Registration Dossier

Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 19, 1999 to July 3, 2003
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Study design is considered to follow OECD guideline Test Guideline No 416 (2001) with insignificant deviations (details on mating procedures lacking, gross observation and histopathology of randomly selected offspring not conducted)

Data source

Referenceopen allclose all

Reference Type:
other: revised final report
Title:
Unnamed
Year:
2003
Report Date:
2003
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
details on mating procedures lacking, gross observation and histopathology of randomly selected offspring not conducted
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
- Storage condition of test material: 5°C protected from light

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: (P) 11 wks; (F1) 11 wks
- Weight at study initiation: (P) Males: 355.6-420.2 g; Females: 220.3-281.6 g; (F1) not reported
- Fasting period before study: no
- Housing: cohoused, 2/cage, polycarbonate cages suspended on stainless-steel racks
- Diet (e.g. ad libitum): Pelleted Harlan Teklad ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23.3
- Humidity (%): 30-70
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: July 13, 1999 To: January 13, 2000

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
VEHICLE
- Justification for use and choice of vehicle (if other than water): not reported.
- Concentration in vehicle: 14, 46, or 140 mg/mL
- Amount of vehicle (if gavage): 1500 mL (total volume)
- Lot/batch no. (if required): not reported
- Purity: 94.9-124.8% (nominal)
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 9 weeks
- Proof of pregnancy: not reported, animals mated on day 8 of dosing
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.: not reported
- Further matings after two unsuccessful attempts: not reported
- After successful mating each pregnant female was caged (how): in cages
- Any other deviations from standard protocol: mating pairs allowed to produced 3 litters, individuals selected from third litter for F1 mating
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dosages were prepared and samples were verified for the first, second, and twenty-sixth mixes. Selected samples were analyzed by high performance liquid chromatography. All dose formulations were within 94.9-124.8% of the nominal concentration
Duration of treatment / exposure:
From 8 days before cohabitation until post natal day 21 after the third litter was born (F1c). F1c selected for F1 cohabitation were administered dosing starting on post natal day 21, with cohabitation at post natal day 81 and the mating pairs were allowed to produce 3 litters
Frequency of treatment:
daily
Details on study schedule:
- F1 parental animals not mated until 61 days after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: 81 days
Doses / concentrationsopen allclose all
Dose / conc.:
70 mg/kg bw/day (actual dose received)
Dose / conc.:
230 mg/kg bw/day (actual dose received)
Dose / conc.:
700 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
20
Control animals:
yes
Details on study design:
- Dose selection rationale: based on range finding study
- Rationale for animal assignment (if not random): random
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once weekly

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly and at littering, as well as Post natal day 4, 7, 14, 18, and 21

FEED CONSUMPTION: Yes
- Time schedule for examinations: weeks 1, 6, 12 (males only), and 14 (males only)
Oestrous cyclicity (parental animals):
Vaginal cytology was performed at 14 days after weaning
Sperm parameters (parental animals):
Parameters examined in all male parental generations:
testis weight, sperm motility, epididymal sperm density, epididymal sperm morphology, spermatids/testis, total spermatids/testis, total spermatid/cauda, epididymis weight.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no, post partum day 16
- If yes, maximum of 4 pups/litter (2/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, body weight, sexual development

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals at study day 178 or 179
- Maternal animals: All surviving animals at study day 178 or 179

GROSS NECROPSY
- Gross necropsy consisted of external surface of the body, all orifices, and the cranial, thoracic, and abdominal cavities and their contents.

HISTOPATHOLOGY / ORGAN WEIGHTS
Organ /tissue histopathology: liver, kidneys, ventral and dorsolateral prostates, seminal vesicles with coagulating glands, spleen, stomach, ovaries, uterus, cervix, vagina, left testis and epididymis, and gross lesions
Organ weights: Liver, spleen, right epididymis, ventral prostate, ovaries, uterus/vagina/cervix, kidneys, right testis, right cauda epididymis
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 21 days of age.
-not examined
Statistics:
Statistical analysis by Analytical Science Inc.
Reproductive indices:
Cochran-Armitage test
Offspring viability indices:
Shirley's or Dunn's test

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical signs noted during the study included abrasions, alopecia, discharge from the eyes and nose, redded area, urine stains, few feces, rales, languid behavior, ulcer, dyspnea, paleness, rough hair coat, swelling, paralysis, malocclusion, small and large stationary tissue masses, and thinness. The incidence of these observations was low to moderate (0-39%), but was not considered to be treatment related. Post-dose observations for clinical signs were performed on all animals. Most observations seen were in the high dose animals and included languid and prostrate behavior and salivation. These observations would continue to be seen until the 1 hour post-dose observation timepoint. At the 2 hour timepoint, all animals would appear normal.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Mortality was observed in four F0 males and ten F0 females. One low dose male was killed moribund on SD138 because of severe clinical signs including dyspnea, rales, few feces, rough haircoat, and languid behavior. Three males (one in each of low-, mid- and high- dose groups) were found dead on SD179, SD10, and SD154, respectively. Each animal appeared normal prior to death. Two mid-dose females were found dead, one on SD114 and one on SD 133. One female appeared normal prior to death. The other female apparently died as a result of gavage error. Eight high dose females were found dead. Two deaths appear to be the result of gavage errors. Three deaths occurred due to parturition difficulties. Three females appeared normal prior to their deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Decreases in body weight (statistically significant decreases of 7.28% and 18.0% at male dose levels of 230 and 700 mg/kg body weight/day and of 9.61%, 11.3%, and 10.8% at female dose levels of 70, 230, and 700 mg/kg body weight/day as measured at Week 26) beginning at week 2 of the study.
Throughout the F0 generation, a dose-related decrease was measured in the mean body weight gain of all adult males compared to control (F0: -8%, -14%* and -34%* at 70, 230 and 700 mg/kg bw/day, respectively). Although not dose related, all F0 females had a reduced mean body weight gain compared to control (-23%*, -24%*, -23%*, for 70, 230 and 700 mg/kg bw/day, respectively).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Decreases in food consumption in mid- and high-dose. Food consumption values were decreased in the high dose F0 males (-27%*, -7%, -12%*, during Week 1, 12 and 14 respectively). During Week 1, mid- and high-dose F0 females had decreased food consumption (-8%* and -20%*). Combined male and female food consumption was not reduced during Week 6.
Food efficiency:
not examined
Description (incidence and severity):
Gavage
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):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
hyperkeratosis and hyperplasia of the squamous epithelium of the non-glandular stomach at all dose levels and in both sexes of the F0 animals. It should be pointed out that the appearance of hyperkeratosis and hyperplasia in non-glandular stomachs and decreased body weight may be due to the route of administration whereby the test material is administered by intubation thereby delivering a bolus of a substance that is shown to have clearly irritant properties at high concentrations.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No changes in number of females with regular cycles, cycle length, number of cycles, and in number of cycling females
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
No changes in sperm motility, epididymal sperm density, percent abnormal sperm, number of spermatids per cauda epididymis, and total number of spermatids per testis
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Decrease in live male pups (21% lower at the high dose compared to controls), decreases in dam body weights at delivery (-8 to -13%), decreases in sire body weights at delivery (-12 to -14%), decrease in absolute female anogenital distance in F1c litter (-8%), dam body weights during lactation in high-dose groups (-7 to -13%); increases in female anogenital distance to body weight ratio in high dose F1a litter (+10%).
The aggregate number of live male pups born during all litters to the F0 parents was decreased by 21%* in the high-dose group (F1a: -22%, F1b: -13%, F1c: -22%). In an outbreeding study, a 42%* increase in the number of live females per litter and a 34%* increase in the number of implantation sites was observed when naïve females were mated with high-dose males. This finding was not reproduced during all litters to the F1 parents and in the outbreeding study, i.e.naïve males mated with high-dose females. Moreover, it occurred only at concentrations greater than parental toxicity. Historical control data are not available, however, in the absence of obvious dose-response reliationship and reproducibility, this finding is considered to be of very low toxicological concern.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
Fertility
Effect level:
>= 700 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effect observed. Expert judgment
Key result
Dose descriptor:
LOAEL
Remarks:
Parental toxicity
Effect level:
<= 70 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Dose descriptor:
NOAEL
Effect level:
>= 230 - < 700 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Expert judgment
Remarks on result:
other: Generation: F1, F2 (migrated information)
Dose descriptor:
LOAEL
Effect level:
700 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on decreased male and female F2 pup weights. Expert judgment. Please see 'Basis for effect level / Remarks".
Remarks on result:
other: Generation: F1, F2 (migrated information)

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical signs noted during the study included abrasions, alopecia, discharge from the eyes, diarrhea, urine stains, few feces, rales, anorexia, hypothermia, dyspnea, paleness, rough hair coat, swelling, hunched posture, malocclusion, small stationary tissue masses, and thinness. The incidence of these observations was low (0-25%), except for alopecia (10-45%), and they were not considered to be treatment related in incidence and severity. Post-dose observations for clinical signs were performed on all animals. Most observations seen were in the mid and high dose animals and included languid and prostrate behavior and salivation. These observations would continue to be seen up to the thirty minute timepoint.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Mortality was observed. One low dose male was killed moribund on PND171 because of severe clinical signs including rouh haircoat, thinness, hunched posture, diarrhea, few feces, red discharge from the mouth, and anorexia. One mid dose male was killed moribund on PND 193 as a result of a gavage error. One high dose male was found dead on PND 171. The animal appeared normal prior to its death. One mid dose female was found dead on PND107 as a result of a gavage error and one on PND124. The second animal appeared normal prior to its death. Two high dose females were found dead on PND109 and PND153. the first animal died immediately after dosing of possible aspiration of compound. The second animal appeared normal prior to its death.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Lower absolute body weights in dams and sires (statistically significant values of 5.39% and 10.0% in mid- and high-dose males; female decreases were not statistically significant).
Throughout the F1 generation, a dose-related decrease was measured in the mean body weight gain of all adult males compared to control (F1: -12%, -25.5%* and -40%* at 70, 230 and 700 mg/kg bw/day, respectively). Similar reduction was only observed in high-dose adult F1 females (-34%*).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption values were decrease in all F1 males during Weeks 12 (-12%*, -13.5* and -15%*, for 70, 230 and 700 mg/kg bw/day, respectively) and Week 14 (-6%, -15% and -23%*, for 70, 230 and 700 mg/kg bw/day, respectively). Combined male and female food consumption was also reduced during Week 3 (-6%, -8%* and -8%*, for 70, 230 and 700 mg/kg bw/day, respectively) and Week 6 (-2%, -5% and --9%*, for 70, 230 and 700 mg/kg bw/day, respectively).
Food efficiency:
not examined
Description (incidence and severity):
gavage
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
Description (incidence and severity):
Organ weight changes included increases in relative weights of the liver, right testis, kidneys, spleen, and cauda epididymis in high-dose males and increases in relative liver and kidney weights in high-dose females compared to controls. 
At the F1 necropsies, some decrease in absolute organ weights and increases in organ-to-body weight ratios were seen in the high-dose males and females. These differences may be attributed to decreased terminal body weights.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Gross findings observed in the males were considered incidental ias not dose-related in incidence and severity with findings spread over the dose groups. There were no gross findings in the females.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings included hyperkeratosis and hyperplasia in the non-glandular stomach at all dose levels and in both sexes of the F1 animals. It should be pointed out that the appearance of hyperkeratosis and hyperplasia in non-glandular stomachs and decreased body weight may be due to the route of administration whereby the test material is administered by intubation thereby delivering a bolus of a substance that is shown to have clearly irritant properties at high concentrations.
Histopathological findings: neoplastic:
no effects observed

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Decreases in dam body weights at delivery of F2 litters (-13 to -16%), decreases in sire body weights at delivery of F2 litters (-18 to -21%), Increase in the female anogenital distance to body weight ratio during the F2a litter by 12%, Decreases in the average absolute F2 combined live male pup weight (-5 to -7%), decreases in the average absolute F2 combined live female pup weight (-4 to -5%), decreases in the average absolute F2 combined live male and female pup weight (-5 to -6%), decreases in the average F2 combined live male pup weight (adjusted for litter size) (-5 to -6%), decreases in the average of F2a litter live female pup weight (adjusted for litter size) (-4 to -5%), decreases in the average combined live male and female pup weight (adjusted for litter size) (-4 to -5%).

Effect levels (P1)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
Fertility
Effect level:
>= 700 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effect observed. Expert judgment.
Key result
Dose descriptor:
LOAEL
Remarks:
Parental toxicity
Effect level:
<= 70 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain

Target system / organ toxicity (P1)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical signs noted during the study included abrasions, alopecia, urine stains, rough hair coat, hunched posture, cyanosis, and thinness. The incidence of these observations was low (0-25%) and they were not considered to be treatment related in incidence or severity. Post-dose clinical observations were performed on all animals. Most observations seen were in the mid and high dose animals and included languid and prostrate behavior and salivation. These observations would continue to be seen up to the thirty minute timepoint.

Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
1 death in mid-dose adolescent male, 2 deaths in high-dose adolescent males, 1 death in high-dose adolescent female. Possible aspiration of compound as cause of deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A decreased body weight was observed in all high-dose litters to the F1 parents compared to control (Combined: -5%, Males: -5.5%, Females: -4%). This effect occurred only at concentrations greater than parental toxicity.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Description (incidence and severity):
Gavage
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
Sexual maturation:
no effects observed
Description (incidence and severity):
No changes were noted in sexual developmental parameters (including testicular descent, preputial separation, and vaginal opening) in any generation. 
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Increases in the female anogenital distance to body weight ratio in the F1a litter compared to control group.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Results: F2 generation

General toxicity (F2)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Slightly lower (i.e., 4 to 5%) pup body weights
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
No changes were noted in sexual developmental parameters (including testicular descent, preputial separation, and vaginal opening). 
Organ weight findings including organ / body weight ratios:
not examined
Description (incidence and severity):
 
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Increases in female F2a litter anogenital distance to body weight ratio compared to controls.

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Effect levels (F2)

open allclose all
Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
>= 230 - < 700 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
LOAEL
Generation:
F2
Effect level:
ca. 700 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
A NOAEL was not reported by the study authors for this study. However, based on expert judgment the following NOAELs/LOAELs are proposed:
NOAEL fertility ≥ 700 mg/kg bw/day.
NOAEL development ≥ 230 mg/kg bw/day / LOAEL = 700 mg/kg bw/day, as a worst-case based on decreased male and female F2 pup weights.
LOAEL parental toxicity ≤ 70 mg/kg bw/day, based on decreased body weight gains at all dose-levels, although probably linked to the bolus effect.
Executive summary:

In a two-generation study conducted similarly to OECD 416 and in compliance with GLP, Isoeugenol was administered to 20 F0 Crl:CD(SD) rat/sex/dose daily via oral gavage at dose levels of 0, 70, 230 or 700 mg/kg bw/day from Study Day (SD) 1 until the day prior to necropsy. The F0 cohabitation began on SD 8. Mating pairs were allowed to produce three litters (F1a, F1b and F1c). Dosing of the F1 generation was initiated on post-natal day (PND) 21 of the F1c animals. On PND 81 ± 10, F1c animals were assigned to mating pairs and allowed to produce three litters (F2a, F2b and F2c).

Endpoints evaluated included body weight, feed consumption, clinical signs, number and weight of pups, anogenital distance, sperm parameters, vaginal cytology, organ weights, and gross and microscopic pathology.

Throughout the F0 and F1 generation, a dose-related decrease was measured in the mean body weight gain of all adult males compared to control (F0: -8%, -14%* and -34%*; F1: -12%, -25.5%* and -40%* during F1, at 70, 230 and 700 mg/kg bw/day, respectively). Although not dose related, all F0 females had a reduced mean body weight gain compared to control (-23%*, -24%*, -23%*, for 70, 230 and 700 mg/kg bw/day, respectively). Similar reduction was only observed in high-dose adult F1 females (-34%*).

Food consumption values were decreased in the high dose F0 males (-27%*, -7%, -12%*, during Week 1, 12 and 14 respectively). During Week 1, mid- and high-dose F0 females had decreased food consumption (-8%* and -20%*). Combined male and female food consumption was not reduced during Week 6. Food consumption values were decrease in all F1 males during Weeks 12 (-12%*, -13.5* and -15%*, for 70, 230 and 700 mg/kg bw/day, respectively) and Week 14 (-6%, -15% and -23%*, for 70, 230 and 700 mg/kg bw/day, respectively). Combined male and female food consumption was also reduced during Week 3 (-6%, -8%* and -8%*, for 70, 230 and 700 mg/kg bw/day, respectively) and Week 6 (-2%, -5% and --9%*, for 70, 230 and 700 mg/kg bw/day, respectively).

The aggregate number of live male pups born during all litters to the F0 parents was decreased by 21%* in the high-dose group (F1a: -22%, F1b: -13%, F1c: -22%). In an outbreeding study, a 42%* increase in the number of live females per litter and a 34%* increase in the number of implantation sites was observed when naïve females were mated with high-dose males. This finding was not reproduced during all litters to the F1 parents and in the outbreeding study, i.e.naïve males mated with high-dose females. Moreover, it occurred only at concentrations greater than parental toxicity. Historical control data are not available, however, in the absence of obvious dose-response reliationship and reproducibility, this finding is considered to be of very low toxicological concern.

A decreased body weight was observed in all high-dose litters to the F1 parents compared to control (Combined: -5%, Males: -5.5%, Females: -4%). This effect occurred only at concentrations greater than parental toxicity.

There were no effects on any other reproductive parameters throughout both generations. Sperm parameters and vaginal cytology were unchanged in the F0 and F1 generations.

At the F0 and F1 necropsies, some decrease in absolute organ weights and increases in organ-to-body weight ratios were seen in the high-dose males and females. These differences may be attributed to decreased terminal body weights. Treatment-related findings included hyperkeratosis and hyperplasia in the non-glandular stomach at all dose levels and in both sexes of the F0 and F1 animals.

It should be pointed out that the appearance of hyperkeratosis and hyperplasia in non-glandular stomachs and decreased body weight may be due to the route of administration whereby the test material is administered by intubation thereby delivering a bolus of a substance that is shown to have clearly irritant properties at high concentrations. (HERA, 2005).

A NOAEL was not reported by the study authors for this study. However, based on expert judgment the following NOAELs/LOAELs are proposed:

NOAEL fertility 700 mg/kg bw/day.

NOAEL development ≥ 230 mg/kg bw/day / LOAEL = 700 mg/kg bw/day, as a worst-case based on decreased male and female F2 pup weights.

LOAEL parental toxicity ≤ 70 mg/kg bw/day, based on decreased body weight gains at all dose-levels, although probably linked to the bolus effect.

* Statistically significant

Reference:

HERA (2005). Risk assessment of Isoeugenol: 4-Hydroxy-3-methoxy-1-propen-1-yl benzene CAS 97-54-1. Human and Environmental Risk Assessment on ingredients of Household Cleaning Products. February 2005.