metabolites to establish an AUC was done in a separate experiment, and a single 6 hour exposure to 80 ppm was considered equivalent to an oral dose of 23 mg/kg/day.
Details on mating procedure:
No information available.
Duration of treatment / exposure:
Days 6 through 15 of gestation.
Frequency of treatment:
Daily
Duration of test:
10 days
Dose / conc.:
0 ppm
Remarks:
Control
Dose / conc.:
40 ppm
Remarks:
Nominal air concerntration
Dose / conc.:
80 ppm
Remarks:
Nominal air concentration
No. of animals per sex per dose:
29-39 females per group
Control animals:
yes, concurrent no treatment
Details on study design:
Acrylonitrile was administered to mated females on days 6-15 of gestation. The animals were killed on day 21 for examination.
Maternal examinations:
Daily clinical examination and periodic determination of body weight, food and water consumption.
Ovaries and uterine content:
The numbers and positions of implantation sites, live, dead and resorbed foetuses were recorded.
Fetal examinations:
All foetuses were examined macroscopically for external abnormalities and cleft palate, one-third were then examined for visceral abnormalities under a dissecting stereo-microscope and the heads were examined by the razor-section technique of Wilson. All remaining foetuses were examined for skeletal alterations.
Statistics:
No information available
Indices:
Incidence of pregnancy, numbers of implantations per dam, live foetuses per litter, resorptions per litter, foetal body weight and crown-rump length
Historical control data:
No information available
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
No treatment-related signs of gross appearance were observed during the exposure period, although maternal body weight was significantly decreased in both the 40 ppm and the 80 ppm groups compared with control between days 6 and 9 of the study and between days 10 and 15. Food consumption was decreased during gestation days 15-17 and increased days 18-20 compared to controls. Statistically significant increased water consumption was observed on gestation days 9-20. No effects on incidence of pregnancy, numbers of implantations per dam, live foetuses per litter, resorptions per litter and foetal body weight was detected at any dose level when acrylonitrile was given by inhalation. Maternal liver weight was unaffected by exposure.
Key result
Dose descriptor:
NOAEL
Effect level:
40 ppm (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
There was no effect on crown-rump length at any dose level tested. The incidence of total major malformations was slightly increased (from 8/421 in controls to 11/416 at 80 ppm, p=0.06), however no single major abnormality occurred at an incidence significantly different than that in the controls. Types of malformations at 80 ppm included short tail, missing vertebrae, short trunk, missing ribs, omphalocele and hemivertebrae. There was a decrease in the incidence of delayed ossification of skull bones at 80 ppm but not at 40 ppm.
Key result
Dose descriptor:
NOAEC
Effect level:
40 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reduced skeletal ossification
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: skull
Description (incidence and severity):
Reduced ossification of skull bones
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
80 ppm (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
no

The NOAEL for maternal and developmental toxicity in this study is 40 ppm.

Selected developmental anomalies and variants

Parameter

0 ppm

40 ppm

80 ppm

External

Number evaluated (foetuses/litters)

421/33

441/36

406/35

Short tail

Fa

0

0

2(0.5)b

L

0

0

2(6)

Short trunk

F

0

0

1(0.2)c

L

0

0

1(3)

Omphalocele

F

0

1(0.2)

1(0.2)

L

0

1(3)

1(3)

Soft tissue

Number evaluated (foetuses/litters)

140/33

148/36

136/35

Anteriorly displaced ovaries

F

0

0

1(0.7)c

L

0

0

1(3)

Skeletal

Number evaluated (foetuses/litters)

421/33

441/36

406/35

Missing vertebra (other than 1 thoracic and 1 lumbar)

F

0

0

2(0.6)c

L

0

0

2(7)

a F = foetuses; L –litters

b number affected (percent affected)

c alteration seen only in foetuses that also showed short or no tail at this dose level

Conclusions:
The NOAEL for maternal and developmental toxicity in this study is 40 ppm.
Executive summary:

Groups of 30 pregnant female Sprague-Dawley rats were exposed to 0, 40, or 80 ppm of acrylonitrile on 6 hrs/day by inhalation on Days 6 -15 of gestation to investigate the effect of maternally inhaled acrylonitrile on embryonic and foetal development.

Mean bodyweights and maternal bodyweight gain was significantly decreased during specific intervals of gestation in both dose exposure groups. Food consumption was decreased during gestation Days 15-17 and increased on Days 18-20 compared to controls. Statistically significant increased water consumption was observed on gestation Days 9-20. Pregnancy incidence, mean litter size, the incidence of resorptions and average foetal body measurements were not affected by exposure at either concentration. Total malformations were significantly increased at 80 ppm. These findings were similar to the findings in the gavage study by Murray et al, 1976, it was therefore considered likely by the authors that the malformations seen at 80 ppm were exposure-related, although the incidence of specific types of malformations was not statistically increased compared to controls. The NOAEL for maternal and developmental toxicity in this study is 40 ppm.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Published study: non-standard route and species, limited reporting on maternal effects
Qualifier:
no guideline followed
Principles of method if other than guideline:
Developmental toxicity was evaluated following intraperitoneal administration to hamsters
GLP compliance:
yes
Remarks:
: published non-standard study
Limit test:
no
Species:
hamster
Strain:
other: Golden
Details on test animals or test system and environmental conditions:
Female pregnant Golden Hamsters
Route of administration:
intraperitoneal
Vehicle:
physiological saline
Details on exposure:
A single i.p. dose of acrylonitrile was administered to pregnant hamsters on gestational day 8 at 80 mg/kg acrylonitrile alone, or at 80, 100 or 120 mg/kg with concurrent administration of 1 g/kg of sodium thiosulfate.
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
No information available
Duration of treatment / exposure:
Single dose
Frequency of treatment:
Once
Duration of test:
single i.p. administration on gestational Day 8; dams killed on Day 14.
Remarks:
Doses / Concentrations:
80, 100 or 120 mg/kg
Basis:
other: actual dose injected
No. of animals per sex per dose:
Not reported
Control animals:
yes, concurrent vehicle
Details on study design:
No further information.
Maternal examinations:
Observation for clinical signs
Ovaries and uterine content:
Numbers of live foetuses, implantation sites and resorptions were recorded
Fetal examinations:
Foetuses were examined macroscopically and after fixation for evidence of malformations.
Statistics:
No information available
Indices:
No information available
Historical control data:
No information available
Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: : at 80 and 120 mg/kg bw

Details on maternal toxic effects:
No signs of toxicity were seen in dams administered up to 65 mg/kg bw acrylonitrile. Animals receiving 80 mg/kg bw showed dyspnoea, gasping, loss of co-ordination, hypothermia, salivation, and convulsions 1-5 hours after the injection, while those administered 120 mg/kg bw all died.
Dose descriptor:
NOAEL
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
The dose of 80 mg/kg bw resulted in encephalocoele (7/51 foetuses), rib fusions and bifurcations in many of the offspring. The percentage of abnormal foetuses was 15.7%, compared with 0.8% in controls.
Abnormalities:
not specified
Developmental effects observed:
not specified

Administration of sodium thiosulphate prevented overt signs of maternal toxicity but developmental effects were still seen in the offspring, indicating that the effects of acrylonitrile seen in this study may be due to the metabolic release of cyanide.

Conclusions:
Developmental toxicity was seen at a dose level causing maternal toxicity. Some evidence of a protective effect of sodium thiosulphate was seen in this study.
Executive summary:

The authors administered doses of 4.8, 10, 25, 65, 80 or 120 mg/kg bw of acrylonitrile in saline via intraperitoneal injection to pregnant golden hamsters on Day 8 of gestation. Separate groups of animals received intraperitoneal injections of 1000 mg/kg bw sodium thiosulphate 20 minutes before and 80 minutes after administration of acrylonitrile. Dams were killed on Day 14 of gestation and the numbers of live foetuses, implantation sites and resorptions were recorded. Foetuses were examined macroscopically and after fixation for evidence of malformations.

No signs of toxicity were seen in dams administered up to 65 mg/kg bw acrylonitrile. Animals receiving 80 mg/kg bw showed dyspnoea, gasping, loss of co-ordination, hypothermia, salivation, and convulsions 1-5 hours after the injection, while those administered 120 mg/kg bw all died. The dose of 80 mg/kg bw resulted in encephalocoele (7/51 foetuses), rib fusions and bifurcations in many of the offspring. Administration of sodium thiosulphate prevented overt signs of maternal toxicity but developmental effects were still seen in the offspring, indicating that the effects of acrylonitrile seen in this study may be due to the metabolic release of cyanide. Overall the study suggests that acrylonitrile may have developmental effects in the hamster, but only at dose levels which are maternally toxic.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Published non-standard study; relevance of findings unclear, dose levels are very low
Qualifier:
no guideline followed
Principles of method if other than guideline:
Investigation of the developmental toxicity of acrylonitrile in rats with a focus on morphological and early behavioural development
GLP compliance:
yes
Remarks:
: non-standard published literature study
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
Rat strain reported as Charles-Wistar
Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on exposure:
Pregnant rats were administered acrylonitrile by gavage from gestational days 5 to 21.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No information available.
Details on mating procedure:
No information available.
Duration of treatment / exposure:
Pregnant rats were administered acrylonitrile by gavage from gestational days 5 to 21.
Frequency of treatment:
Daily, from gestational days 5 to 21
Duration of test:
17 days
Remarks:
Doses / Concentrations:
5 mg/kg bw/d
Basis:
actual ingested
gavage
No. of animals per sex per dose:
30 females
Control animals:
yes, concurrent vehicle
Details on study design:
No further information.
Maternal examinations:
Body weights, food and water consumption, clinical examinations, length of gestation.
Ovaries and uterine content:
Numbers and positions of implantation sites, live, dead and resorbed foetuses.
Fetal examinations:
At parturition litters were culled to 8, with equal numbers of males and females where possible. Pups were evaluated post-partum for morphological development and functional teratology using a screening protocol suggested by Vorhees (1979) including timing of developmental landmarks, reflex development, grip strength, open field motor activity and a passive avoidance test. On day 21 post-partum the pups were killed and a range of neurochemical analyses were carried out on the brain.
Statistics:
No information available
Indices:
Not investigated
Historical control data:
No information available
Details on maternal toxic effects:
Maternal toxic effects:no effects. Remark: : none observed

Details on maternal toxic effects:
There were no effects on maternal body weight, length of gestation or numbers of offspring delivered.
Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: : biochemical alterations

Details on embryotoxic / teratogenic effects:
There were no treatment-related effects in pups on timing of developmental landmarks, reflex development, grip strength, open field motor activity or on a passive avoidance test of learning and memory. There was no effect on sex within litters and in pup weight at parturition and post partum. There were slight, but statistically significant alterations in brain levels of noradrenaline, 5-hydroxytryptamine and monoamine oxidase, which the authors suggested could be indicative of a derangement in synaptic transmission.
Abnormalities:
not specified
Developmental effects observed:
not specified

No effects were seen on maternal body weight, offspring weight, length of gestation, number of offspring delivered, sex within the litters, the onset of pinna detachment, eye opening, incisor eruption, or fur appearance. Pups had no abnormalities in the development of righting reflex, cliff avoidance, grip strength, spontaneous motor activity or learning ability. Two weeks post partum, 5-hydroxytryptamine was decreased in the corpus striatum and hypothalamus and increased in the pons/medulla while noradrenaline concentrations were slightly increased in the hippocampus and significantly reduced in the pons/medulla. Monoamine-oxidase levels were decreased in the 3 week old treated pups compared to controls. Also at 3 weeks there were no significant differences in acetylcholinesterase concentrations or sodium/potassium ATPase activity between treated and control pups.

Conclusions:
No evidence of maternal toxicity was seen in this study at the only dose level investigated (5 mg/kg bw/d). Some changes in neurotransmitter levels were seen in pups at 3 weeks of age, however, the biological significance of the changes, in the absence of any evidence of neurotoxicity, is unknown.
Executive summary:

This study investigated the biochemical and developmental effects of acrylonitrile in rats exposed in utero.

 

Pregnant Wistar rats were gavaged with 0 or 5mg/kg bw/d acrylonitrile on gestation Days 5-21. Treatment with acrylonitrile had no effect on maternal of pup bodyweight, gestation length, litter size, sex, the onset of pinna detachment, eye opening, incisor eruption or fur appearance. Pups had no abnormalities in the development of righting reflex, cliff avoidance, grip strength, spontaneous motor activity or learning ability. Two weeks post partum, biochemical investigation showed reduced 5-hydroxytryptamine in the corpus striatum and hypothalamus, while levels were increased in the pons/medulla. Noradrenaline concentrations were slightly increased in the hippocampus and significantly reduced in the pons/medulla. MAO levels were decreased in treated pups compared to controls. At 3 weeks there were no significant differences in acetylcholinesterase concentrations or sodium/potassium ATPase activity between treated and control pups.  The authors concluded that although exposure to low levels of acrylonitrile do not effect functional development, there are changes in biogenic amines that may lead to future neurological effects.

This study provides some information exposure to a relatively low level of acrylonitrile during gestation is unlikely to result in overt behavioural deficits in the offspring of exposed dams. The study is limited by the single dose tested, and by the lack of maternal toxicity at the dose tested.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Published study; very limited investigation performed as part of an in vitro study
Qualifier:
no guideline followed
Principles of method if other than guideline:
The effects of the compound on embryonic development were investigated as part of an in vitro study
GLP compliance:
no
Remarks:
: non-standard published study
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
Four pregnant Sprague-Dawley rats
Route of administration:
oral: gavage
Details on exposure:
Four pregnant rats were given a single oral dose of acrylonitrile by gavage at 100 mg/kg, on gestational day 10.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No information available
Details on mating procedure:
No information available
Duration of treatment / exposure:
Single dose administered on gestational day 10
Frequency of treatment:
Single dose
Duration of test:
Embryos evaluated on gestational day 12.
Remarks:
Doses / Concentrations:
100 mg/kg bw
Basis:
actual ingested
gavage dose
No. of animals per sex per dose:
Four pregnant rats, the number of embryos per litter was not reported
Control animals:
no
Details on study design:
No further information; limited investigation as part of an in vitro study
Maternal examinations:
Clinical observations and body weights
Ovaries and uterine content:
None
Fetal examinations:
Embryos were evaluated for abnormalities on gestational day 12.
Statistics:
No information available
Indices:
No information available
Historical control data:
No information available
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Maternal toxicity was evidenced by decreased body weight, tremors, piloerection and prostration.
Dose descriptor:
NOAEL
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Embryos showed misdirected allantois or caudal extremities in 3 of the 4 litters evaluated.
Abnormalities:
not specified
Developmental effects observed:
not specified

The study also had an in vitro phase in which effects were investigated in a rat limb bud micromass assay.

Conclusions:
Maternal toxicity was evident following a single oral dose of 100 mg/kg bw acrylonitrile, and 3 out of 4 litters evaluated showed abnormalities.
Executive summary:

Four pregnant Sprague-Dawley rats were exposed to a single high gavage dose of acrylonitrile (100 mg/kg bw) on gestational day 10. Embryos were evaluated on gestational day 12. Maternal toxicity was evidenced by decreased body weight, tremors, piloerection and prostration. Embryos showed misdirected allantois or caudal extremities in 3 of the 4 litters evaluated. The study is of unconventional design, and only a small number of litters were evaluated. However, the results suggest that single high-dose exposures to acrylonitrile may prove developmentally toxic. The embryotoxic effects were seen in the presence of severe maternal toxicity; lower doses were not evaluated in this study.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Published study: non-standard in vitro method
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
Pregnant rats were exposed by inhalation to evaluate developmental toxicity as part of a comparative evaluation of aliphatic mononitriles.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
Female pregnant Sprague-Dawley rats
Route of administration:
inhalation
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
Rats were exposed to 0, 12, 25, 50 or 100 ppm acrylonitrile by inhalation for 6 hours/day on gestation days 6 -15.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentrations were determined analytically by hourly sampling and gas-liquid chromatography.
Details on mating procedure:
No information available
Duration of treatment / exposure:
6 hours/day
Frequency of treatment:
Daily (gestation Day 6-15)
Duration of test:
10 days
Remarks:
Doses / Concentrations:
0, 12, 25, 50 or 100 ppm
Basis:
nominal conc.
No. of animals per sex per dose:
20-23
Control animals:
yes
Details on study design:
The rats were exposed to the test article in 200 liter stainless steel chambers (23 C, 50% rel. humidity) with dynamic and adjustable laminar air flow (10-20 m3/hr). Vapour was generated by bubbling air through a flask containing acrylonitrile, the concentration in the chamber being calculated from the ratio of the amount of acrylonitrile vaporised to the total chamber air flow during the test period.
Maternal examinations:
Clinical signs, body weight, feed consumption, gross necropsy.
Ovaries and uterine content:
Average number of implantations, live foetuses, non-surviving implants, resorptions.
Fetal examinations:
Sex and body weight, external abnormalities, skeletal and soft-tissue anomalies.
Statistics:
No information available
Indices:
Pregnancy rate, foetal sex ratio
Historical control data:
No information available
Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: Reduced body weight gain was evident in the three highest dose groups in a concentration-dependent manner.

Details on maternal toxic effects:
Reduced body weight gain was evident in the three highest dose groups in a concentration-dependent manner.
Dose descriptor:
NOAEC
Effect level:
12 ppm
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEC
Effect level:
12 ppm
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
LOAEC
Effect level:
25 ppm
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEC
Effect level:
25 ppm
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: : reduced weight

Details on embryotoxic / teratogenic effects:
A concentration related reduction in foetal weights was observed with statistically significant differences in the 25 ppm and higher exposure concentrations. One foetus from a 25 ppm litter had a missing thoracic centre, but there was no other evidence of major malformation in any acrylonitrile-exposed litter.
Abnormalities:
not specified
Developmental effects observed:
not specified

There were no maternal deaths, but a concentration-dependent decreased absolute body weight gain was observed (significant at p<0.01 at three highest dose groups; 25.1 g, 16.1 g, -0.1 g, -7.8 g, and -24.3 g, respectively for the 0, 12, 25, 50, and 100 ppm groups). There was no adverse effect on pregnancy rate, average number of implantations or number of live fetuses, incidences of non-surviving implants and resorptions, or fetal sex ratio. A statistically significant (p<0.01 to 0.005) exposure-related reduction in fetal weights was observed at 25 ppm and higher concentrations. Evaluation of external, visceral and skeletal variations in the foetuses revealed no treatment-related effects.

Conclusions:
The NOAEL for maternal and developmental toxicity in this study was 12 ppm, based on bodywieght effects
Executive summary:

Pregnant Sprague-Dawley rats were exposed to 0, 12, 25, 50 or 100 ppm acrylonitrile by inhalation for 6 hours/day on gestation Day 6 -20 to evaluate developmental toxicity.

Maternal toxicity was evident at doses of 25 ppm and higher as reduced body weight gain. Acrylonitrile exposure did not result in any adverse effects on pregnancy rate, average number of implantations and live foetuses, incidences of non-surviving implants and resorptions, or foetal sex ratio. A concentration-related reduction in foetal weight was observed with statistically significant differences in the 25 ppm and higher exposure concentrations. No adverse effects due to acrylonitrile exposure were observed in the evaluations of external, visceral and skeletal variations in the foetuses.

The NOAEL for maternal and developmental toxicity in this study is 12 ppm.

Endpoint:
developmental toxicity
Type of information:
other: review
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Published review of the available animal data on the developmental toxicity of acrylonitrile.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Critical review of the available (proprietary and published) studies of the developmental toxicity of acrylonitrile.
GLP compliance:
no
Remarks:
: some individual studies are GLP compliant
Limit test:
no
Species:
other: rat, hamster
Strain:
other: various
Route of administration:
other: gavage, inhalation, intraperitoneal
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on mating procedure:
The reviewed studies used different methodology
Duration of treatment / exposure:
The reviewed studies used different methodology
Frequency of treatment:
The reviewed studies used different methodology
Duration of test:
The reviewed studies used different methodology
Remarks:
Doses / Concentrations:
0, 10, 25, or 65 mg/kg/day
Basis:
actual ingested
Rat gavage developmental toxicity (Toxicological Research Laboratory, 1976; Murray et al., 1978)
Remarks:
Doses / Concentrations:
0, 40, or 80 ppm
Basis:
other: inhalation: Rat inhalation developmental toxicity (Toxicological Research Laboratory, 1978; Murray et al., 1978)
Remarks:
Doses / Concentrations:
0, 12, 25, 50, or 100 ppm
Basis:
other: inhalation: Rat inhalation developmental toxicity (Saillenfait et al., 1993a)
Remarks:
Doses / Concentrations:
100 mg/kg bw
Basis:
actual ingested
Rat gavage developmental toxicity (Saillenfait and Sabate, 2000)
Remarks:
Doses / Concentrations:
80, 100, or 120 mg/kg
Basis:
other: ip injection: Hamster intraperitoneal developmental toxicity (Willhite et al., 1981a, 1981b)
No. of animals per sex per dose:
The reviewed studies used different methodology
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOAEL
Effect level:
< 40 ppm
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
40 ppm
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOAEL
Effect level:
12 ppm
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
12 ppm
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOAEL
Effect level:
< 100 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
< 100 mg/kg bw/day
Basis for effect level:
other: developmental toxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Rat gavage developmental toxicity (Toxicological Research Laboratory, 1976; Murray et al, 1978)

Severe maternal toxicity at 65 mg/kg bw/d included lethality (in a single dam), hyperexcitability, salivation, decreased body weight gain, decreased feed consumption, increased water consumption, increased liver weight, and gastric wall thickening.  At 25 mg/kg/ bw/d there were decreased feed consumption and gastric wall thickening. There were no increases in number of dead fetuses per litter, resorptions, or changes in foetal sex ratio. Fetotoxicity included decreased fetal body weight and crown–rump length at 65 mg/kg bw/d. Developmental anomalies included statistically significantly increased incidence, on a fetal basis, of fetuses with missing or short tails or trunk, or missing vertebrae at 65 mg/kg bw/d. There were two instances of imperforate anus among the fetuses with tail malformations. Litter incidences were not evaluated statistically. There were two foetuses with short tails at 25 mg/kg bw/d (possibly treatment related based on dose response). A single control fetus also had a short tail.  Findings are linked to skeletal variations/malformation. There was a low incidence of fetuses with right-sided aortic arch and anteriorly displaced ovaries at 25 and 65 mg/kg bw/d.  Statistically significant increases in skeletal alterations were observed at 65 mg/kg/day, including one or more missing vertebrae, ribs, and delayed ossification.

Rat inhalation developmental toxicity (Toxicological Research Laboratory, 1978; Murray et al, 1978)

No treatment-related clinical signs were observed during the exposure period. Mean body weight and maternal body weight gain were significantly decreased during specific intervals of gestation in both dose groups. Feed consumption was inconsistently affected; water consumption was statistically significant increased on gestation days 9–20. Maternal liver weight was unaffected by exposure. Pregnancy incidence, mean litter size, incidence of resorptions, and average fetal body measurements (body weight and crown–rump length) were not affected. Total malformations were increased at 80 ppm (not statistically significant). Types of malformations at 80 ppm included short tail, missing vertebrae, short trunk, and omphalocele. These high-dose findings are considered possibly exposure related, even though the incidence of specific types of malformations was not statistically significantly increased, because of similarities of several of these findings to the dose-related malformations in the gavage study by the same investigators (Toxicological Research Laboratory, 1976; Murray et al, 1978).

 

Rat inhalation developmental toxicity (Saillenfait et al, 1993a)

Decreased maternal body weight gain was seen at ≥25 ppm in a concentration-dependent manner. There were no effects on pregnancy rate, implantations, live fetuses, non-surviving implants and resorptions, or fetal sex ratio. There was a concentration-related reduction in fetal weights, statistically significant at ≥25 ppm. There were no fetal malformations attributable to exposure.

 

Rat gavage developmental toxicity (Saillenfait & Sabate, 2000)

Maternal toxicity included decreased body weight, tremors, piloerection and prostration. Embryos were reported to show 'misdirected allantois and/or trunk and caudal extremity' in three of the four litters evaluated. This study is limited by the unconventional design, single dose level, and small number of litters evaluated. The developmental toxicity was seen in the presence of severe maternal toxicity. This study also had an in vitro phase that showed embryotoxic and teratogenic effects of acrylonitrile to cultured embryos.

 

Hamster intraperitoneal developmental toxicity (Willhite et al, 1981)

The incidence of encephalocele and possibly axial skeletal defects was increased by acrylonitrile alone, but not by acrylonitrile at the same dose level (80 mg/kg) in the presence of sodium thiosulfate (which protects against cyanide toxicity). No protective effect was evident at higher doses.

Rat postnatal developmental toxicity (Mehrotra et al, 1988)

No maternal toxicity was seen in this study. There were no effects on offspring weight, length of gestation, number of offspring delivered, development of developmental landmarks or reflexes, grip strength, open field motor activity, or on a passive avoidance test of learning and memory. Acetylcholinesterase was not affected. Slight but statistically significant differences (increases or decreases in different brain regions) were found for 5-hydroxytryptamine and noradrenaline concentrations. Monamine oxidase levels were significantly decreased compared to control.

Conclusions:
Acrylonitrile has the potential to cause developmental toxicity and teratogenicity at high exposure levels. There was no evidence of developmental toxicity in any study in the absence of maternal toxicity.
Executive summary:

Evaluation of developmental toxicity and malformations in eight animal studies leads to the conclusion that very high (maternally toxic) exposure to acrylonitrile results in foetotoxicity, and may result in teratogenicity. Teratogenicity appears to be most likely following oral gavage exposure, which is not a relevant route of exposure to humans. The studies considered to be of highest quality do not show clear evidence of teratogenicity. The Saillenfait et al (1993) rat inhalation developmental toxicity study (which tested to the highest inhalation concentration) showed decreased foetal body weights at a maternally toxic dose, but no exposure-related malformations. The Nemec et a l(2008) rat inhalation reproductive toxicity study showed only a single high-dose malformation. Although it is not a study design ideal to detect low level teratogenicity, it is howeverconsidered at most equivocally related to treatment. It is notable that there was no evidence of developmental toxicity in any study in the absence of maternal toxicity.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
25 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
A guideline-compliant study in the rat is supported by non-standard investigative studies
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
40 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
Proprietary study, guideline comparable and well reported
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Rat inhalation developmental toxicity study (Murray et al., 1978)

In this study, groups of 30 pregnant Sprague-Dawley rats were exposed to 0, 40, or 80 ppm of acrylonitrile on 6 hours/day by inhalation on Days 6 -15 of gestation to investigate the effect of maternally inhaled acrylonitrile on embryonic and foetal development. Mean bodyweights and maternal bodyweight gain was significantly decreased during specific intervals of gestation in both dose exposure groups. Food consumption was decreased during gestation Days 15-17 and increased on Days 18-20 compared to controls. Statistically significant increased water consumption was observed on gestation Days 9-20. Pregnancy incidence, mean litter size, the incidence of resorptions and average foetal body measurements were not affected by exposure at either concentration. Total malformations were significantly increased at 80 ppm. These findings were similar to the findings in the gavage study by Murray et al, 1976, it was therefore considered likely by the authors that the malformations seen at 80 ppm were exposure-related, although the incidence of specific types of malformations was not statistically increased compared to controls. The NOAEL for maternal and developmental toxicity in this study is 40 ppm.

 

Rat inhalation developmental toxicity study (Saillenfait et al., 1993)

In this study, pregnant Sprague-Dawley rats were exposed to 0, 12, 25, 50 or 100 ppm acrylonitrile by inhalation for 6 hours/day on gestation Day 6 -20 to evaluate developmental toxicity. Maternal toxicity was evident at doses of 25 ppm and higher as reduced body weight gain. Acrylonitrile exposure did not result in any adverse effects on pregnancy rate, average number of implantations and live foetuses, incidences of non-surviving implants and resorptions, or foetal sex ratio. A concentration-related reduction in foetal weight was observed with statistically significant differences in the 25 ppm and higher exposure concentrations. No adverse effects due to acrylonitrile exposure were observed in the evaluations of external, visceral and skeletal variations in the foetuses. The NOAEL for maternal and developmental toxicity in this study is 12 ppm (26 mg/m3).

 

Rat gavage developmental toxicity study (Murray et al., 1976)

Mated female Sprague-Dawley rats (29 -39/group) were gavaged with acrylonitrile (in water) at dose levels of 0, 10, 25 or 65 mg/kg bw/d on Days 6 -15 of gestation. Dams were observed for clinical signs, bodyweight, food and water consumption. Dams were killed on Day 21 and the uterine contents examined. All foetuses were examined for external abnormalities, one third for visceral abnormalities and two thirds for skeletal findings. Mortality, signs of toxicity (hyperactivity and salivation), reduced weight and food consumption and increased water consumption were noted in the high dose group. Necropsy revealed gastric irritation in most animals at 65 mg/kg bw/d and a small number at 25 mg/kg bw/d. Post-implantation loss was increased at the high dose level; reduced foetal weight and length was also apparent in this group. No effects were seen in the other treated groups. A higher incidence of short tail was seen in foetuses at 65 mg/kg bw/d, with other abnormalities and skeletal variations also increased in this group. No effects were seen in the lower dose groups. Some evidence of developmental toxicity was seen in this study at the highest (and maternally toxic) dose level of 65 mg/kg bw/d. The NOAEL for maternal toxicity is therefore 10 mg/kg bw/d based on the signs of gastric irritation at 25 mg/kg bw/d; the NOAEL for developmental toxicity is 25 mg/kg bw/d.

 

Rat gavage developmental toxicity study (Mehrotra et al., 1988)

This study specifically investigated the biochemical and developmental effects of acrylonitrile in rats exposed in utero. Pregnant Wistar rats were gavaged with 0 or 5mg/kg bw/d acrylonitrile on gestation Days 5-21. Treatment with acrylonitrile had no effect on maternal of pup bodyweight, gestation length, litter size, sex, the onset of pinna detachment, eye opening, incisor eruption or fur appearance. Pups had no abnormalities in the development of righting reflex, cliff avoidance, grip strength, spontaneous motor activity or learning ability. Two weeks post partum, biochemical investigation showed reduced 5-hydroxytryptamine in the corpus striatum and hypothalamus, while levels were increased in the pons/medulla. Noradrenaline concentrations were slightly increased in the hippocampus and significantly reduced in the pons/medulla. MAO levels were decreased in treated pups compared to controls. At 3 weeks there were no significant differences in acetylcholinesterase concentrations or sodium/potassium ATPase activity between treated and control pups.  The authors concluded that although exposure to low levels of acrylonitrile do not effect functional development, there are changes in biogenic amines that may lead to future neurological effects. This study provides some information exposure to a relatively low level of acrylonitrile during gestation is unlikely to result in overt behavioural deficits in the offspring of exposed dams. The study is limited by the single dose tested, and by the lack of maternal toxicity at the dose tested.

 

Rat gavage study (Saillenfait & Sabate, 2000)

This study was performed as part of an in vitro investigation of the developmental toxicity of various nitriles and is of limited value. Four pregnant Sprague-Dawley rats were exposed to a single high gavage dose of acrylonitrile (100 mg/kg bw) on Gestation Day 10. Embryos were evaluated on Gestation Day 12. Maternal toxicity was evidenced by decreased body weight, tremors, piloerection and prostration. Embryos showed misdirected allantois or caudal extremities in 3 of the 4 litters evaluated. The study is of unconventional design, and only a small number of litters were evaluated. However, the results suggest that single high-dose exposures to acrylonitrile may prove developmentally toxic. The embryotoxic effects were seen in the presence of severe maternal toxicity; lower doses were not evaluated in this study.

 

Hamster intraperitoneal study (Willhite et al., 1981)

This study is considered to be of limited value, given the non-standard exposure route and deficiencies in design and reporting. The authors administered doses of 4.8, 10, 25, 65, 80 or 120 mg/kg bw of acrylonitrile in saline via intraperitoneal injection to pregnant golden hamsters on Day 8 of gestation. Separate groups of animals received intraperitoneal injections of 1000 mg/kg bw sodium thiosulphate 20 minutes before and 80 minutes after administration of acrylonitrile. Dams were killed on Day 14 of gestation and the numbers of live foetuses, implantation sites and resorptions were recorded. Foetuses were examined macroscopically and after fixation for evidence of malformations. No signs of toxicity were seen in dams administered up to 65 mg/kg bw acrylonitrile. Animals receiving 80 mg/kg bw showed dyspnoea, gasping, incoordination, hypothermia, salivation, and convulsions 1-5 hours after the injection, while those administered 120 mg/kg bw all died. The dose of 80 mg/kg bw resulted in encephalocoele (7/51 foetuses), rib fusions and bifurcations in many of the offspring. Administration of sodium thiosulphate prevented overt signs of maternal toxicity but developmental effects were still seen in the offspring, indicating that the effects of acrylonitrile seen in this study may be due to the metabolic release of cyanide. Overall the study suggests that acrylonitrile may have developmental effects in the hamster, but only at dose levels which are maternally toxic.

 

Weight of evidence review (Neal et al., 2009)

The authors of this review consider the available data from animal developmental toxicity studies and relevant findings from the reproductive toxicity studies and discuss the potential of acrylonitrile to cause reproductive toxicity in exposed humans.

 

Overview

The Toxicological Research Laboratory (1976) gavage developmental toxicity study and Toxicological Research Laboratory (1978) inhalation developmental toxicity study (both published by Murray et al., 1978) and the Saillenfait et al. (1993) inhalation developmental toxicity study in rats are considered to be complete, reasonably well-conducted and well-reported evaluations, although these evaluations were not performed in compliance with GLP. It is also noted that the Murray gavage study was potentially compromised by SDA infection. The Murray gavage study and the Saillenfait inhalation study both show developmental toxicity (decreased foetal body weight) in the presence of maternal toxicity. The gavage study additionally shows exposure-related evidence of teratogenicity, whereas developmental toxicity in the Saillenfait inhalation study was limited to effects on foetal bodyweight. Evidence of possible concordant teratogenic effects was also seen at the high dose in the Murray inhalation study; however, the findings in this study were not statistically significantly increased compared to control and foetal body weights were not affected. The developmental findings in the Murray inhalation study are attributed to acrylonitrile exposure primarily because of the similarity of the nature of the findings to those in the gavage study by the same workers. In contrast to the results in these gavage and inhalation studies, there were no teratogenic effects in the Saillenfait inhalation developmental toxicity study, although the high concentration administered was greater (100 ppm versus 80 ppm) and duration of exposure longer, and maternal toxicity was evident in both inhalation studies. There was no increase in post-implantation loss in the Saillenfait study, so a treatment-related increase in malformations was not obscured by post-implantation loss

 

Significance of tail malformations

Tailless or short-tailed foetuses, as reported in the Murray study, are not common findings in available developmental toxicity study historical control data. Mean foetal control incidence of agenesis of caudal vertebrae was reported from 222 developmental toxicity studies with Sprague-Dawley rats to be 0.014%, with a maximum incidence of 0.58% (Hood, 1996). Charles River control data for CD rats show external tail malformations in an average of 0.05% fetuses and 0.67% litters. Higher foetal incidences were reported by Toxicological Research Laboratory (1976 and 1978) at the high dose in both the gavage and inhalation developmental toxicity studies. No historical control data are available from that laboratory for the timeframe in which those studies were conducted. A single control group fetus from the Toxicology Research Laboratory (1976) gavage study was 'acaudate or short tail' for a foetal incidence of 0.2% and litter incidence of 3%. Acrylonitrile administered intraperitoneally to hamsters may be associated with encephalocele and possibly axial skeletal defects, based on the study by Willhite. This study is not considered particularly reliable, because of low numbers of animals tested and the extensive range of background developmental defects typically seen with Syrian golden hamsters. Willhite speculates that cyanide mediates the developmental toxicity of acrylonitrile (and other aliphatic nitriles), and cites an earlier study of sodium cyanide in hamsters using infusion pump administration, which was reported to induce a wide spectrum of developmental malformations including axial skeletal disorders, ectopic hearts, hydropericardium, and tail malformations.

 

Mechanistic considerations

Based on the studies by Willhite, Saillenfait & Sabate (2000) tested the hypothesis that cyanide contributed to or caused acrylonitrile-related malformation. They reported similar findings for acrylonitrile and cyanide based on extremely high single-dose gavage studies in rats on GD 10 and on in vitro embryoculture evaluations. The findings in these studies were reported only as 'misdirected allantois and/or trunk and caudal extremity. Embryos were examined in the in vivo study by Saillenfait & Sabate on GD 12. Both the immature state of the embryos and the lack of descriptive details on the nature of the findings make it difficult to evaluate the conclusion that the findings between cyanide and acrylonitrile are similar. Importantly, these findings cannot be reliably compared to the more standard developmental evaluations conducted by Saillenfait (1993), or those by Murray et al. In contrast to the findings of Saillenfait & Sabate (2000), neither a gavage developmental study of acetone cyanohydrin nor a drinking-water developmental study of potassium cyanide showed developmental malformations in rats at dose levels toxic to the dams (ECETOC, 2007). Thus, the pattern of developmental findings in rats is not consistent between cyanide and acrylonitrile. For both chemicals, it is clear that developmental toxicity in rats has not been seen in the absence of maternal toxicity. There is biological plausibility to an association of developmental effects from aliphatic nitriles with cyanide-mediated toxicity, with a possible mechanism of glutathione depletion as noted by Saillenfait et al. (1993b). There is also a possibility, however, that acrylonitrile or a reactive epoxide metabolite may be involved, acting either through glutathione depletion or reactivity with other proteins.

 

Relevant findings from reproductive toxicity studies

No clearly exposure-related developmental malformations were observed in the reproductive toxicity studies with acrylonitrile and, based on litter size, there was no evidence of increased post-implantation loss or resorptions that might have obscured such a finding. Developmental malformations, however, may not be fully evaluated in reproductive toxicity studies, because of potential cannibalism prior to observation of the litters or the lack of comprehensive internal pup evaluations. Pups with tail malformations would be likely to be observed if they were present at the first clinical observation interval (generally on the day of delivery, or on the day following delivery). The one-generation reproductive toxicity study reports findings in pups at a low incidence lacking dose response (one litter at all three dose levels, foetal incidence not described). These findings were not characterised by histopathological examination of the affected pups, so it is unknown if these observations were due to developmental malformation or to postnatal amputation. No historical control data were available to assist with interpretation of this finding, but the low incidence and absence of dose response argue against a treatment relationship, as does the short-tailed foetus observed in the control group in the almost contemporaneous (1976) gavage developmental toxicity study conducted by the same laboratory. One tailless pup was observed at the high dose level in the 1980 drinking-water three-generation reproductive toxicity study reported by Freidman & Beliles (2002); this observation was attributed to an amputation and is not to be considered treatment related. The high concentration of acrylonitrile in drinking water in this study was the same as that tested in the drinking-water one-generation reproductive toxicity study. A single F1 pup with a 'threadlike' tail and absence of caudal vertebrae on histopathological evaluation was reported in the 2006 inhalation reproductive toxicity study at 90 ppm. This finding was observed in the high-dose group, and it was similar in nature to the most predominant finding in the Murray gavage developmental toxicity study. However, because there was only a single incidence, the relationship to treatment is considered to be weak and equivocal. Overall, the incidence of tailless pups in the reproductive toxicity studies is considered too low and sporadic to make a definitive ascription to treatment.

 

Relationship to maternal toxicity

The studies showing clearly compound-related teratogenic effects were primarily high-dose gavage studies, which only showed malformations in the presence of maternal toxicity, although the latter study was limited by severe maternal toxicity and the single dose tested. Gavage is not a particularly relevant route of exposure for human risk assessment, even though it is frequently used to predict oral toxicity, because the bolus dose regimen is dissimilar to anticipated human exposure patterns and may result in exaggerated toxicity. The Willhite hamster study also showed possible teratogenicity; this study was very limited, and the intraperitoneal route of exposure is clearly not relevant to humans. Foetotoxicity in the Saillenfait inhalation developmental toxicity study and the decreased pup weight gain in the 2006 inhalation reproductive toxicity study demonstrate that inhalation exposure to acrylonitrile may result in adverse fetal and pup outcomes at high and maternally toxic concentrations. The high-quality inhalation reproductive toxicity study is not considered to confirm a treatment relationship for malformations, and malformations were not observed in the moderately high quality inhalation developmental toxicity study by Saillenfait, which tested the highest inhalation concentration during the period of major organogenesis. The statistically not significant increased incidence of malformations in the Murray inhalation study is difficult to evaluate in the absence of historical control data, and in the face of the lack of similar findings in the Saillenfait study, which tested a higher concentration for a longer duration. No unique foetal susceptibility was identified in any of these studies, with effects seen only at high and overtly maternally toxic doses. The foetotoxicity (reduced foetal weights) seen in the Saillenfait inhalation developmental toxicity study and in the Murray gavage study is commonly seen as a consequence of maternal stress-related toxicity, and it may be due to maternal toxicity in these studies also. The Murray inhalation study did not show decreased foetal body weight, although the high exposure concentration in this study (80 ppm) was higher than the lowest dose (25 ppm, 6 h/day) that caused decreased fetal body weight in the Saillenfait inhalation developmental toxicity study. The malformations seen in the Murray gavage study, however, are not characteristic of foetal findings due to stress-induced teratogenicity. There was no apparent correlation between the affected litters and the degree of toxicity for individual dams. Maternal toxicity was most evident at the start of gavage dosing (GD 6–9), whereas the affected structures (posterior portion of the axial skeleton) would be forming from approximately GD 10–11 onward, which shows some temporal correspondence. The findings in this study show some similarity to findings in the in vitro embryotoxicity study (Saillenfait & Sabate, 2000), although the characterization of the in vitro findings in the Saillenfait & Sabate study is very limited. The very high dose gavage also reported by Saillenfait & Sabate (2000), which evaluated a single dose administered on GD10, also showed malformations in the presence of very severe maternal toxicity, showing a temporal correspondence between the occurrence of malformations and the dosing gestational interval. All of these factors support that there may have been direct developmental toxicity to the foetus in the Murray gavage study. It should be noted, however, that this study was compromised by concurrent SDA infection, which could have increased both maternal and foetal susceptibility. The Murray inhalation study showed a very slight response (not statistically significant); the difference in response from that seen in the gavage study may be due to differences in kinetics or may have been influenced by the concurrent infection present in the first study. The most contemporary of the developmental toxicity studies, Saillenfait (1993a), by the most relevant route of exposure (inhalation), did not show any evidence of exposure-related malformations, even though maternal and fetotoxicity were both evident, and a higher exposure was tested than in the Murray inhalation study.

 

Species tested

Although acrylonitrile has been assessed thoroughly in only one species for developmental toxicity, this assessment included several well-conducted studies with generally concordant NOAELs for maternal and developmental toxicity. One hamster study was conducted; this study is not considered adequate for evaluation. The rabbit is not likely to prove a suitable species for assessment of the developmental toxicity of acrylonitrile. Because of the gastric ulceration induced by acrylonitrile and the likely effects on the rabbit gastrointestinal flora from high concentrations of this reactive compound, exposure is likely to lead to stress-related abortion or significant nutrient deficiencies at maternally toxic dose levels, compromising assessment of developmental toxicity.

 

Investigation of post-natal effects

Data supporting the evaluation of potential post-natal developmental effects are limited. The one- and three-generation reproductive toxicity drinking-water studies show potential effects on pup body weight, which may have been contributed to by the extremely poor palatability to the dams potentially limiting milk production through compromising drinking-water intake. The three-generation reproductive toxicity drinking-water study also showed some evidence of an adverse high dose effect on pup survival; this finding was limited to one generation and is considered unlikely to be treatment related. The inhalation two-generation reproductive toxicity study showed effects on pup growth, and growth-related changes in development of sexual landmarks, but no effects on pup survival. Other than general clinical signs, endpoints other than survival, body weight, and, for the inhalation study, development of sexual landmarks were not evaluated for pups in these studies. The Mehrotra et al (1988) study provides some data suggesting that low-level exposure would not be expected to have adverse consequences on morphological or behavioral development; as discussed earlier, this study, conducted at a single relatively low dose level, is limited.

 

Conclusion

Overall, it is concluded that the animal studies indicate that very high exposure levels of acrylonitrile resulting in maternal toxicity result in foetal toxicity and, potentially, teratogenicity. Teratogenicity appears to be most likely following oral gavage exposure, which is not a relevant route of exposure for humans and the highest quality studies do not indicate clear evidence of teratogenicity.

Toxicity to reproduction: other studies

Description of key information

A number of non-standard studies are available which investigate the testicular toxicity of acrylonitrile in the rat and mouse.

Link to relevant study records

Referenceopen allclose all

Endpoint:
toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Unknown
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Published study of non-standard design
Qualifier:
no guideline followed
Principles of method if other than guideline:
Investigations of sperm count, selected enzyme measurements and testicular histopathology in mice following 60 days acrylonitrile exposure by gavage
GLP compliance:
no
Type of method:
in vivo
Specific details on test material used for the study:
No details
Species:
mouse
Strain:
CD-1
Sex:
male
Route of administration:
oral: gavage
Vehicle:
physiological saline
Details on exposure:
Male mice were dosed by gavage administration at 1 or 10 mg/kg bw/d for 60 days.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
Not reported
Duration of treatment / exposure:
60 days
Frequency of treatment:
Daily
Duration of test:
60 days
Dose / conc.:
0 mg/kg bw/day
Remarks:
Vehicle control
Dose / conc.:
1 mg/kg bw/day
Dose / conc.:
10 mg/kg bw/day
No. of animals per sex per dose:
10 (males)
Control animals:
yes, concurrent vehicle
Details on study design:
No further information available
Key result
Dose descriptor:
NOAEL
Effect level:
1 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
There were no overt signs of toxicity or changes in body weight. At 10 mg/kg bw/d the authors reported decreased sorbitol DH and acid phosphatase, increased LDH and B-glucuronidase (according to the study authors, these findings suggest degeneration of germinal epithelium). Decreased epidydimal sperm count was reported. Histopathological evaluations showed seminiferous tubule degeneration.
Leydig cells were not affected.

Testicular damage was observed in mice gavaged with 10 mg/kg bw/d acrylonitrile, consisting of tubular atrophy and degeneration in approximately 40% of seminiferous tubules, with cytolysis and nuclear pyknosis of spermatids, formation of multinucleate giant cells and interstitial oedema. These changes were accompanied by a decrease in testicular sorbitol dehydrogenase (22% decrease, p<0.05) and acid phosphatase (16% decrease p < 0.05) and an increase in lactate dehydrogenase (12% increase, p < 0.05) and B-glucuronidase (36.7% increase, p < 0.05). Glucose-6-phosphatase was unaffected. These changes were seen in the absence of overt signs of toxicity or any effect on body weight or testicular weight. No effects were seeen in mice gavaged with 1 mg/kg bw/d acrylonitrile.

Conclusions:
Gavage administration of acrylonitrile at 10 mg/kg bw/d caused testicular effects in the absence of overt toxicity; no effects were seen at a dose level of 1 mg/kg bw/d.
Executive summary:

Daily oral administration of acrylonitrile (10 mg/kg bw/d) to mice for a period of 60 days caused a significant decrease in the activity of testicular sorbitol dehydrogenase and acid phosphatase, and an increase in that of lactate dehydrogenase and beta-glucuronidase. Histopathological studies revealed degeneration of the seminiferous tubules. A decrease in the sperm counts of the epididymal spermatozoa was also observed in the animals of the acrylonitrile-exposed group. The authors suggest that acrylonitrile may affect male reproductive function by causing testicular injury. No effects were seen at a dose level of 1 mg/kg bw/d.

Endpoint:
toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Published investigative study; non-standard guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
Investigation of the testicular toxicity of acrylonitrile in rats in vivo and the protective effects of the anti-oxidant beta-carotene.
GLP compliance:
not specified
Type of method:
in vivo
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Breeding Unit of the Egyptian Organization for Biological and Vaccine production
- Age at study initiation: Not reported
- Weight at study initiation: 240 +/- 10 g
- Fasting period before study: Not reported
- Housing: housed in stainless steel cages after grouping in groups of five
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: One week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23-27
- Humidity (%): 50-60
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Rats were allocated into four groups of ten as follows:

Group I: (Control) pre-treated with corn oil (2 mL/kg bw) daily for 25 days; treatment continued with distilled water (2 mL/kg bw) daily for an additional 5 days.

Group II: (Acrylonitrile group) pre-treated with corn oil (2 mL/kg bw) daily for 25 days; treatment continued with acrylonitrile (30 mg/kg bw) in distilled water (2 mL/kg bw) daily for an additional 5 days.

Group III: (Beta-carotene group) pre-treated with beta-carotene (40 mg/kg bw) in corn oil (2 mL/kg bw) daily for 25 days; treatment continued with distilled water (2 mL/kg bw) for an additional 5 days.

Group IV: (Beta-carotene and acrylonitrile group) pre-treated with beta-carotene (40 mg/kg bw) for 25 days; treatment continued with acrylonitrile (30 mg/kg bw) for an additional 5 days.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
Not applicable
Duration of treatment / exposure:
5 days (acrylonitrile treatment)
Frequency of treatment:
Daily
Duration of test:
30 days
Remarks:
Doses / Concentrations:
30 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
10 males
Control animals:
yes, concurrent vehicle
Statistics:
Results were expressed as means (±SE). Multiple comparisons were done using one-way ANOVA followed by Tukey–Kramer as a post-ANOVA test. Statistical significance was accepted at P < 0.001, P < 0.01 and P <0.05.

Levels of circulating testosterone, androsterone, FSH and LH were significantly reduced in acrylonitrile-treated rats. Pre-treatment with beta-carotene was shown to reduce the effect of acrylonitrile on circulating hormone levels; treatment with beta-carotene alone slightly (but significantly) increased hormone levels

Serum hormone levels

Group

Treatment

Testosterone (ng/mL)

Androsterone (pg/mL)

FSH (mIU/mL)

LH (mIU/mL)

I

Control

7.05

49.41

10.64

8.96

II

Acrylonitrile

3.00**

30.41**

4.09**

4.51 **

III

β-carotene

7.63**

52.40**

12.61**

9.26**

IV

β-carotene + acrylonitrile

4.62**

41.61**

8.32**

7.48**

**significantly different to controls (p<0.001)

Treatment with acrylonitrile was shown to significantly reduce the levels of testicular glutathione and glutathione-S-transferase and significantly increase the level of malondialdehyde.

Group

Treatment

GSH (mmol/g)

GST (mmol/g)

MDA (mmol/g)

I

Control

3.93

5.05

17.22

II

Acrylonitrile

1.51**

2.46**

43.55**

III

β-carotene

4.30**

5.78**

18.13**

IV

β-carotene + acrylonitrile

3.28**

3.39**

30.91**

**significantly different to controls (p<0.001)

Conclusions:
The results of this study indicate that repeated administration of a high dose of acrylonitrile produced significant reductions of GSH and GST levels, increased malondialdehyde levels, reduced hormone levels and testicular toxicity. Pre-treatment with beta-carotene results in a degree of protection against the effects of acrylonitrile. Findings indicate an association between oxidative damage and testicular toxicity.
Executive summary:

This study was designed to examine the effects of pre- and co-treatment with the anti-oxidant beta-carotene on the testicular toxicity of acrylonitrile in adult male rats. Groups of 10 rats were administered acrylonitrile by gavage at a dose level of 30 mg/kg bw for five days. Treatment with acrylonitrile significantly reduced the levels of serum testosterone, androsterone, follicle-stimulating hormone and luteinising hormone, indicating testicular toxicity. Significantly decreased serum and testicular glutathione content and glutathione-S-transferase were also seen in acrylonitrile-treated rats, and were accompanied by significantly elevated malondialdehyde levels, indicating lipid peroxidation. Acrylonitrile treatment also resulted in marked microscopic findings in the seminiferous tubules including germ cell depletion, tubular atrophy, maturation arrest, complete necrosis and multinucleated giant cell formation. Expansion of intertubular spaces and interstitial haemorrhage were also observed. Ultrastructural examination of the seminiferous tubules by TEM revealed thickened boundary tissue, pyknosis of Sertoli cell nuclei, damaged mitochondria and smooth endoplasmic reticulum-derived vacuoles. Spermatogenic cells also demonstrated altered cytoplasmic organelles, vacuoles of varying sizes and deformed spermatids. Mitochondrial disruption and a decrease in the amount of smooth endoplasmic reticulum were observed in Leydig cells. Pre-treatment with beta-carotene and its co-administration with acrylonitrile daily at a dose level of 40 mg/kg bw showed a remarkable degree of protection as evidenced by less marked changes in hormone levels, serum and testicular glutathione, GST and malondialdehyde levels and considerably less marked changes in testicular morphology and cell structure. The results of the study therefore indicate that the repeated administration of high dose levels of acrylonitrile (30 mg/kg bw compared to a reported oral LD50 of 81 mg/kg bw) causes testicular toxicity in the rat associated with oxidative stress.

Endpoint:
toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Qualifier:
no guideline followed
Principles of method if other than guideline:
Two studies are reviewed in the EU RAR that investigate effects on testes and sperm parameters in the rat following acrylonitrile gavage exposure.
GLP compliance:
no
Type of method:
in vivo
Specific details on test material used for the study:
Not reported
Species:
rat
Strain:
not specified
Sex:
male
Details on test animals or test system and environmental conditions:
No further information
Route of administration:
oral: gavage
Vehicle:
physiological saline
Details on exposure:
Acrylonitrile was administered to rats orally by gavage in saline
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
2 and 4 weeks
Frequency of treatment:
Daily
Duration of test:
2-4 weeks
Dose / conc.:
11.5 mg/kg bw/day
Dose / conc.:
23 mg/kg bw/day
Dose / conc.:
46 mg/kg bw/day
Dose / conc.:
0 mg/kg bw/day
Remarks:
Control group
No. of animals per sex per dose:
Not reported
Control animals:
yes
Details on study design:
Male rats were gavaged with acrylonitrile for 2 or 4 weeks. Testicular weight, sperm count and motility were reported. Measurement of LDH-X and flow cytometry of testicular aspirates also investigated.
Key result
Dose descriptor:
LOAEL
Effect level:
11.5 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Effects on bodyweight, organ weights, sperm count and sperm motility were seen at this dose level
Dose descriptor:
NOAEL
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested
There was a a dose-dependent decrease in body weight gain and in testicular weight. Decreases in testicular weight were paralleled by decreases in weight of the cauda epididymis and caput epididymis, however there was no significant effect on the weights of ventral prostate or seminal vesicles. Histopathological examination of testes indicated that spermatogenesis was affected after 4 weeks treatment with 23 or 46 mg/kg bw acrylonitrile, as evidenced by a decreased number of spermatocytes and spermatids. Sperm count and sperm motility were significantly decreased at all dose levels, and testicular LDH-X, a marker of pachytene spermatocytes, was inhibited at dose levels of 23 and 46 mg/kg bw. Flow cytometric analysis of testicular aspirates from rats treated with 46 mg/kg bw showed a reduction in the proportion of haploid cells (22% reduction after 4 weeks) and tetraploid cells (65% reduction), while diploid cells were increased (83%).

The results of this study suggest that acrylonitrile may be able to alkylate testicular DNA and induce DNA repair. It should be noted, however, that the dose level used in this study is very high, approaching the oral LD50 for the rat.

Conclusions:
The results of this study indicate that the administration of high doses of acrylonitrile has the potential to cause testicular effects, and potentially therefore also to adversely affect male fertility
Executive summary:

In this non-standard investigative study reviewed in the EU RAR, acrylonitrile was administered to male rats by gavage at dose levels of 11.5,

23 and 46 mg/kg bw/d in saline daily over periods of 2 or 4 weeks. There was a dose-dependent decrease in body weight gain and reductions in in testicular weight. Histopathological examination of testes indicated that spermatogenesis was affected after 4 weeks treatment with 23 or

46 mg/kg bw/d acrylonitrile. Overall the results in this study indicate that repeated administration of acrylonitrile produces testicular damage in the rat. The dose levels are however relatively high, approaching the acute oral lethal dose, and the effects seen in the study may well have been secondary to systemic toxicity. The limited study report provides no detail on the condition of the animals. The dose level of 11.5 mg/kg bw/d was a

LOAEL in this study, since effects on sperm count and sperm motility were seen at this dose level.

Endpoint:
toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Qualifier:
no guideline followed
Principles of method if other than guideline:
Evaluation of sperm aberrations in rats exposed to acrylonitrile by inhalation
GLP compliance:
no
Type of method:
in vivo
Specific details on test material used for the study:
No details
Species:
rat
Strain:
other: Kunming
Sex:
male
Details on test animals or test system and environmental conditions:
Seven week old male Kunming rats
Route of administration:
inhalation: aerosol
Type of inhalation exposure (if applicable):
not specified
Vehicle:
not specified
Details on exposure:
No further details
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
7, 14 or 28 days.
Frequency of treatment:
6 days/week, 2 hours/day for 28 days. Additional animals in the high dose group were also exposed for 7, 14 or 28 days.
Duration of test:
7, 14 or 28 days.
Dose / conc.:
60 mg/m³ air
Dose / conc.:
90 mg/m³ air
Dose / conc.:
120 mg/m³ air
No. of animals per sex per dose:
Not reported
Control animals:
yes
not specified
Details on study design:
Rats were exposed to acrylonitrile for 6 days/week, 2 hours/day for 28 days. Further groups were exposed to the high dose for 7, 14 or 28 days.
Positive control: IP cylophosphamide for 2 days
Key result
Dose descriptor:
NOAEC
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested
It is stated that most of the animals showed no obvious intoxication, however clinical signs and bodyweight changes were not reported. The study reports a dose-dependent increase in the number of "distorted" sperm compared to controls, with the rate being statistically significantly increased compared to control at all three exposure levels of acrylonitrile, although not as high as that in the positive control group. However, there were no dose-related increases in the percentage of sperm with any particular aberration, with 83, 84 and 87.5% of the observed sperm for the 60, 90 or 120 mg/m³ acrylonitrile group rats being described as "no fixed shape," which was also the description for 82% of the control sperm. No sperm were characterized as normal shape, and the high percentage of "no fixed shape" sperm in the controls suggests either misclassification of findings or an artifact of sample preparation, rather than a treatment-related effect. The positive control, in contrast, showed marked increases in specific sperm anomalies, e.g., no hook, two heads, "banana." Analyses of distortion rates after 7 or 14 days of exposure to 120 mg/m³ showed no statistically significant increases in rates of distortion compared to control. The 28-day exposure duration 120 mg/m³ group showed a significant increase in sperm aberrations compared to control (types of aberrations were not reported). This appears to replicate the previous finding at 120 mg/m³.

It is noteworthy that the number of distorted sperm reported at 120 mg/m³ for the 28-day exposure duration in both study phases is identical. The methods section of the report suggests that the phase of the study designed to assess the effect of varying exposure durations used a separate repeat dose group of 120 mg/m³. The exact duplication of results, however, suggests the possibility that results from the first phase were incorporated into the second phase.

Conclusions:
The study indicates that inhalation exposure to acrylonitrile causes sperm aberrations in male rats, however the design and reporting of the study are considered to be limited.
Executive summary:

Sperm aberrations were evaluated in seven-week old Kunming rats exposed by inhalation to 60, 90 or 120 mg/m3 acrylonitrile on 6 days/week, 2 hours/day for 28 days. A positive control group received cyclophosphamide by intraperitoneal injection for 2 days. Groups were also exposed to 120 mg/m3 acrylonitrile for exposure durations of 7, 14, or 28 days. Although this study suggests that exposure to acrylonitrile by inhalation may result in increased sperm aberrations, only limited confidence can be

placed in the study findings due to extensive limitations.

Additional information

A number of published studies of non-standard design have investigated the testicular toxicity of high doses of acrylonitrile.

Abd-El Azeim et al. (2012) investigated the effects of pre- and co-treatment with the anti-oxidant beta-carotene on the testicular toxicity of acrylonitrile in adult male rats. Groups of 10 rats were administered acrylonitrile by gavage at a dose level of 30 mg/kg bw for five days. Treatment with acrylonitrile significantly reduced the levels of serum testosterone, androsterone, follicle-stimulating hormone and luteinising hormone, indicating testicular toxicity. Significantly decreased serum and testicular glutathione content and glutathione-S-transferase were also seen in acrylonitrile-treated rats, and were accompanied by significantly elevated malondialdehyde levels, indicating lipid peroxidation. Acrylonitrile treatment also resulted in marked microscopic findings in the seminiferous tubules including germ cell depletion, tubular atrophy, maturation arrest, complete necrosis and multinucleated giant cell formation. Expansion of intertubular spaces and interstitial haemorrhage were also observed. Ultrastructural examination of the seminiferous tubules by TEM revealed thickened boundary tissue, pyknosis of Sertoli cell nuclei, damaged mitochondria and smooth endoplasmic reticulum-derived vacuoles. Spermatogenic cells also demonstrated altered cytoplasmic organelles, vacuoles of varying sizes and deformed spermatids. Mitochondrial disruption and a decrease in the amount of smooth endoplasmic reticulum were observed in Leydig cells. Pre-treatment with beta-carotene and its co-administration with acrylonitrile daily at a dose level of 40 mg/kg bw showed a remarkable degree of protection as evidenced by less marked changes in hormone levels, serum and testicular glutathione, GST and malondialdehyde levels and considerably less marked changes in testicular morphology and cell structure. The results of the study therefore indicate that the repeated administration of high dose levels of acrylonitrile (30 mg/kg bw compared to a reported oral LD50 of 81 mg/kg bw) causes testicular toxicity in the rat associated with oxidative stress.

Abdel Naim et al. (1994) report that the gavage administration of acrylonitrile to rats at dose levels of 23 and 46 mg/kg bw for 2-4 weeks caused reductions in testicular weight. The decreases in testicular weight were paralleled by decreases in weight of the cauda epididymis and caput epididymis, however there was no significant effect on the weights of ventral prostate and seminal vesicles. Histopathological examination of testes indicated that spermatogenesis was affected after 4 weeks treatment with 23 or 46 mg/kg acrylonitrile, as evidenced by a decreased number of spermatocytes and spermatids. Sperm count and sperm motility were significantly decreased at all dose levels, and testicular LDH-X, a marker of pachytene spermatocytes, was inhibited at dose levels of 23 and 46 mg/kg. Flow cytometric analysis of testicular aspirates from rats treated with 46 mg/kg showed a reduction in the proportion of haploid cells (22% reduction after 4 weeks) and tetraploid cells (65% reduction), while diploid cells were increased (83%). Weight gain and mean testicular weight were seen in all groups. The results of this study suggest that acrylonitrile may be able to alkylate testicular DNA and induce DNA repair. It should be noted, however, that the dose level used in this study is very high, approaching the oral LD50 for the rat.

 

Wang et al. (1995) evaluated sperm aberrations in seven-week old Kunming rats exposed by inhalation to 60, 90 or 120 mg/m3 acrylonitrile on 6 days/week, 2 hours/day for 28 days. Groups were also exposed to 120 mg/m3 acrylonitrile for exposure durations of 7, 14, or 28 days. It is stated that most of the animals showed no obvious intoxication, however clinical signs and body weight changes were not reported. The study reports a dose-dependent increase in the number of "distorted" sperm compared to control, with the rate being statistically significantly increased compared to control at all three exposure levels of acrylonitrile, although not as high as that in the positive control group. However, there were no dose-related increases in the percentage of sperm with any particular aberration, with 83, 84 and 87.5% of the observed sperm for the 60, 90 or 120 mg/m³ acrylonitrile group rats being described as "no fixed shape", which was also the description for 82% of the control sperm. No sperm were characterized as normal shape, and the high percentage of "no fixed shape" sperm in the controls suggests either misclassification of findings or an artifact of sample preparation, rather than a treatment-related effect. The positive control, in contrast, showed marked increases in specific sperm anomalies, e.g., no hook, two heads, "banana." Analyses of distortion rates after 7 or 14 days of exposure to 120 mg/m³ showed no statistically significant increases in rates of distortion compared to control. The 28-day exposure duration 120 mg/m³ group showed a significant increase in sperm aberrations compared to control (types of aberrations were not reported). This appears to replicate the previous finding at 120 mg/m³. Although this study suggests that exposure to acrylonitrile by inhalation may result in increased sperm aberrations, only limited confidence can be placed in the study findings due to its extensive limitations.

 

Tandon et al. (1988) report that the daily oral administration of acrylonitrile (10 mg/kg bw/d) to mice for a period of 60 days caused a significant decrease in the activity of testicular sorbitol dehydrogenase and acid phosphatase, and an increase in that of lactate dehydrogenase and beta-glucuronidase. Histopathological studies revealed degeneration of the seminiferous tubules. A decrease in the sperm counts of the epididymal spermatozoa was also observed in the animals of the acrylonitrile-exposed group. The authors suggest that acrylonitrile may affect male reproductive function by causing testicular injury. No effects were seen at a dose level of 1 mg/kg bw/d.

Mode of Action Analysis / Human Relevance Framework

The existing animal dataset on the reproductive toxicity of acrylonitrile does not raise significant concerns and does not demonstrate a significant reproductive hazard. Decreased pup growth was observed at maternally toxic dose levels in both drinking-water and inhalation reproductive toxicity studies.  Developmental toxicity studies in animals show no evidence for unique susceptibility of the developing foetus to acrylonitrile. There appears to be a potential for developmental toxicity, including teratogenicity, at maternally toxic dose levels, particularly by the gavage route of exposure.

 

Chinese epidemiological studies have reported increased rates of birth defects, stillbirths, premature delivery, infertility and spontaneous abortions. These findings may be artifacts of differential ascertainment, reporting bias, confounding exposures, or other factors. The epidemiological studies are not considered to be sufficiently robust, free of potential confounders, or adequately documented for exposure to be an appropriate basis for causal assessment. Based on multiple animal studies, acrylonitrile is not expected to be a developmental or reproductive toxicant in the absence of significant parental toxicity.

Justification for classification or non-classification

There is no convincing evidence that acrylonitrile can cause effects on fertility or reproduction; non-standard studies report high dose effects on sperm parameters, however similar findings are not apparent in more reliable standard studies.

There is some evidence from animal studies that exposure to high levels of acrylonitrile (sufficient to cause overt maternal toxicity) can cause developmental toxicity. Neal et al. (2009) have reviewed other earlier studies which investigated potential reproduction effects. In the most reliable studies they found some evidence of foetal malformations at high concentrations (80 ppm and above); short tail, missing vertebrae, short trunk etc. One tailless pup was found in the industry sponsored Friedman & Beliles (2002) three generation study, again at the high dose. One pup at the high dose in Nemec et al. (2008) reportedly had a threadlike tail and absence of caudal vertebrae. While the numbers of affected foetuses were not significant in these studies; it is proposed that the effects are recognised in the classification of acrylonitrile as an indication of potential hazard. However given the low level incidences of findings in the studies available, classification of acrylonitrile for reproductive toxicity (pre-natal developmental toxicity) according to CLP in Category 2 is considered to be appropriate.  Classification in this category is proposed as there is some evidence from experimental animals of an adverse effect on foetal development.

Additional information