N-(butoxymethyl)acrylamide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The toxicity to reproduction of the read-across material, acrylamide, was assessed according to OECD Test Guideline 416 under GLP conditions using male and female rats. The NOAEL for general toxicity was 0.5 mg/kg bw/day (nominal) for the parental generation. The NOAEL for reproductive toxicity was 0.2 mg/kg bw/day for the parental generation. For the F1 generation, the NOAEL for reproductive toxicity was 2 mg/kg bw/day based on developmental neurotoxicity. The NOAEL for general toxicity in the F1 generation was 0.5 mg/kg bw/day.

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

See the Analogue Approach Report attached in Section 13 of the IUCLID dossier.

Reason / purpose for cross-reference:

other: Read across to target substance

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

yes

Remarks:

Inclusion of F0 males after first mating in a Dominant Lethal study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston, NY, USA
- Age at study initiation: (P) 4 wks; (F1) 5-6 wks (16-17 weeks at initiation of mating period)
- Weight at study initiation: (P) Males: approximately 75 g; Females: approximately 65 g; (F1) Males: 121-130 g; Females: 97 -100 g
- Fasting period before study: no
- Housing: Singly (except for cohabitation and lactational periods) in stainless steel, wire-mesh cages. From gestational day 18, through parturition, and lactation until weaning, female rats were housed in plastic shoebox cages with Alpha-Dri bedding
- Diet: ad libitum (Certified Ground Rodent Chow)
- Water: ad libitum (tap water)
- Acclimation period: 14 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12:12
IN-LIFE DATES: From: 12/02/1985 To: 05/12/1985

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

Fresh concentrated premix and dosing solutions were prepared twice weekly (Mondays and Thursdays). Dosing solutions prepared on Mondays were administered to the animals on Tuesdays; those prepared on Thursdays were presented to the animals on Fridays. Prior to changing cage bottles on Tuesdays, postdosing samples were obtained from randomly selected cage bottles for analysis (after being administered to the animals for four days).
Test animals were maintained on drinking water containing sufficient acrylamide to provide dose levels during the prebreed exposure periods of the F0 (10 weeks) and F1 (11 weeks) generations. The concentrations were adjusted weekly based on the mean body weights and water consumption per sex per group. During mating, gestation, and the first week of lactation, females from each treatment group were provided with the water containing the appropriate concentration of acrylamide given during the week before initiation of mating. Because the males were cohabited with the females during mating, the males were exposed to acrylamide concentrations, based on female body weights, to prevent overexposure of the females. During the second and third week of lactation, females received 1/2 and 1/3, respectively, of the water concentration of acrylamide given during breeding and gestation to avoid overdosing of the neonates, because maternal water consumption increased during lactation and as pups began to drink the water. During week 4 of lactation, nursing females received 1/2 of the water concentration of acrylamide given during breeding and gestation. This provided the offspring with a period of acclimation to one-half strength acrylamide concentrations before the start of their prebreed exposure to the dose given F0 animals during their first week of treatment. Until all F1 litters were weaned, weanlings chosen for the F1 generation received water containing the same concentration of acrylamide that was given to the F0 rats during their first week of treatment.

Details on mating procedure:

After the 70 day pre-breeding exposure period was completed, the F0 animals were mated on the basis of one male to one female selected randomly within each dose group for a period of 14 days. The observation of dropped copulation plug and/or vaginal sperm was considered evidence of successful mating. Females were examined daily during the cohabitation period for dropped copulation plugs. Any female not exhibiting a dropped copulation plug was examined for the presence of sperm in the vaginal tract. The day a copulation plug or vaginal sperm was observed was designated gestational day (GD) 0. Once a plug or vaginal sperm was observed, the male and female from that mating pair were individually housed. If after 7 days of cohabitation the female did not exhibit a copulation plug, she was removed from the mating cage and placed in the cage of a male which had already impregnated a female (if possible) for 7 more days or until evidence of copulation was found, whichever came first. For any mating pairs which did not show evidence of successful mating (i.e., no copulation plug or vaginal sperm was observed) the last scheduled mating day was considered gd 0 for that female and the animals were treated accordingly for subsequent events. Mated females were weighed on gd 0, 6, 13 and 20. On gd 18, each female was transferred to a shoe-box-cage-. Females were observed twice daily beginning on gd 20- for evidence of littering. Dams with litters were weighed on postnatal days 1, 4, 7, 14, 21 and 28. The dams were allowed to rear their young to day 28 postpartum. On day 28 postpartum, each litter was weaned. When the last Fl litter reached day 35 postpartum, 30 male and 30 female pups per dose group were randomly selected to produce the F2 generation. These selected animals continued on dosed water at the same dosage level of acrylamide as their parents. Each litter was represented at least once per sex if possible, and a second pup of the same sex was chosen from a given litter only after one pup per sex had been chosen from all possible litters

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The premixes were analyzed twice weekly for the first two weeks of the study, then once weekly for the rest of the study. The dosing solutions were analyzed (all four concentrations per sex per preparation) for the first two weeks of the study, then all four concentrations per sex from one preparation every other week for the remainder of the study. Cage bottle samples were analyzed for the three doses (5 bottles/sex/group) for the first two weeks and then for low and high doses (5 bottles/sex/group) from one preparation monthly for the remainder of the study. These samples were collected on Tuesdays following four days of administration.

Duration of treatment / exposure:

Premating (F0) through weaning (F2)
Premating exposure period - Male : 10 weeks, F0
Premating exposure period - Female : 10 weeks, F0

Frequency of treatment:

Continuous

Details on study schedule:

See above

Remarks:

Doses / Concentrations:
0, 0.5, 2.0, or 5.0 mg/kg
Basis:
nominal in water

No. of animals per sex per dose:

F0: 30 rats/sex/group
F1: 30 F1 weanlings/sex/group

Control animals:

yes, concurrent no treatment

Positive control:

No

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality and morbidity
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily for any clinical signs of toxicity
BODY WEIGHT: Yes
- Time schedule for examinations: weighed weekly (food consumption measured at the same time)
WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: weekly (measured at the time the body weights were taken)

Oestrous cyclicity (parental animals):

No data

Sperm parameters (parental animals):

No data

Litter observations:

STANDARDISATION OF LITTERS:
On day 4 after birth, the size of each litter was adjusted by eliminating extra pups by computerized random selection to yield, as nearly as possible, four males and four females per litter. 30 males and 30 females from each group were then selected from Fl litters on a random basis to become parents of the next generation. All pups were available for selection except those not expected to survive because of physical abnormalities. The parentage of each weanling was ascertained to avoid brother-sister-matings if possible.
PARAMETERS EXAMINED:
In F1 / F2 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities.
GROSS EXAMINATION OF DEAD PUPS:
External and internal abnormalities; possible cause of death was/was not determined for pups born or found dead

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: F0 males were sacrificed after completion of the concurrent dominant lethal assay. F1 males were sacrificed after F1 or F2 were weaned.
- Maternal animals: No data given for all surviving animals. F0 females were sacrificed after F1 or F2 were weaned.
GROSS NECROPSY
All F0 and F1 parental animals in all groups were subjected to full gross necropsy which included: examination of the external surfaces; all orifices; cranial cavity; carcass; external and cut surfaces of-the brain and spinal cord; the thoracic, abdominal and pelvic cavities and their viscera; and cervical tissues and organs
HISTOPATHOLOGY / ORGAN WEIGHTS
30 male and 30 female F1 adults from the control and high-dose group were subjected to histologic examination of vagina, cervix, uterus, oviducts, ovaries (females), and testes, epididymides, seminal vesicles, coagulating glands, prostate (males), and target tissues (peripheral nerves; sciatic and tibial), brain (five coronal sections), and spinal cord (cervical and lumbar enlargements) for both sexes. After the initial histologic evaluation was performed, peripheral nerve sections (sciatic and tibial) from six high-dose male and three control male F1 adults, and spinal cord sections from three high dose and two control female F1 adults were stained with Bodian's method (Bodian's/Luxol fast blue stain) and examined histologically. The fixed (buffered neutral 1090 formalin) uterus from any female of the F0 or F1 generations failing to produce a litter was stained with potassium ferricyanide for confirmation of pregnancy status; implantation sites (if any) were recorded. (This method, developed and validated at the performing laboratory, does not interfere with subsequent histopathologic evaluations.)

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 4 days of age.
- These animals were not subjected to postmortem examinations (macroscopic and/or microscopic examination).
GROSS NECROPSY
Fl Pups: 20/sex from control and 5.0 mg/kg/day; 10/sex from 0.5 and 2.0 mg/kg/day
F2 Pups: 10/sex/group
- Gross necropsy consisted of same organs and tissues examined in adults..
HISTOPATHOLOGY / ORGAN WEIGTHS
Fl Pups: 20/sex from control and 5.0 mg/kg/day (10/sex perfused, 10/sex non-perfused); 10/sex from 0.5 and 2.0 mg/kg/day (non-perfused) only if lesions found in high dose animals
F2 Pups: 0/sex (non-perfused) from control and 5.0 mg/kg/day.
OTHER:
A gross internal examination was also performed on any pup appearing abnormal or dying on test.

Statistics:

The unit of comparison was the male, the pregnant female, or the litter (Weil, 1970). Results of the quantitative continuous variables (eg., body weights, food consumption, organ weights, etc.) were intercompared for the three treatment groups and one control group by use of Levene's test for equal variances (Levene, 1960); analysis of variance (ANOVA), and t-tests. When Levene's test indicated homogeneous variances and the ANOVA was significant, the pooled t-test was used for pairwise comparisons. When Levene's test indicated heterogeneous variances, all groups were compared by an ANOVA for unequal variances (Brown and Forsythe, 1974) fo11owed; when necessary, by the separate variance t-test for pairwise comparisons. Nonparametric data were statistically evaluated using the Kruskal-Wallis test (Sokal and Rohlf, 1969) followed by the Mann-Whitney U test for pairwise comparisons (Sokal and Rohlf, 1969) when appropriate. Frequency data (such as the various indices) were compared using the Fisher's exact test (Sokal and Rohlf, 1969). For all statistical tests, the fiducial limit of 0.05 (two-tailed) was used as the criterion for statistical significance.

Reproductive indices:

No. F0 pairs at mating: 1 per dose group (4 in all)
No (%) plug/sperm-positive females
No. (%) males impregnating\
No. (%) pregnant females
No. implantations/dam
No. live litters on PND
Gestational length (d)
Mating index (females)
Mating index (males)
Fecundity index
Fertility index
Gestational index

Offspring viability indices:

No. live pups/litter (PND 0)
No. live litters on PND 4
Postimplantation loss

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Neurotoxicity

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

effects observed, treatment-related

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Pre-breeding exposure of the F0 animals resulted in evidence of toxicity to both sexes: reduced body weights for F0 rats of either sex given 2.0 or 5.0 mg/kg/d and males at 0.5 mg/kg/d. At 5.0 mg/kg/d, head tilt and leg splay were observed in F0 males and leg splay was observed in F0 females. One adult animals died on study: one F0 female at 5.0 mg/kg/day( found dead on study day 100) which died from intrauterine haemorrhage subsequent to pregnancy.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
F0 females exhibited reduced weights and weight gain during gestation at 5.0 mg/kg/d. During lactation, maternal body weights remained lower at 5.0 mg/kg/d than in controls. However, lactational weight gain was increased at 5.0 mg/kg/d.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS) The calculated intake in mg/kg/day was close to the target doses for all weeks examined.
The amount of acrylamide in the drinking water was changed weekly for each dose group based on the mean body weights and water consumed for each dose group. Therefore, the calculated dosage of acrylamide ingested dropped consistently from start of exposures to day 70 in all treated groups: 0.568-0.476 mg/kg/day at 0.5 mg/kg/day, 2.197-2.012 mg/kg/day at 2.0 mg/kg/day and 5.556-4.786 mg/kg/day at 5.0 mg/kg/day because of increasing body weight and decreasing water intake relative to body weight over the ten (10) week period. The target concentrations in mg/kg/day were maintained throughout the study by these adjustments.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Reproductive indices, relating to mating and pregnancy were unaffected by treatment. Gestational length was also unchanged across groups. However, the number of implantàtions per female (detected at scheduled necropsy after weaning of the Fl litters), and the number of live pups per litter at birth (postnatal day 0) were significantly reduced at 5.0 mg/kg/day. The percent postimplantation loss was increased at 5.0 mg/kg/day. Lower dose groups were unaffected.
In the Dominant Lethal study: The mating parameters for the dominant lethal phase (one treated male:two naive females) exhibited no treatment-related effects at any dosage level. At 5.0 mg/kg/day, the total implants per litter were reduced (the number of ovarian copora lutea of pregnancy were unchanged); the percent preimplantation loss was therefore increased. The number of live implantations per Utteri was reduced, so the percent postimplantation loss was increased, and the number of nonlive implants per litter was increased. In addition, calculation of the frequency of dominat lethal factors, FL% indicated a 20% frequency at 5.0 mg/kg/day. Lower dose groups were unaffected.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Top dose F0 maternal body weights during gestation were reduced on gestational days 13 and 20 and overall gestational weight gain was reduced. F0 lactational body weights were reduced on postnatal days 4, 7, 14 and 21 at 5.0 mg/kg/day, but not on postnatal day 1 or 28. Weight gain over the lactation period was increased at 5.0 mg/kg/day

GROSS PATHOLOGY (PARENTAL ANIMALS)
All F0 adults indicated no treatment-related lesions

HISTOPATHOLOGY (PARENTAL ANIMALS)
No treatment-related histopathological lesions were seen in the F0 parents of the F1 weanlings.

Dose descriptor:

NOAEL

Effect level:

0.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: General toxicity

Dose descriptor:

NOAEL

Effect level:

0.2 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: Reproductive toxicity

Clinical signs:

effects observed, treatment-related

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

VIABILITY (OFFSPRING)
Survival of F1 pups was unaffected by treatment. During the latter half of the lactation period during the time when pups begin to self feed and drink, male pup weight per litter was significantly reduced at 5.0 mg/kg/d. One F1 male at 5.0 mg/kg/day with a renal mass diagnosed as embryonal nephroma was sacrificed moribund on study day 122
CLINICAL SIGNS (OFFSPRING)
Treatment-related clinical signs for F1 males included only head tilt, observed only in 4 males at 5.0 mg/kg/day. F1 breeding (to produce F2 offspring) produced results essentially identical to those observed in the F0 breeding. F2 pup survival indices were unaffected by treatment.
BODY WEIGHT (OFFSPRING)
Pup body weights per litter (males, females or total) were unaffected by treatment until postnatal day 14. On postnatal days 14, 21 and 28, male pup weight (but not female or total pup weights) per litter was significantly reduced at 5.0 mg/kg/day. Female and total pup weights per litter were slightly reduced at 5.0 mg/kg/day at these time points but these differences were not statistically significant. F1 animals, selected as parents for F2 generation, had reduced body wieghts at 2.0 and 5.0 mg/kg/d for both sexes during the 11 week prebreed exposure. Maternal gestational weights were depressed at 5.0 mg/kg/d and gestational weight gain was depressed at 2.0 and 5.0 mg/kg/d. Maternal lactational body weight gain was increased at 5.0 mg/kg/d.
SEXUAL MATURATION (OFFSPRING)
Reproductive indices and gestational length were unaffected. The number of implantations and live pups per litter were decreased with the percent post-implantation loss therefore increased at the 5.0 mg/kg/d dose level. F2 litter sizes were reduced at 5.0 mg/kg/d due entirely to the initial reduction observed at birth.
GROSS PATHOLOGY (OFFSPRING)
Gross necropsy of selected F2 weanlings and all F1 adults indicated no treatment related effects.
HISTOPATHOLOGY (OFFSPRING)
Histopathology of reproductive and nervous system tissues of the necropsied F1 pups also disclosed no treatment related lesions. Peripheral nerve sections (sciatic and tibial) from randomly selected F1 males from the high dose group revealed minimal to mild axonal fragmentation and/or swelling. Spinal cord sections from three high dose females did not reveal any abnormal findings.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

2 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

water consumption and compound intake

developmental neurotoxicity

Remarks on result:

other: Reproductive toxicity

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

0.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: General toxicity

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

2 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

Remarks on result:

other: General and reproductive toxicity

Reproductive effects observed:

not specified

This effect was primarily a "jack pot" with a few litters effected to a large extent and most uneffected. The only significant effect in this study was litter size.

Conclusions:

Long term exposure to acrylamide in the drinking water, over two generations in Fischer 344 rats, resulted in parental toxicity (reduced bodyweight, clinical signs of toxicity, histologic evidence of axonal swelling and/or degeneration in peripheral nerves) at 5.0 mg/kg/day, accompanied by prenatal lethality. Exposure to 2.0 mg/kg/day resulted in similar but lesser adult toxicity but no prenatal lethality. Exposure to 2.0 mg/kg/day resulted in no change to reproductive parameters in either generation except for reduced body weights and weight gain in F0 males in the prebreed exposure period and reduced body weight and weight gain in F0 females late in the prebreed exposure period. The only significant reproductive event induced by acrylamide was decreased litter size as a result of dominant lethal mutations.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

0.2 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

The key study was conducted on the read-across substance in vivo in accordance with an internationally recognised guideline and in compliance with GLP.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The toxicity to reproduction of the read-across material, acrylamide, was assessed according to OECD Test Guideline 416 under GLP conditions using male and female rats at 0, 0.5, 2.0 or 5.0 mg/kg.

After the 70 day pre-breeding exposure period was completed, the F0 animals were mated on the basis of one male to one female selected randomly within each dose group for a period of 14 days. The observation of dropped copulation plug and/or vaginal sperm was considered evidence of successful mating. Females were examined daily during the cohabitation period for dropped copulation plugs. Any female not exhibiting a dropped copulation plug was examined for the presence of sperm in the vaginal tract. The day a copulation plug or vaginal sperm was observed was designated gestational day (GD) 0. Once a plug or vaginal sperm was observed, the male and female from that mating pair were individually housed. If after 7 days of cohabitation the female did not exhibit a copulation plug, she was removed from the mating cage and placed in the cage of a male which had already impregnated a female (if possible) for 7 more days or until evidence of copulation was found, whichever came first. For any mating pairs which did not show evidence of successful mating (i.e., no copulation plug or vaginal sperm was observed) the last scheduled mating day was considered gd 0 for that female and the animals were treated accordingly for subsequent events. Mated females were weighed on gd 0, 6, 13 and 20. On gd 18, each female was transferred to a shoe-box-cage-. Females were observed twice daily beginning on gd 20- for evidence of littering. Dams with litters were weighed on postnatal days 1, 4, 7, 14, 21 and 28. The dams were allowed to rear their young to day 28 postpartum. On day 28 postpartum, each litter was weaned. When the last Fl litter reached day 35 postpartum, 30 male and 30 female pups per dose group were randomly selected to produce the F2 generation. These selected animals continued on dosed water at the same dosage level of acrylamide as their parents. Each litter was represented at least once per sex if possible, and a second pup of the same sex was chosen from a given litter only after one pup per sex had been chosen from all possible litters.

The animals were checked for mortality and morbidity twice daily, once daily for any clinical signs of toxicity and body weight, food and consumption were assessed once weekly.

On day 4 after birth, the size of each litter was adjusted by eliminating extra pups by computerized random selection to yield, as nearly as possible, four males and four females per litter. 30 males and 30 females from each group were then selected from Fl litters on a random basis to become parents of the next generation. All pups were available for selection except those not expected to survive because of physical abnormalities. The parentage of each weanling was ascertained to avoid brother-sister-matings if possible. In F1 / F2 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities.

F0 males were sacrificed after completion of the concurrent dominant lethal assay. F1 males were sacrificed after F1 or F2 were weaned. F0 females were sacrificed after F1 or F2 were weaned. All F0 and F1 parental animals in all groups were subjected to full gross necropsy which included: examination of the external surfaces; all orifices; cranial cavity; carcass; external and cut surfaces of-the brain and spinal cord; the thoracic, abdominal and pelvic cavities and their viscera; and cervical tissues and organs.

30 male and 30 female F1 adults from the control and high-dose group were subjected to histologic examination of vagina, cervix, uterus, oviducts, ovaries (females), and testes, epididymides, seminal vesicles, coagulating glands, prostate (males), and target tissues (peripheral nerves; sciatic and tibial), brain (five coronal sections), and spinal cord (cervical and lumbar enlargements) for both sexes. After the initial histologic evaluation was performed, peripheral nerve sections (sciatic and tibial) from six high-dose male and three control male F1 adults, and spinal cord sections from three high dose and two control female F1 adults were stained with Bodian's method (Bodian's/Luxol fast blue stain) and examined histologically. The fixed (buffered neutral 1090 formalin) uterus from any female of the F0 or F1 generations failing to produce a litter was stained with potassium ferricyanide for confirmation of pregnancy status; implantation sites (if any) were recorded. The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 4 days of age. These animals were not subjected to post-mortem examinations (macroscopic and/or microscopic examination).

Long term exposure to acrylamide in the drinking water, over two generations in Fischer 344 rats, resulted in parental toxicity (reduced bodyweight, clinical signs of toxicity, histologic evidence of axonal swelling and/or degeneration in peripheral nerves) at 5.0 mg/kg/day, accompanied by prenatal lethality. Exposure to 2.0 mg/kg/day resulted in similar but lesser adult toxicity but no prenatal lethality. Exposure to 2.0 mg/kg/day resulted in no change to reproductive parameters in either generation except for reduced body weights and weight gain in F0 males in the prebreed exposure period and reduced body weight and weight gain in F0 females late in the prebreed exposure period. The only significant reproductive event induced by acrylamide was decreased litter size as a result of dominant lethal mutations.

Survival of F1 pups was unaffected by treatment. During the latter half of the lactation period during the time when pups begin to self-feed and drink, male pup weight per litter was significantly reduced at 5.0 mg/kg/d. One F1 male at 5.0 mg/kg/day with a renal mass diagnosed as embryonal nephroma was sacrificed moribund on study day 122.

Treatment-related clinical signs for F1 males included only head tilt, observed only in 4 males at 5.0 mg/kg/day. F1 breeding (to produce F2 offspring) produced results essentially identical to those observed in the F0 breeding. F2 pup survival indices were unaffected by treatment.

Pup body weights per litter (males, females or total) were unaffected by treatment until postnatal day 14. On postnatal days 14, 21 and 28, male pup weight (but not female or total pup weights) per litter was significantly reduced at 5.0 mg/kg/day. Female and total pup weights per litter were slightly reduced at 5.0 mg/kg/day at these time points but these differences were not statistically significant. F1 animals, selected as parents for F2 generation, had reduced body weights at 2.0 and 5.0 mg/kg/d for both sexes during the 11 week pre-breed exposure. Maternal gestational weights were depressed at 5.0 mg/kg/d and gestational weight gain was depressed at 2.0 and 5.0 mg/kg/d. Maternal lactational body weight gain was increased at 5.0 mg/kg/d.

Reproductive indices and gestational length were unaffected. The number of implantations and live pups per litter were decreased with the percent post-implantation loss therefore increased at the 5.0 mg/kg/d dose level. F2 litter sizes were reduced at 5.0 mg/kg/d due entirely to the initial reduction observed at birth.

Gross necropsy of selected F2 weanlings and all F1 adults indicated no treatment related effects.

Histopathology of reproductive and nervous system tissues of the necropsied F1 pups also disclosed no treatment related lesions. Peripheral nerve sections (sciatic and tibial) from randomly selected F1 males from the high dose group revealed minimal to mild axonal fragmentation and/or swelling. Spinal cord sections from three high dose females did not reveal any abnormal findings.

In this study, rats were more sensitive to neurotoxicity, which had a NOAEL of 2.0 mg/kg based on head tilt in males than any reproductive parameter (NOAEL 5.0 mg/kg day for litter size).

Effects on developmental toxicity

Description of key information

Developmental toxicity of the read-across material, acrylamide, was assessed using methodology equivalent to OECD Test Guideline 414 under GLP conditions using rats. The NOAEL for maternal toxicity (reduced body weight gain) was 2.5 mg/kg/d and the NOAEL for developmental toxicity was 15 mg/kg/d.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 27 - March 10, 1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

See the Analogue Approach Report attached in Section 13 of the IUCLID dossier.

Reason / purpose for cross-reference:

other: Read across to target substance

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: no data
- Weight at study initiation: 201-263 g
- Fasting period before study: no
- Housing: 1 per cage
- Diet: Purina 502 ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C
- Humidity (%): 48-49%
- Air changes (per hr):12-14 per hour
- Photoperiod (hrs dark / hrs light): 12:12 (0700-1900)
IN-LIFE DATES: January 27 - March 18, 1987

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Concentration in vehicle: 0, 0.5, 1.5 and 3 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

IR, UV, MS, NMR and TLC and GC, KF conducted on receipt of test article and following completion of study

Details on mating procedure:

- Impregnation procedure: cohoused
- Proof of pregnancy: sperm positive referred to as GD0

Duration of treatment / exposure:

Gestational days 6 to 20

Frequency of treatment:

Once/day

Duration of test:

20 days

Dose / conc.:

2.5 mg/kg bw/day (nominal)

Dose / conc.:

7.5 mg/kg bw/day (nominal)

Dose / conc.:

15 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Groups of 29-30

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on preliminary study
- Rationale for animal assignment: body weight

Maternal examinations:

Animals were observed daily for clinical signs.
CAGE SIDE OBSERVATIONS: No data
DETAILED CLINICAL OBSERVATIONS: Yes (neurotoxicty)
- Time schedule: daily up to GD6 the twice daily
BODY WEIGHT: Yes
- Time schedule for examinations: daily
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20
- Organs examined: uterus

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

Fetuses were thoroughly examined macroscopically for visceral and skeletal abnormalities (including the head).
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Statistics:

GLM procedures were applied for the ANOVA of maternal and fetal parameters. GLM analysis determined the significance of dose-response relationships and the significance of dose effects, replicate effects and dose x replicate interactions. If the ANOVA was significant, William’s or Dunnett’s Multiple Comparison Tests compared ACRL exposed to control groups. One tailed tests were used for all pairwise comparisons except maternal body and organ weights and fetal body weight. Nominal scale measures were analysed by chi square test for independence and by a test for linear trend on proportions. When a chi square showed significant group differences, a one-tailed Fisher’s exact probability test was used for pairwise comparisons of ACRL and control groups

Indices:

Percent foetuses malformed per litter, percent litters with malformed foetuses, percent foetuses with variations per litter, percent litters with variations, percent foetuses with extra ribs per litter, percent litters with extra ribs

Historical control data:

Percent litters with 1 or more:
- malformed foetuses: 22.9
- non-live implants: 33.8
- dead foetuses: 1.1
- resorptions: 33.2
Percent malformed foetuses: 3.2

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
There were no maternal mortalities and no clear clinical signs of toxicity. When corrected for gravid uterine weight, maternal body weight gain was decreased amongst animals receiving 7.5 and 15 mg/kg/day (12% and 18% reductions respectively).

Dose descriptor:

NOAEL

Effect level:

2.5 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

15 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: No developmental toxicity observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no apparent effects on embryo/fetal viability, growth or malformations. There was a slight, but not statistically significant, increase in the incidence of skeletal variations (percentage of litters with variations - 61% in controls, 92% at 15 mg/kg/day, and percentage of foetuses with variations per litter - 14% in controls, 24% at 15 mg/kg/day). The most frequently observed variation was the presence of a rudimentary extra lumbar rib. This finding is considered likely to be an indirect consequence of maternal toxicity or stress and is of limited toxicological importance.

Dose descriptor:

NOAEL

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: There were no apparent effects on embryo/fetal viability, growth or malformations.

Abnormalities:

no effects observed

Developmental effects observed:

no

DEVELOPMENTAL TOXICITY IN PREGNANT CD RATS FOLLOWING MATERNAL EXPOSURE TO ACRYLAMIDE BY GAVAGE ON GESTATIONAL DAYS 6 THROUGH 20
	Acrylamide (mg/kg/day, po)
	0	2.5	7.5	15
All litters^a	23	26	26	24
No. implantation sites per litter	13.5±0.9	14.6±0.8	14.8±0.7	14.5±0.8
% resorptions per litter	3.4±1.4	3.2±1.4	3.7±1.6	2.0±0.9
% litters with resorptions	30.4	26.9	26.9	20.8
Live litters^b
No. live foetuses per litter	13.0±0.9	14.1±0.8	14.2±0.6	14.3±0.8
Average male foetal body weight (g) per litter	3.72±0.17	3.44±0.12	3.38±0.14	3.52±0.12
Average female foetal body weight per litter (g)	3.44±0.13	3.29±0.11	3.20±0.13	3.29±0.11
% foetuses malformed per litter	0.3±0.3	1.7±0.6	0.2±0.2	0.8±0.6
% litters with malformed foetuses	4.4	23.1	3.8	8.3
% foetuses with variations per litter	14.3±3^+	16.5±3.6	18.3±4.5	24.0±3.9
% litters with variations	60.9^+	69.2	73.1	91.7
% foetuses with extra ribs per litter^c	11.1±3.6	11.1±2.9	13.5±4.5	16.4±4.0
% litters with extra ribs	47.8	57.7	53.8	70.8

 

a Includes all dams pregnant at termination; litter size = No. implantation sites per dam; means ± SE.

b Includes only dams with live foetuses; litter = No. live foetuses per dam; means ± SE.

c Includes foetuses with one or more rudimentary or full extra lumbar rib.

+ Linear trend test,p< 0.05.

Conclusions:

2.5 mg/kg/d was considered to be the NOAEL for maternal toxicity (reduced body weight gain) and 15 mg/kg/d was considered to be the NOAEL for developmental toxicity.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

15 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The key study was conducted on the read-across substance using methodology equivalent to an internationally recognised guideline and in compliance with GLP.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

The developmental toxicity of the read-across substance, acrylamide, was assessed using methodology equivalent to OECD Test Guideline 414. Pregnant female rats were administered the read-across material at 2.5, 7.5 and 15 mg/kg bw/day by oral gavage during gestational days 6 to 20. Animals were observed daily for clinical signs. Sacrifice on gestation day 20 was followed by post-mortem examination during which ovaries and uterine contents were examined.

There were no maternal mortalities and no clear clinical signs of maternal toxicity. When corrected for gravid uterine weight, maternal body weight gain was decreased amongst animals receiving 7.5 and 15 mg/kg/day (12 and 18 % reductions, respectively).

There were no apparent effects on embryo/foetal viability, growth or malformations. There was a slight, but not statistically significant, increase in the incidence of skeletal variations (percentage of litters with variations – 61 % in controls, 92 % at 15 mg/kg/day, and percentage of foetuses with variations per litter – 14 % in controls, 24 % at 15 mg/kg/day). The most frequently observed variation was the presence of a rudimentary extra lumbar rib. This finding is considered likely to be an indirect consequence of maternal toxicity or stress and is of limited toxicological importance.

The NOAEL for maternal toxicity was 2.5 mg/kg bw/day based on reduced body weight gain. The NOAEL for developmental toxicity was 15 mg/kg bw/day.

Justification for classification or non-classification

The read-across substance, acrylamide, has a harmonised classification as Category 2 for reproductive toxicity.