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Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Peer-reviewed publication suitable for assessment; acrolein is a major metabolite of allyl alcohol

Data source

Reference
Reference Type:
publication
Title:
Reproductive study of acrolein on two generations of rats
Author:
Parent, R.A., Caravello, H.E., Hoberman, A.M.
Year:
1992
Bibliographic source:
Fundamental and applied toxicology 19: 228-237

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
not specified
Limit test:
no

Test material

Constituent 1
Reference substance name:
Acrolein
IUPAC Name:
Acrolein
Details on test material:
Two samples were supplied by Baker Performance Chemicals, Houston, Texas.
Purity: 96.05 - 96.72% (stabilised with 0.25 hydroquinone; test samples also contained approximately 3% water).

Test animals

Species:
rat
Strain:
other: Crl:CD (SD)BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc.
- Age at study initiation: 58 days
- Weight at study initiation: Males: 278-375 g; Females: 187-251 g
- Housing: 1-2 rats/sex/cage. All adult rats were housed in suspended stainless-steel cages above absorbent paper liners. Cages were changed every other week and cage pan liners were changed three times a week. During the cohabiting period (maximum period of 21 days), male and female rats of the same dosage group were housed together (1:1). No later that day 14 of presumed gestation, female rats were transferred to a nesting box (1 female per nesting box). During the 21-day postpartum period, each dam was housed in a common nesting box. Nesting boxes contained Bed-o'cobs bedding which was changed three times a week.
- Diet (e.g. ad libitum): certified rodent chow 5002 in meal form ad libitum
- Water (e.g. ad libitum): reverse osmosis membrane treated water (with up to 1 ppm chlorine) ad libitum.
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 74 +/-4
- Humidity (%): 40 - 70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Dosing solutions of acrolein were prepared daily with reverse osmosis membrane-processed deionized water.
These were analysed prior to dosing using a spectrophotometer and found to be within 1% of the desired concentration.
The stability of acrolein in deionized water at concentrations of 0.2 and 1.2 mg/ml were investigated at 6 hours postpreparation.
A loss of 7% and 4% for the low-dose and high-dose respectively was observed.

Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 21 days
- Proof of mating: vaginal plug or sperm in vaginal smear referred to as day 0 of presumed gestation.
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility from the same dosage group.
- After successful mating each pregnant female was individually housed.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Verification of doses was carried out using a Cary 118 twin beam spectrophotometer set at 211 nm.
Duration of treatment / exposure:
F0 females: 96 - 130 daily doses
F0 males: 93 - 94 daily doses
F1 females: 104 - 149 daily doses
F1 males: 106 - 125 daily doses
Frequency of treatment:
Daily
Details on study schedule:
All rats (male and female) were dosed with acrolein at 0, 1, 3 and 6 mg/kg at a volume of 5 ml/kg for 70 days. After this dosing period, rats within the same dosage group were housed together at a ratio of 1 male rat to 1 female rat for a minimum period of 14 days. Female rats that had not mated within the 14-day period were housed with a different male rat from the same dosage group for an additional 7 days. When copulatory plugs or spermatozoa in vaginal smears are observed, this was regarded as day 0 of the presumed gestation period. All female rats received their respective daily dose of acrolein throughout cohabitation, gestation and lactation; this continued for all female rats until either day 25 of presumed gestation (for females who failed to deliver offspring) or day 21 of lactation (for females that successfully delivered offspring). F0 males were given daily doses up until the scheduled termination date.

F1 pups (40/sex from as many litters as possible/group, at least 1M + 1F/litter where possible) were selected for continuation of the study on day 21 of lactation. These pups received doses beginning on day 21 postpartum. F1 Female rats that had not mated within the 14-day period were housed with a different male rat from the same dosage group for an additional 7 days. When copulatory plugs or spermatozoa in vaginal smears are observed, this was regarded as day 0 of the presumed gestation period. All female rats received their respective daily dose of acrolein throughout cohabitation, gestation and lactation; this continued for all female rats until either day 25 of presumed gestation (for females delivering no offspring) or day 21 of lactation (for females which successfully delivered offspring). F1 males were given daily doses up until the scheduled termination date.

F2 pups were not directly dosed with acrolein (but may have been indirectly exposed).
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 1, 3 or 6 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
30
Control animals:
yes
Details on study design:
A dose range-finding study was carried out with 6 groups of 16 rats (8 male, 8 female). All rats were dosed 7 days prior to cohabitation up to day 25 of presumed gestation or day 4 postpartum. At the higher doses (10, 15 and 20 mg/kg) many mortalities were observed. At doses of 5 mg/kg, clinical signs (including excess salivation, reduced motor activity and respiratory difficulty) were observed. No effect on mating or fertility parameters were seen at 2.5 mg/kg/day. Based on the results of the dose range-finding study, doses of 0, 1, 3, and 6 mg/kg were used in the main study.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice a day during study period

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice a day

BODY WEIGHT: Yes
- Time schedule for examinations: daily during dosage period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- For males, this was recorded weekly (except during cohabitation - was recorded but not tabulated) For females, food consumption was recorded weekly prior to cohabitation on days 0, 6, 12, 15, 20 and/or 25 of presumed gestation; in addition on days 1, 4, 7, 10, 14, 16, 18 and 21 of lactation.

OTHER:
For all F0 and F1 female rats:
- The nursing behaviour, caring of pups and other dam-pup interactions were observed on a daily basis, any abnormal maternal behaviour was recorded.
- mating performance was assessed daily during the cohabitation period, confirmed by observed delivery of offspring, confirmed mating date or implantation site at necropsy.
- The duration of gestation and pup viability was assessed.
- Those that did not deliver offspring were killed on day 25 of presumed gestation and examined for implantation sites and gross lesions.

Necropsies were conducted on any F0 and F1 rats that were found dead.
Oestrous cyclicity (parental animals):
- The oestrous cycle were assessed during the cohabitation period (maximum of 21 days) up until day 0 of presumed gestation.
Sperm parameters (parental animals):
No data
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were 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:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead (provided that autolysis or cannabalisation had not taken place).
Postmortem examinations (parental animals):
SACRIFICE
- Maternal animals: All surviving animals on day 21 of lactation (for females that successfully delivered offspring) or day 25 of presumed gestation period (for females that did not produce offspring).

GROSS NECROPSY and histopathology
- Gross necropsy and histopathology was carried out on testes, seminal vesicles, epididymides, prostate, ovaries uterus, vagina, kidneys, lungs heart, liver, spleen, stomach, thyroid, pituitary glands, adrenal glands, spinal cord and brain.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed on day 21 post partum

GROSS NECROPSY and histopathology
- Gross necropsy and histopathology was carried out on testes, seminal vesicles, epididymides, prostate, ovaries uterus, vagina, kidneys, lungs heart, liver, spleen, stomach, thyroid, pituitary glands, adrenal glands, spinal cord and brain.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
See details on results below
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See details on results below
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
See details on results below
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See details on results below
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
At 6 mg/kg/day, P and F1 mortality was increased in both sexes (statistically significant except for P males). In both P and F1 parental generations, at the high dose level, the following significant clinical effects were observed: rales, gasping, hyperpnoea and irregular breathing. Other clinical effects observed (although not always significant) include, chromorrhinorrhea, yellow/red-brown perioral substance, bradypnoea, alopecia, excess salivation, abdominal distension, red/brown urine, soft/liquid stools or none at all, paleness, cold to the touch, emaciated appearance and head tilt.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
In P and F1 generation males in the highest dose group, a significant decrease in weight gain was observed throughout the study . Highest dose F0 females also showed a decrease in body weight gain during the premating, gestation and lactation periods. High-dose F1 females also showed some significant decreases in premating body weight.

Food consumption in the P and F1 generation males was reduced in the first 3 weeks of the study, but then increased throughout the remainder of the study (increased significantly compared to the control group). A similar effect was also seen with F0 and F1 females in the premating period.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
No effect

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No effects on P and F1 generation male rats. However in F0 females, the reduced F1 pup body weights (high dose group) during the lactation period could be regarded as a reproductive effect.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No effects

GROSS PATHOLOGY (PARENTAL ANIMALS)
In P and F1 high-dose males, F1 females and F1 mid-dose females glandular and forestomach lesions were observed.

HISTOPATHOLOGY (PARENTAL ANIMALS)
In P and F1 high-dose males, F1 females and F1 mid-dose females lesions observed were hyperplasia/hyperkeratosis of the forestomach mucosa, increases of erosion(s), ulcer(s) and haemorrhage of the glandular mucosa and focal hyperplasia of the glandular mucosa. Increases in submucosal inflammation and oedema with mononuclear-cell infiltrates were also seen in areas of erosion and ulcers. No effects were seen in reproductive organs

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Stomach lesions and decrease in body weight gains seen at higher dosage
Dose descriptor:
NOAEL
Remarks:
Reproductive
Effect level:
6 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Maximum tested dosage
Remarks on result:
other: Generation: P, F1 (migrated information)
Dose descriptor:
NOAEL
Remarks:
Reproductive
Effect level:
3 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Assumes reduction in bodyweights for F1 during lactation period are a female reproductive performance effect.
Remarks on result:
other: Generation: P, F1 (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:
not specified
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

VIABILITY (OFFSPRING)
No effects

BODY WEIGHT (OFFSPRING)
F1 pup weights were significantly reduced in the high-dose group.

GROSS PATHOLOGY (OFFSPRING)
No effects

HISTOPATHOLOGY (OFFSPRING)
No effects

Effect levels (F1)

open allclose all
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Stomach lesions and decrease in body weight gain seen at higher dosage
Dose descriptor:
NOAEL
Remarks:
Offspring
Generation:
F1
Effect level:
3 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: F1 pup bodyweights reduced during lactation at 6 mg/kg/day

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the reproductive NOAEL for males is >6 mg/kg/day and for females 3 mg/kg/day.
Executive summary:

In a 2-generation reproduction study acrolein was administered to 30 rats (Crl:CD (SD)BR strain)/sex/dose by oral gavage at dose levels of 0, 1, 3 or 6 mg/kg. In both P and F1 parental generations, at the high dose level, mortality was increased and various clinical signs were observed (rales, gasping, hyperpnoea, irregular breathing and other, minor signs).

 

No reproductive performance effects on P and F1 generation male rats were observed. However for F0 females (of the high dose group) reduced F1 pup body weights during the lactation period could be regarded as a reproductive effect.

 

No significant changes in reproductive organs were seen.

Gross pathology findings in P and F1 high-dose males, F1 females and F1 mid-dose females were glandular and forestomach lesions. Histopathology findings in P and F1 high-dose males, F1 females and F1 mid-dose females were hyperplasia/hyperkeratosis of the forestomach mucosa, increases of erosion(s), ulcer(s) and haemorrhage of the glandular mucosa and focal hyperplasia of the glandular mucosa. Increases in submucosal inflammation and oedema with mononuclear-cell infiltrates were also seen in areas of erosion and ulcers. 

 

The parental NOAEL was 1 mg/kg/day (based on stomach lesions and reduction in body weight gain). Male parental and F1 reproductive NOAEL was >6 mg/kg/day, female parental and F1 reproductive NOAEL was 3 mg/kg/day (assuming reduced body weight in P offspring during lactation is considered a reproductive performance effect) and male/female offspring NOAEL is 3 mg/kg/day (based on reduced pup weights).