Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

When the test material was administered to rats continuously in the diet through 2 generations (2 litters per generation) at concentrations up to and including 800 ppm, the only treatment-related effects were decreased body weights, body weight gains and food consumptions in males and females at 400 and 800 ppm of the F0 and F1b generations. Based on these results, the no-observable-effect level was 80 ppm for systemic effects and greater than 800 ppm for male and female reproduction performance.

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:
11th April 1998 to 16th June 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EPA OPP 83-4 (Reproduction and Fertility Effects)
Deviations:
no
Remarks:
There were a number of protocol deviations but none were expected to affect the results of the study
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl: CD BR
Details on species / strain selection:
The rat is sensitive to a number of agents that interfere with reproduction and is frequently used in safety evaluation studies as a representative of a rodent species.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Portage, Michigan, USA
- Age at study initiation: (P) 6 wks
- Weight at study initiation: (P) 123-236 g. The weight variation of the animals of each sex selected for the F0 generation of the study were within ±2 standard deviations of the mean body weight for each sex, and group mean body weights for each sex were not statistically different at the 5 % level.
- Housing: Animals were housed individually in suspended, stainless steel, screen-bottom cages except during breeding periods, when each female was housed with a male in a double-sized, stainless steel cage. Pregnant rats (beginning at approximately Day 15 of gestation), rats with pups, and females that did not show evidence of positive mating were housed in polycarbonate cages. A bedding material consisting of heat-treated hardwood chips covered the bottom of the polycarbonate cages and was changed at least once weekly.
- Diet: Certified Rodent Chow® #5002 ad libitum
- Water: ad libitum from an automatic system or in water bottles (during the last week of gestation and during lactation).
- Acclimation period: (P) two weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 72 ± 3 ºF
- Humidity: 50 ± 20 %
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES
- From: (P) 28 March 1988
- To: (P) Week of 07 November 1988
Route of administration:
oral: feed
Vehicle:
corn oil
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Diets were prepared weekly.
- Mixing appropriate amounts with basal diet: Dietary concentrations were based on the test material after correction for purity. Each dose level was prepared independently in order of increasing concentration. A specified amount of basal diet was weighed into a labelled Hobart® mixing bowl, from which approximately 1 kg was removed and placed in an N-50 mixing bowl. The required amount of test material was weighed in a glass beaker, and a specified amount of corn oil was weighed and added to the test material. This test material and corn oil mixture was stirred, transferred to the N-50 mixing bowl, then thoroughly mixed; this premix was transferred to the Hobart mixing bowl. The contents of the Hobart mixing bowl were then thoroughly mixed. Control diets were prepared using the maximum amount of corn oil that was used to prepare the test diets.
- Storage temperature of food: Diets were stored refrigerated in covered containers until dispensed into food jars.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: Maximum of 21 days.
- Proof of pregnancy: Vaginal plug / sperm in vaginal smear referred to as day 0 of gestation. The date of mating (i.e., date that positive sperm or vaginal plug was observed) and male number were recorded. Vaginal smears were taken daily during cohabitation, and the presence of a copulatory plug or sperm in the vaginal smear was considered evidence of positive mating.
- After successful mating each pregnant female was caged (how): Once mating had occurred, the males and females were separated and females housed individually. On approximately Day 15 of gestation, the mated female was placed in a plastic, solid-bottom nesting cage. Females that showed no evidence of mating were placed in nesting boxes after the mating period.
- Assignment of animals for breeding: Breeding was initiated by selecting 1 male and 1 female at random from the same treated group and placing each pair of animals in a separate, double-sized, screen-bottom cage. The first female in the randomization sequence was placed with the first male in the randomization sequence from the same group. The next female was placed with the next male, and this sequence was repeated until all available animals were paired. Sibling matings were avoided.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples for dose analyses were taken directly from the mixing bowl during diet preparation.
Assays for the test material in the basal diet were done by the testing facility using an analytical method developed and validated in house. Homogeneity was determined for all dietary concentrations. Samples taken from the top, bottom and two opposing sides of the mix were assayed in duplicate for test material content.
To evaluate the stability of the test material in basal diets, four sets of samples were taken from the lowest and highest test material concentrations used for the study. One set was analysed after being stored 7 days in the animal room, then 7 days in a freezer; and one set was analysed after being stored refrigerated for 1 day, 8 days in the animal room, then 27 days in a freezer.
The remaining sets were stored in a freezer; one set was analysed after 18 days and the other set was analysed after 57 days.
All diets for the first 4 weeks of the study were assayed. Each week thereafter, one test material concentration was selected sequentially for analysis. These samples and a sample of the control diet were analysed as a group approximately every 4 weeks. For Weeks 24 and 51, all dose levels were also analysed. All samples were stored in a freezer until analysed.

ANALYTICAL METHOD
The samples are extracted with hexane by shaking for 30 minutes. The extract is centrifuged and cleaned up on a florisil column. The final extract is concentrated by evaporating under nitrogen, transferred into chloroform, and injected on a GC-FID.

- Precision, Sensitivity, Accuracy
The limit of detection for this method was established at 20 ppm. When validated, thirty-two feed samples were spiked and assayed at two separate levels (2000 and 100 ppm). The mean percent recovery for these samples was 95 % with a coefficient of variation of 0.041.

- Interference
A temperature program is recommended because of the presence of late eluting peaks

- Procedure
Extraction: Weigh 10.0 grams of feed directly into a glass stoppered 250 mL Erlenmeyer flask. Add 50 mL of hexane, secure stopper, and shake on a wrist-action shaker for 30 minutes. Centrifuge hexane extract for 10 minutes at 2000 rpm.
Florisil Chromatography: Prepare a "wet packed" florisil column by adding 10 g of activated florisil to a 1.2 x 27 cm glass chromatographic column which is filled with hexane. After the column has settled cap the column with 5.0 grams of Na2SO4 and drain the excess hexane to the top of the Na2SO4 layer. Place an aliquot of hexane extract on the florisil column. Extract the flask with two additional 25 mL rinses and place on the column. Drain to the top of the Na2SO4 layer and discard effluents. Rinse the florisil column with an additional 50 mL of hexane and discard. Elute the test material from the column with 100 mL of 5 % acetone in hexane and collect in a 250 mL boiling flask.
Concentration and GC Analysis: Evaporate the 5 % acetone: hexane to dryness using a nitrogen stream. Extract the dry flask with chloroform rinses, transfer quantitatively to a 10 or 20 mL volumetric flask and dilute to volume. Inject a 2 µL aliquot into a GC-FID under the following conditions:
Column: 3 %, SP 2100 6’x 4 mm ID
Temperature: Injector: 300 °C; Column: 235 °C 8 min, 32 °C/min, 280 °C 8 min; Detector: 300 °C
Carrier: He at 60 mL/min

Due to the nature of the test material, inconsistent responses upon injection were observed. To deter from this the glass wool was removed from the front of the GC column.

- Calculation
C x (V / F) x D = µg/g, ppm test material
where:

C = concentration of extract from standard curve (µg/mL)
V = final volume (mL)
F = weight of feed (g)
D =dilution factor, if any

RESULTS
Mean values for method validation assay results ranged from 88 to 104 % of the theoretical levels. These results indicate that the method was suitable for the range of dietary concentrations in this study.
The results of homogeneity assays at 80, 400 and 800 ppm of diets mixed before initiation of treatment ranged from 104 to 106 %, 99 to 102 % and 97 to 104 % of the theoretical levels, respectively. These results indicate that the mixing procedure produced homogeneous mixtures.
The results for stability analyses of diets mixed at the low (80 ppm) and high (800 ppm) dose levels ranged from 95 to 102 % of the theoretical levels under various storage conditions. Stability assay results (duplicate assay means) for the 80- and 800-ppm dose levels, were as follows: diets stored for 7 days under animal room conditions, followed by 7 days in a freezer were 96 and 95 %, respectively, of the theoretical levels; diets stored refrigerated for 1 day, followed by 8 days under animal room conditions then 27 days in a freezer were 99 and 102 %, respectively, of the theoretical levels; diets stored in a freezer for 18 days were 99 and 98 %, respectively, of the theoretical levels; and diets stored in a freezer for 57 days were 99 and 99 %, respectively, of the theoretical levels.
Control diets were all below the limit of detection of the assay. The results indicate that the test material was stable when mixed in the basal diet. Overall mean dose results for the study were 96, 95, and 96 % of the theoretical levels of 80, 400, and 800 ppm, respectively.
The results of the dose verification analyses from Weeks 1 through 62 indicate that the diets were prepared at the proper levels, except Week 24, when assay results indicated that diets for Groups 3 (400 ppm) and 4 (800 ppm) were inadvertently switched. The in-life data indicate that the inadvertent switch of diets for Week 24 appeared to be insignificant, because decreased body weight, body weight gain, and food consumption were observed in the 400- and 800-ppm groups, and there were no adverse reproductive effects at any dose level.
Duration of treatment / exposure:
Over two generations
Frequency of treatment:
Continuous in the diet
Details on study schedule:
DOSE ADMINISTRATION
The F0 males and females received the test material in the diet continuously from initiation of treatment (11 April 1988) for 10 weeks before mating; throughout mating, gestation, and lactation for the F1a and F1b litters; and until necropsy on 07 to 09 November 1988.
The F1b males and females were exposed to the test material post-weaning for at least 10 weeks before mating; throughout mating, gestation, and lactation for F2a and F2b litters; and until necropsy on 13 to 16 June 1989.

BREEDING
Breeding of the F0 adults for F1a litters was initiated on 20 June 1988, after test diets had been fed to the animals for 10 weeks. After weaning of the F1a litters, the F0 adults were rested for at least 2 weeks. Breeding to produce the F1b litters was initiated on 05 September 1988.
Breeding of the F1b adults for F2a litters was initiated on 24 January 1989, after test diets had been fed to the animals for at least 10 weeks. After weaning of the F2a litters, the F1b adults were rested for at least 2 weeks. Breeding to produce the F2b litters was initiated on 11 April 1989. Breeding pairs used to produce the F1a and F2a litters were not the same pairs used to produce the F1b and F2b litters, respectively.
On Day 21 of F1b lactation, 1 male and 1 female pup were selected at random from each litter to continue as F1b generation adult animals. For litters without a male or female pup, 1 weanling was selected at random from the pups of the available sex. When sufficient pups or litters were not available, male and female pups were selected at random from the remaining litters to provide 25 male and 25 female weanlings/group.
Dose / conc.:
80 ppm (nominal)
Dose / conc.:
400 ppm (nominal)
Dose / conc.:
800 ppm (nominal)
No. of animals per sex per dose:
Twenty-five animals per sex per dose
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Dose levels were selected based on results from a chronic study in rats in which 800 ppm was shown to be the maximum­tolerated dose, and minimal effects were seen at 100 ppm and based on results from previous studies in which effects had been seen at 2000 ppm but not at 900 ppm.
- Rationale for animal assignment: Animals were assigned to the F0 generation of the study using a computer­ generated randomization.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Twice daily (mortality and moribundity); once daily (signs of toxicity). During gestation, females were observed closely for signs of abortion, excessive bleeding, premature delivery or difficult and prolonged parturition.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed physical examination was given on the first day of treatment, weekly thereafter and on the day of necropsy. Females were also examined weekly after the lactation phase.

BODY WEIGHT: Yes
- Time schedule for examinations: Males were weighed on the first day of treatment, weekly thereafter and on the day of necropsy. Females were weighed on the first day of treatment, weekly during premating, on presumed days 0, 7, 14 and 20 of gestation, on days 0, 4, 7, 14 and 21 of lactation and on the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule: Individual food consumption data were recorded weekly during premating phase of the F0 and F1b generations (F1a and F2a litters).

WATER CONSUMPTION: No
Sperm parameters (parental animals):
Parameters examined in all adult males: Weight of testes, epididymides, coagulation gland and seminal vesicles
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded. Any pup with an external abnormality was culled. Culled pups were sacrificed using T-61® euthanasia solution.

PARAMETERS EXAMINED
- Birth (Day 0 of Lactation): As soon as possible after birth, the sex of each pup was determined and the litter size (total number of pups born live or found dead) was recorded. Each live pup was examined for external abnormalities and weighed.
- Day 4: The sex of each pup was determined, and the litter size (number of live pups) was recorded. The pups were examined for external abnormalities and weighed.
- Days 7, 14 and 21: Litter size (number of live pups) was recorded. Live pups were examined for external abnormalities and were weighed individually.

GROSS EXAMINATION OF DEAD PUPS:
Whenever possible, any offspring found dead were examined externally and internally.
- Birth (Day 0 of Lactation): Dead pups were examined macroscopically for cervical, thoracic, and abdominal visceral abnormalities and possible cause of death, then discarded.
- Day 4: Culled pups were examined for cervical, thoracic, or abdominal visceral abnormalities then discarded.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No
Postmortem examinations (parental animals):
SACRIFICE
A necropsy was done to the fullest extent possible on all adult animals that died or were sacrificed in a moribund condition.
After the F1b and F2b pups were weaned, the respective parental (F0 and F1b) adult males and females were anesthetized with methoxyflurane, weighed, exsanguinated, and necropsied.

GROSS NECROPSY
The necropsy included a macroscopic examination of the external surface of the body, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the nasal cavity and paranasal sinuses and the thoracic, abdominal, and pelvic cavities and viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
At the scheduled necropsy, the following (when present), or representative samples thereof, were preserved in 10 % phosphate-buffered formalin: Cervix, coagulation gland, gross lesions, epididymides, ovaries, prostate, pituitary, seminal vesicles, testes, uterus and vagina.
These were embedded in paraffin, sectioned, stained with haematoxylin and eosin, and examined microscopically from all F0 and F1b adults in the control and high-dose groups.
Postmortem examinations (offspring):
SACRIFICE
On Day 21 of F1a lactation, all F1a pups were euthanatized by carbon dioxide asphyxiation and discarded. On Day 21 of F1b lactation, all F1b pups not selected as future parents were euthanatized by carbon dioxide asphyxiation and discarded. On Day 21 of F2a and F2b lactation, or as soon as possible thereafter, all F2a and F2b pups were euthanatized by carbon dioxide asphyxiation and discarded.

GROSS NECROPSY
Whenever possible, any offspring found dead were examined externally and internally.
Statistics:
Levene's test was done to test for variance homogeneity. In the case of heterogeneity of variance at p≤0.05, rank transformation was used to stabilize the variance.
Analysis of variance (ANOVA) was done on the homogeneous or transformed data. If the ANOVA was significant, Dunnett's t-test, Student's t-test or Games and Howell Modified Tukey-Kramer test was used for pairwise comparisons between groups.
Standard one-way ANOVA was used to analyze body weight, body weight gain, food consumption, litter data, days to mate, and length of gestation. Reproduction indices were analyzed by the Cochran-Armitage test for trend and departure and by a Fisher-Irwin exact test.
Group comparisons were found to be statistically significant at the 5.0 and 1.0 % two-tailed probability levels.
As appropriate, for percentages calculated to analyze litter data or mean pup weight data, values were first derived within the litter, and the group mean values were derived as a mean of individual litter percentages.
Reproductive indices:
Mating index = (Number of females mated / Number of females placed with males) x 100
Female fertility index = (Number of females pregnant / Number of females mated) x 100
Male fertility index = (Number of males shown to be fertile / Number of males placed with females) x 100
Gestation index = (Number of live litters born/ Number of pregnant females) x 100
Offspring viability indices:
Viability index = (Number of pups surviving to Day 4 / Number of pups born alive) x 100
Weaning index = (Number of pups surviving to Day 21/ Number of pups alive at Day 4 post-culling) x 100
Sex ratio (Days 0 and 21) = (Number of live male pups / Total number of live pups) x 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test material-related clinical observations were noted.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were no treatment related effects on survival.
In the F0 generation (F1a Litter), 1 male at the 80-ppm level was sacrificed in a moribund condition during Week 17 after showing signs of genital problems (bleeding from the penis) and body weight loss. In the F0 generation (F1b Litter), 1 female in the control group was sacrificed on Day 16 of gestation after showing evidence of abortion; 1 female at the 80-ppm level was found dead 6 days post-copulation; and 1 female at the 80-ppm level was sacrificed in a moribund condition on Day 1 of lactation after a prolonged parturition and subsequent loss of pups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
BODY WEIGHT
In the F0 generation (F1a Litter), mean body weights were significantly (approximately 5 %) lower for the 400-ppm males at Weeks 3 through 8 and 10 of the premating phase and were significantly (9 to 18 %) lower for the 800-ppm males beginning at Week 1 of the premating phase and continuing throughout the postmating phase. The mean body weights were significantly (approximately 5 %) lower for the 400-ppm females for Weeks 7 through 10 of the premating phase and for Days 0 and 7 of gestation and were significantly (approximately 5 to 15 %) lower for the 800-ppm females beginning at Week 1 and continuing throughout the premating, gestation, and lactation phases.
In the F0 generation (F1b Litter), mean body weights were significantly (6 to 7 %) lower for the 400-ppm males throughout most of the postmating phase and were significantly (17 to 19 %) lower for the 800-ppm males throughout the entire premating and postmating phases. The mean body weights were significantly (approximately 7 %) lower for the 400-ppm females throughout the premating and gestation phases and on Days 0 and 4 of lactation. The mean body weights were significantly (7 to 18 %) lower for the 800-ppm females throughout the premating, gestation, and lactation phases.

BODY WEIGHT CHANGES
In the F0 generation (F1a Litter), the mean cumulative body weight gains for the 400- and 800-ppm males were significantly (7 to 10 % and 27 to 41%, respectively) lower throughout most of the premating and postmating phases. Although the mean cumulative body weight gains were significantly (17 %) higher for the 400-ppm females during Weeks 0 to 1 of the premating phase, they were significantly (approximately 10 %) lower for these animals during Weeks 7, 8 and 10 of premating. Cumulative body weight gains were significantly (20 to 29 %) lower for the 800-ppm females throughout the entire premating phase. The overall mean cumulative body weight gains during lactation for the 400- and 800- ppm females were significantly higher than those of the controls.
In the F0 generation (F1b Litter), the mean cumulative body weight gains were significantly lower for the 400-ppm males throughout most of the postmating phase and lower for the 800-ppm males during Week 1 of the premating phase and throughout most of the postmating phase. The mean cumulative body weight gains were significantly (33 %) lower for the 400-ppm females for Days 7 to 14 of gestation. The overall mean cumulative body weight gains during lactation for the 400- and 800-ppm females were significantly higher than those of the controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In the F0 generation, mean food consumptions were significantly (5 to 8 %) lower for the 400-ppm males during Weeks 0 to 1, 4 to 5 and 8 to 9 of the premating phase and were significantly (8 to 21 %) lower for the 800-ppm males throughout the entire premating phase. The mean food consumptions were significantly (approximately 7 %) lower for the 400-ppm females during Weeks 6 to 7 and were significantly (11 to 21 %) lower for the 800-ppm females throughout the entire premating phase.
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test material-related microscopic changes. All microscopic findings were considered to be incidental and unrelated to the dietary intake of the test material.
Histopathological findings: neoplastic:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
- Males: There were no adverse significant differences for male fertility indices during breeding for the F1a and F1b litters.
- Females: There were no adverse significant differences observed for mating, female fertility, or gestation indices or mean days to mate for the F1a and F1b litters. There was a statistically significant decrease in the mean duration of F1a gestation for the 400- and 800-ppm females; however, this difference was very slight and is not considered to be biologically significant.
Key result
Dose descriptor:
NOEL
Remarks:
Systemic effects
Effect level:
80 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Key result
Dose descriptor:
NOEL
Remarks:
Reproductive performance
Effect level:
> 800 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Key result
Critical effects observed:
no
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test material-related clinical observations were noted.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were no treatment related effects on survival.
No deaths occurred during the F1b generation (F2a Litter) phase of the study. In the F1b generation (F2b Litter), 1 male at the 800-ppm level was sacrificed in a moribund condition during Week 6 after showing signs of paralysis in the hind limbs; 1 female at the 80-ppm level was sacrificed in a moribund condition 7 days post-copulation after a movable mass became ulcerated; and 1 female at the 400-ppm level was sacrificed in a moribund condition on Day 2 of lactation after showing signs of staggered gait, red stains at the eyes, mouth, forelimbs, and genital area and emaciation (the entire litter was stillborn).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
BODY WEIGHT
In the F1b generation (F2a Litter), mean body weights were significantly (5 to 9 %) lower for the 400-ppm males throughout most of the premating phase and continuing throughout the postmating phase and were significantly (26 to 29 %) lower for the 800-ppm males throughout the entire premating and postmating phases. The mean body weights were significantly (approximately 5 to 10 %) lower for the 400-ppm females beginning Week 1 and continuing throughout the premating and gestation phases and on Days 0, 4, 7, and 14 of lactation. The mean body weights were significantly (18 to 22 %) lower for the 800-ppm females beginning at Week 0 and continuing throughout the premating, gestation, and lactation phases.
In the F1b generation (F2b Litter), mean body weights for the 400- and 800-ppm males were significantly (8 to 10 % and 26 to 27 %, respectively) lower throughout the entire premating and postmating phases. The mean body weights were significantly (5 to 10 % and 15 to 22 %) lower for the 400- and 800-ppm females, respectively, throughout the premating, gestation, and lactation phases.

BODY WEIGHT CHANGES
In the F1b generation (F2a Litter), the mean cumulative body weight gains were significantly (10 to 12 %) lower for the 400-ppm males throughout most of the premating phase, during mating and Weeks 11, 12, and 16 postmating and were significantly (25 to 29 %) lower for the 800-ppm males throughout the entire premating and postmating phases. The mean cumulative body weight gains were significantly (18 to 24 % and 19 to 22 %) lower for the 400- and 800-ppm females throughout the entire premating phase. The mean cumulative body weight gains for the 800-ppm females were significantly (approximately 18 %) lower throughout most of gestation. The overall mean cumulative body weight gains during lactation for the 400-ppm females were significantly higher than those of the controls.
In the F1b generation (F2b Litter), the mean cumulative body weight gains were significantly (21 to 24 % and 27 to 34 %) lower for the 400- and 800-ppm males, respectively, during the last 2 weeks of the postmating phase. Although the mean cumulative body weight gains for the 80-ppm females were significantly higher during the 2-week period before mating, no significant changes were seen during gestation or lactation for these animals. The mean cumulative body weight gains were significantly (28 %) lower for the 400-ppm females for Days 0 to 7 of gestation and significantly (16 to 36 %) lower for the 800-ppm females throughout most of gestation. The overall mean cumulative body weight gains during lactation for the 400- and 800-ppm females were significantly higher than those of the controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In the F1b generation, mean food consumptions were significantly (5 to 8 %) higher for the 80-ppm males starting at Week 0 and continuing through Week 6 of premating; however, these differences were not considered biologically meaningful because there were no significant differences in body weight or body weight change data for this time period. The mean food consumptions were significantly (6 to 8 %) lower for the 400-ppm males starting at Week 2 and continuing throughout the premating phase and significantly (19 to 25 %) lower for the 800-ppm males throughout the entire premating phase. The mean food consumptions for the 400-ppm females were significantly (9 to 19 %) lower throughout most of the premating phase and significantly (17 to 31 %) lower for the 800-ppm females throughout the entire premating phase.
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test material-related macroscopic changes. All macroscopic findings were considered to be incidental and unrelated to the dietary intake of the test material.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test material-related microscopic changes. All microscopic findings were considered to be incidental and unrelated to the dietary intake of the test material.
Histopathological findings: neoplastic:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
- Males: There were no adverse significant differences for male fertility indices during breeding for the F2a and F2b litters.
- Females: There were no adverse significant differences observed for mating, female fertility, or gestation indices or mean days to mate for the F2a and F2b litters.
Key result
Dose descriptor:
NOEL
Remarks:
Systemic effects
Effect level:
80 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Key result
Dose descriptor:
NOEL
Remarks:
Reproductive performance
Effect level:
> 800 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Key result
Critical effects observed:
no
Clinical signs:
not examined
Mortality / viability:
not specified
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Pup weights were significantly lower for the F1a litters at the 400-ppm dose level for Lactation Days 7, 14, and 21 and at the 800-ppm dose level for Lactation Days 0, 4, 7, 14, and 21 and significantly lower for the F1b litter at the 400-ppm dose level for Lactation Days 7, 14, and 21 and at the 800-ppm dose level for Lactation Days 4, 7, 14, and 21.
At Lactation Day 21, F1a and F1b pup weights in the 400-ppm group were 12 and 8 % lower, respectively, than those of the controls; pup weights in the 800-ppm group were 37 and 36 % lower, respectively, than those of the controls.
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:
not examined
Description (incidence and severity):
Although not specifically examined, the F1b animals were bred to produce the F2a and F2b litters with no effects being seen on reproduction parameters.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test material-related findings noted during the necropsy of culled pups or pups found dead.
Histopathological findings:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
In general, there were no adverse significant differences observed for the number of pups per litter or sex ratio for the F1a and F1b litters.
Key result
Dose descriptor:
NOEL
Generation:
other: F1a and F1b
Effect level:
80 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Critical effects observed:
no
Clinical signs:
not examined
Mortality / viability:
not specified
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Pup weights were significantly lower for the F2a litter at the 400-ppm dose level for Lactation Days 14 and 21 and at the 800-ppm dose level for Lactation Days 0, 4, 7, 14 and 21; and significantly lower for the F2b litter at the 400-ppm dose level for Lactation Days 7, 14, and 21 and at the 800-ppm dose level for Lactation Days 0, 4, 7, 14, and 21.
At Lactation Day 21, F2a and F2b pup weights in the 400-ppm group were 11 and 14 % lower, respectively, than those of the controls; pup weights in the 800-ppm group were 43 and 38 % lower, respectively, than those of the controls.
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:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test material-related findings noted during the necropsy of culled pups or pups found dead.
Histopathological findings:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
In general, there were no adverse significant differences observed for the number of pups per litter or sex ratio for the F2a and F2b litters.
Key result
Dose descriptor:
NOEL
Generation:
other: F2a and F2b
Effect level:
80 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
When the test material was administered to rats continuously in the diet through 2 generations (2 litters per generation) at concentrations up to and including 800 ppm, the only treatment-related effects were decreased body weights, body weight gains and food consumptions in males and females at 400 and 800 ppm of the F0 and F1b generations. Based on these results, the no-observable-effect level was 80 ppm for systemic effects and greater than 800 ppm for male and female reproduction performance. These values are equivalent to 4 and 40 mg/kg bw/day, respectively.
Executive summary:

The purpose of this study was to assess the effects of the test material on male and female gonadal function, mating behaviour, fertility, gestation, parturition and lactation and the growth and development of offspring from two consecutive generations of rats (2 litters per generation) that were continuously exposed to the test material. The F0 generation had the F1a and F1b litter; the F1b generation had the F2a and F2b litters.

Groups of 25 male and 25 female immature Crl:CD®BR albino rats (F0 animals) were fed diets that contained 0, 80, 400 or 800 ppm for 10 weeks premating and throughout mating, gestation, lactation and weaning of the F1a pups. After weaning of the F1a litters, the F1a pups were sacrificed and discarded, and the F0 animals were then re-mated to produce F1b litters. After weaning, the F1b animals were assigned at random to 4 groups (25/sex/group) and the F0 adults were sacrificed and necropsied. The selected F1b animals were fed diets that contained 0, 80, 400 or 800 ppm for at least 10 weeks premating and throughout gestation, lactation and weaning of the F2a pups. After weaning of the F2a litters, the F2a pups were sacrificed and discarded, and the F1b animals were then re-mated to produce F2b litters. After weaning of the F2b litters, the F2b pups were sacrificed and discarded and the F1b adults were sacrificed and necropsied.

Test diets were fed continuously throughout the study. Antemortem data (i.e., clinical observations, body weights and food consumptions), reproduction data, and litter data were recorded. All F0 and F1b adults were examined macroscopically; microscopic examinations were done on the reproductive organs from the 0- and 800-ppm animals.

There were no test material-related clinical observations for F0 or F1b adults. Survival for F0 generation was 100 % for males at 0, 400 or 800 ppm and for females at 400 or 800 ppm; 96 % for males at 80 ppm and females at 0 ppm; and 92 % for females at 80 ppm. Survival for F1b generation was 100 % for males at 0, 80 and 400 ppm and for females at 0 or 800 ppm and 96 % for males at 800 ppm and females at 80 or 400 ppm.

In general, body weights and cumulative body weight gains were significantly lower than those of the controls at the 400- and 800-ppm dose levels during both generations for males and females premating, for males postmating, and for females during gestation and lactation. Body weights for males and females in the 400-ppm groups were generally 5 to 10 % lower than those of the controls. When body weights for animals in the 800-ppm groups were compared with those of the controls, F0 males were 9 to 19 % lower, F0 females were 5 to 18 % lower, F1b males were 26 to 29 % lower and F1b females were 15 to 22 % lower.The differences in cumulative body weight gains were similar to those found in body weights, except that the reductions from the controls were of a greater magnitude.

During the F0 generation, food consumptions were significantly (approximately 7 %) lower than those of the controls for the 400-ppm males (Weeks 0 to 1, 4 to 5, and 8 to 9) and for the 400-ppm females (Weeks 6 to 7).           Food consumptions for the 800-ppm F0 males and females were significantly (approximately 10 to 20 %) lower than those of the controls throughout most of the premating phase. During the F1b generation, food consumptions were significantly (approximately 7 %) lower than those of the controls for the 400-ppm males (Weeks 2 to 10) and significantly lower than those of the controls for the 400-ppm females (9 to 19 %) and 800-ppm males (19 to 25 %) and females (17 to 31 %) throughout most of the premating phase.

There were no adverse significant differences from controls in mating or male or female fertility indices during breeding for the F1a, F1b, F2a, and F2b litters. There were no significant differences from controls in female gestation indices or mean days to mate for the F1a, F1b, F2a, and F2b litters.

During the F1a, F1b, F2a, and F2b litters, there were no significant differences from controls for number of pups per litter or for pup (male and female) sex ratios. In general, the mean pup weights for most litters were significantly (approximately 10 %) lower than those of the controls at the 400-ppm dose level for Lactation Days 7, 14, and 21 and significantly lower than those of the controls at the 800-ppm dose level for Lactation Days 0, 4, 7, 14, and 21. Pup weights in the 800-ppm group on Lactation Day 21 were 36 to 43 % lower than those of the controls.

There were no test material-related macroscopic or microscopic changes.

When the test material was administered to rats continuously in the diet through 2 generations (2 litters per generation) at concentrations up to and including 800 ppm, the only treatment-related effects were decreased body weights, body weight gains and food consumptions in males and females at 400 and 800 ppm of the F0 and F1b generations. Based on these results, the no-observable-effect level was 80 ppm for systemic effects and greater than 800 ppm for male and female reproduction performance. These values are equivalent to 4 and 40 mg/kg bw/day, respectively.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
58.5 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The key study was performed to GLP and has a Klimisch score of 1. Although conducted to the contemporaneous iteration of OECD 416, some may consider that there are deficiencies in this study compared to current requirements. However it is considered that the deficiencies can be met from other existing data and a new study is not required. Further discussion is provided below and in the attached document.
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 purpose of this study was to assess the effects of the test material on male and female gonadal function, mating behaviour, fertility, gestation, parturition and lactation and the growth and development of offspring from two consecutive generations of rats (2 litters per generation) that were continuously exposed to the test material. The F0 generation had the F1a and F1b litter; the F1b generation had the F2a and F2b litters.

Groups of 25 male and 25 female immature Crl:CD®BR albino rats (F0 animals) were fed diets that contained 0, 80, 400 or 800 ppm for 10 weeks premating and throughout mating, gestation, lactation and weaning of the F1a pups. After weaning of the F1a litters, the F1a pups were sacrificed and discarded, and the F0 animals were then re-mated to produce F1b litters. After weaning, the F1b animals were assigned at random to 4 groups (25/sex/group) and the F0 adults were sacrificed and necropsied. The selected F1b animals were fed diets that contained 0, 80, 400 or 800 ppm for at least 10 weeks premating and throughout gestation, lactation and weaning of the F2a pups. After weaning of the F2a litters, the F2a pups were sacrificed and discarded, and the F1b animals were then re-mated to produce F2b litters. After weaning of the F2b litters, the F2b pups were sacrificed and discarded and the F1b adults were sacrificed and necropsied.

Test diets were fed continuously throughout the study. Antemortem data (i.e., clinical observations, body weights and food consumptions), reproduction data, and litter data were recorded. All F0 and F1b adults were examined macroscopically; microscopic examinations were done on the reproductive organs from the 0- and 800-ppm animals.

There were no test material-related clinical observations for F0 or F1b adults. Survival for F0 generation was 100 % for males at 0, 400 or 800 ppm and for females at 400 or 800 ppm; 96 % for males at 80 ppm and females at 0 ppm; and 92 % for females at 80 ppm. Survival for F1b generation was 100 % for males at 0, 80 and 400 ppm and for females at 0 or 800 ppm and 96 % for males at 800 ppm and females at 80 or 400 ppm.

In general, body weights and cumulative body weight gains were significantly lower than those of the controls at the 400- and 800-ppm dose levels during both generations for males and females premating, for males postmating, and for females during gestation and lactation. Body weights for males and females in the 400-ppm groups were generally 5 to 10 % lower than those of the controls. When body weights for animals in the 800-ppm groups were compared with those of the controls, F0 males were 9 to 19 % lower, F0 females were 5 to 18 % lower, F1b males were 26 to 29 % lower and F1b females were 15 to 22 % lower.The differences in cumulative body weight gains were similar to those found in body weights, except that the reductions from the controls were of a greater magnitude.

During the F0 generation, food consumptions were significantly (approximately 7 %) lower than those of the controls for the 400-ppm males (Weeks 0 to 1, 4 to 5, and 8 to 9) and for the 400-ppm females (Weeks 6 to 7).           Food consumptions for the 800-ppm F0 males and females were significantly (approximately 10 to 20 %) lower than those of the controls throughout most of the premating phase. During the F1b generation, food consumptions were significantly (approximately 7 %) lower than those of the controls for the 400-ppm males (Weeks 2 to 10) and significantly lower than those of the controls for the 400-ppm females (9 to 19 %) and 800-ppm males (19 to 25 %) and females (17 to 31 %) throughout most of the premating phase.

There were no adverse significant differences from controls in mating or male or female fertility indices during breeding for the F1a, F1b, F2a, and F2b litters. There were no significant differences from controls in female gestation indices or mean days to mate for the F1a, F1b, F2a, and F2b litters.

During the F1a, F1b, F2a, and F2b litters, there were no significant differences from controls for number of pups per litter or for pup (male and female) sex ratios. In general, the mean pup weights for most litters were significantly (approximately 10 %) lower than those of the controls at the 400-ppm dose level for Lactation Days 7, 14, and 21 and significantly lower than those of the controls at the 800-ppm dose level for Lactation Days 0, 4, 7, 14, and 21. Pup weights in the 800-ppm group on Lactation Day 21 were 36 to 43 % lower than those of the controls.

There were no test material-related macroscopic or microscopic changes.

When the test material was administered to rats continuously in the diet through 2 generations (2 litters per generation) at concentrations up to and including 800 ppm, the only treatment-related effects were decreased body weights, body weight gains and food consumptions in males and females at 400 and 800 ppm of the F0 and F1b generations. Based on these results, the no-observable-effect level was 80 ppm for systemic effects and greater than 800 ppm for male and female reproduction performance.

Despite the study not being conducted to the most recent iteration of the OECD 416 guideline, alleged deficiencies can be addressed from other existing data. In addition there are no triggers in existing data that would require the conduct of additional immuno- or neuro-toxicity cohorts in an EOGRTS study.

 

In addition :-

Reprotoxicity or fertility were not identified as a critical area of concern by EFSA in the peer review conclusion report (EFSA Journal 2011; 9(5):2087) when considering propargite for use as a plant protection product.

 

The CORAP report of 17/03/2015 did not consider a new multi-generation study or EOGRTS was required to further clarify endocrine concerns.

 

Overall existing data is sufficient to address all reproductive endpoints required for an up-to-date risk assessment, within which there is no indication that the NOAELs derived from Kehoe 1990 should be altered.

 

Full explanation can be found in the attached document 'CH-D-2114311551-64-01_D_DD Response', supported by 'US EPA memorandum EDSP WoE propargite 2015-06-29', also attached.

As reported in the WoE document, a series of studies were conducted in line with those recommended in Tier 1 of the US EPA Endocrine Screening strategy. These did not indicate any endocrine activity that would impact on the human health risk assessment for propargite. Further detail is provided in section 7.9.3.

Effects on developmental toxicity

Description of key information

NOAEL for maternal toxicity was determined to be 4 mg/kg bw/day. NOAEL for developmental toxicity was determined to be 6 mg/kg bw/day; study conducted in accordance with OECD 414 and EPA OPP 83-3; Schardein (1989)

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10th July 1989 to 11th August 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPP 83-3 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Hazleton Research Products, Denver, Pennsylvania, USA
- Age at study initiation: approximately 5-6 months
- Weight at study initiation: 3049-4528 g on gestation day 0
- Housing: individually in suspended stainless steel cages
- Diet: Certified Rabbit Chow® #5322 ad libitum
- Water: tap water ad libitum
- Acclimation period: 25-67 days

ENVIRONMENTAL CONDITIONS
- Temperature: 67-76 ºF (mean 71 ± 2.3 ºF)
- Humidity: 54-70 % (mean 62 ± 4.4 %)
- Photoperiod: 12 hrs dark / 12 hrs light
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- The appropriate amount of test material was weighed into a beaker and the appropriate amount of corn oil added. The test material was stored at room temperature with fresh material prepared weekly.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
Prior to test material administration, the 10 day stability of the test material stored at room temperature was established.

To ensure correct concentrations of the test material, analyses of the prepared dosing suspension was conducted prior to dosing on days 7 and 12.
Details on mating procedure:
- Impregnation procedure: artificial insemination
Duration of treatment / exposure:
Days 7 to 19 of gestation
Frequency of treatment:
Daily
Duration of test:
Up to gestation day 29.
Dose / conc.:
2 mg/kg bw/day (nominal)
Dose / conc.:
4 mg/kg bw/day (nominal)
Dose / conc.:
6 mg/kg bw/day (nominal)
Dose / conc.:
8 mg/kg bw/day (nominal)
Dose / conc.:
10 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Twenty-five
Control animals:
yes, concurrent vehicle
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked: mortality and overt changes in appearance and behaviour.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily on days 7 to 29 of gestation.

BODY WEIGHT: Yes
- Time schedule for examinations: days 0, 7, 13, 20, 24 and 29 of gestation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29. The abdominal and thoracic cavities and organs of the females were examined for grossly evident morphological changes and the carcasses discarded.
- Females not surviving to scheduled sacrifice were necropsied in an attempt to determine the cause of death.

OTHER:
- Any female showing signs of abortion or premature delivery was sacrificed on the day such evidence was seen and the aborted tissue examined and preserved.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: location of viable and non-viable foetuses
Fetal examinations:
- External examinations: Yes [all per litter]
- Soft tissue examinations: Yes [all per litter]
- Skeletal examinations: Yes [all per litter]
- Head examinations: No
Statistics:
Sex ratios of offspring compared using chi-squared test and/or Fisher's exact probability test. Proportion of resorbed/dead foetuses and post-implantation losses compared using Mann-Whitney test. Mean maternal body weight, corpora lutea, total implantations, number of live foetuses and pups compared with one-way analysis of variance using Bartlett's test for homogeneity of variance and the appropriate t-test to determine significance of differences.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Antemortem signs observed prior to abortion included hair loss, aborted material, anogenital staining, decreased defecation and emaciation
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were no treatment-related effects on survival. One animal in each of the 6 and 8 mg/kg bw/d groups died although the cause of death was not established.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was a significant reduction in body weight and in body weight gain of the 8 and 10 mg/kg animals but only during gestation days 7-20 (see Tables 1 and 2).
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
effects observed, treatment-related
Description (incidence and severity):
Abortions occurred in three, one and four females in the 4, 8 and 10 mg/kg bw/d groups, respectively. The abortions at 4 and 8 mg/kg bw/d were not considered to be treatment-related due to a lack of dose-response (no abortions at 6 mg/kg bw/d). The abortions at 10 mg/kg bw/d, however, were deemed to be treatment-related by virtue of the other signs of toxicity observed at this dose level.
Pre- and post-implantation loss:
not specified
Description (incidence and severity):
Following caesarean section, pre- and post-implantation losses and total implantations in treated animals were all comparable to the control group.
Total litter losses by resorption:
not specified
Early or late resorptions:
not specified
Dead fetuses:
no effects observed
Description (incidence and severity):
Following caesarean section, foetal viability in treated animals was comparable to the control group.
Changes in pregnancy duration:
not specified
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified
Changes in number of pregnant:
not specified
Other effects:
no effects observed
Description (incidence and severity):
Following caesarean section, corpora lutea in treated animals were comparable to the control group.
Key result
Dose descriptor:
NOAEL
Remarks:
maternal toxicity
Effect level:
4 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical signs
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Following caesarean section, foetal body weight in treated animals was comparable to the control group.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Following caesarean section, foetal body weight in treated animals was comparable to the control group.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Following caesarean section, foetal viability in treated animals was comparable to the control group.
Changes in sex ratio:
not examined
Changes in litter size and weights:
not specified
Changes in postnatal survival:
not specified
External malformations:
not specified
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Fused sternebrae was observed in kits from females treated at dose levels of 8 and 10 mg/kg bw/d (in 2/125 and 9/116 foetuses, respectively; 8 % of foetuses, 56 % of litters at 10 mg/kg/day). Fused skull bones were also noted in kits from these dose groups but there was no dose-response (2.4 % of foetuses, 17 % of litters at 8 mg/kg/day). There were no other treatment related malformations or developmental variations.
Visceral malformations:
not specified
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
6 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Table 1: Summary of body weight values (g)

 0 mg/kg/day  2 mg/kg/day  4 mg/kg/day  6 mg/kg/day  8 mg/kg/day  10 mg/kg/day
 Day of study  Mean  S.D.  Mean  S.D.  Mean  S.D.  Mean  S.D.  Mean  S.D.  Mean  S.D.
 0  3471  329.0  3570  360.7  3632  352.4  3664  358.2  3574  414.0  3530  329.0
 7  3674  350.9  3784  398.1  3824  373.6  3811  376.3  3752  435.0  3733  347.5
 13  3760  353.1  3872  374.4  3910  344.3  3824  343.3  3792  399.7  3679  248.8
 20  3787  276.7  3950  361.3  3943  331.0  3849  365.6  3760  401.4  3648  272.3
 24  3885  274.0  4001  419.6  4006  378.5  3904  371.4  3918  423.0  3781  227.2
 29  3958  250.2  4110  358.3  4093  337.4  3985  334.8  4014  406.0  3907  261.3

Table 2: Summary of body weight gain values (g)

 0 mg/kg/day  2 mg/kg/day  4 mg/kg/day  6 mg/kg/day  8 mg/kg/day  10 mg/kg/day
 Day of study  Mean  S.D.  Mean  S.D.  Mean  S.D.  Mean  S.D.  Mean  S.D.  Mean  S.D.
 0-7  203  107.6  214  84.8  193  91.0  147  60.5  177  73.0  203  73.5
 7-13  87  100.3  88  69.7  86  98.8  13  116.6  41  119.4  -55  171.8
 13-20  27  147.1  78  89.9  33  201.8  25  203.0  -32  198.2  -4  209.2
 7-20  114  188.3  165  133.2  119  253.4  38  290.7  9  267.5  -20  308.4
 20-24  98  77.6  52  92.7  55  158.6  54  101.3  139  118.4  101  89.3
 24-29  73  254.9  108  144.6  49  129.7  46  150.7  97  94.2  126  129.8
 0-29  487  254.9  540  135.0  493  205.8  318  369.9  447  201.6  464  188.9
Conclusions:
Under the conditions of the test, dose levels of 8 and 10 mg/kg bw/day led to maternal toxicity as evidenced by abortions at the 10 mg/kg level and significantly reduced body weight/body weight gain at both dose levels. The only potential developmental toxicity was manifested by fused sternebrae in a small percentage of kits from the 8 and 10 mg/kg bw/d females (i.e. from groups treated with a maternally toxic dose). However, this is a spontaneously occurring variation that is commonly seen in kits delivered by control animals (as supported by a large historical control database). Moreover, there were no other signs of developmental toxicity as evidenced by an absence of adverse effects on foetal viability, foetal body weight, pre- and post implantation losses, total implantations and corpora lutea. Detailed analysis of the litters containing kits with fused sternebrae indicate that only two litters contained >1 kit with the condition (3 and 2 foetuses in each, respectively), the other litters each containing just one kit exhibiting fused sternebrae, an incidence which would not be considered treatment-related. Taken with the absence of any other indications of developmental toxicity in this study, this very slight increase in incidence of a normally spontaneously occurring variation in just two litters is not considered to indicate a teratogenic potential for the test material. In addition, this developmental variation was not observed at dose levels below the maternally toxic dose of 8 mg/kg bw/d. Based on these results, the NOEL for maternal toxicity was determined to be 6 mg/kg bw/d; this value also covers the increased incidence of fused sternebrae seen in the 8 mg/kg bw/d animals.
Executive summary:

New Zealand White rabbits were dosed with 0, 2, 4, 6, 8 and 10 mg/kg/day test material by oral gavage as a single daily dose on days 7 to 19 of gestation. Caesarean examinations were performed on all surviving females on gestation day 29 followed by teratologic examinations.

8 and 10 mg/kg/day does exhibited evidence of maternal toxicity with respect to abortions at the 10 mg/kg/day level and body weight inhibition/loss during the treatment period at both levels. There were malformed (fused) sternebrae in 2 and 9 kits respectively in the 8 and 10 mg/kg/day groups. This was the only evidence of developmental toxicity.

The NOEL was determined to be 6 mg/kg/day for both maternal and developmental toxicity.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
6 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Quality of whole database:
One GLP study with a Klimisch score of 1 and 2 each are available; two non-GLP studies with Klimisch scores of 2 are available. The quality of the database is therefore high.
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

Administration of technical propargite by oral gavage to rabbits from days 7 to 19 of gestation at 8 and 10 mg/kg bw/day lead to maternal toxicity as evidenced by abortions at 10 mg/kg and significantly reduced body weight and body weight gain at both dose levels. Fused sternebrae in a small percentage of kits from these dose levels was also observed; however, this is a spontaneously seen variation that is commonly seen. Moreover, there were no other signs of developmental toxicity as evidenced by an absence of adverse effects on foetal viability, foetal body weight, pre- and post-implantation losses, total implantations and corpora lutea. This very slight increase in the incidence of a normally spontaneously occurring variation is not considered to indicate a teratogenic effect. Based on these results, the NOEL for maternal toxicity was found to be 4 mg/kg bw/day (which covers the increased incidence of fused sternebrae at 8 mg/kg bw/day). This study was performed to GLP and in line with standardised guidelines and so is considered to be the key study for this endpoint.

Supporting information includes three studies on rats performed in line with sound scientific principles, one in line with GLP and two not to GLP.

Toxicity to reproduction: other studies

Description of key information

A series of studies were conducted in line with those recommended in Tier 1 of the US EPA Endocrine Screening strategy.

These did not indicate any endocrine activity that would impact on the human health risk assessment for propargite.

Further detail is provided in section 7.9.3.

Additional information

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay battery as defined by the US EPA is designed to provide the necessary empirical data to evaluate the potential of chemicals to interact with the estrogen (E), androgen (A) or thyroid (T) signalling pathways. This interaction includes agonism and antagonism at the estrogen and androgen receptosr, altered steroidogenesis, as well as hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary thyroid (HPT) axes.

In addition to the available Tier 1 assay data, other scientifically relevant information (OSRI), including general toxicity data and open literature studies of sufficient quality were considered in a weight of evidence (WoE) assessment by the US EPA. The studies considered are owned by the registrant and summarised in this IUCLID dossier. 

In determining whether propargite interacts with E, A or T hormone pathways, the number and type of effects induced, the magnitude and pattern of responses observed across studies, taxa, and sexes were considered. Additionally, the conditions under which effects occur were considered, in particular, the dose(s) at which responses happen and whether overt toxicity was present. The conclusions from the WoE evaluation are presented by pathway (E, A and then T) beginning with the results of the Tier 1 in vitro assays followed by in vivo mammalian and wildlife results, then the results of the cited literature for mammalian and wildlife studies. For propargite, for the estrogen pathway, the Tier 1 in vitro assays were negative. In the female pubertal assay, the only estrogen-related effect observed in the absence of overt toxicity was an increases in the incidence and severity of interstitial cell hyperplasia of the ovaries (7/16 rats versus 0/16 in controls) at the low dose (62.5 mg/kg/day). There were no estrogen-related effects observed in the mammalian toxicity studies. In the fish short-term reproduction assay (FSTRA), while fecundity was lower than guideline criteria and ovarian histopathological alterations were observed in both the controls and treatments which lessen the degree of confidence in this study, there were limited effects observed in this study. A delay in ovary maturation stage was observed at the high concentration and a decrease in gonadal somatic index (GSI) at the low treatment. In the Part 158 mallard duck and bobwhite quail reproduction studies, there was a decrease in egg laying at the high concentration however this was coincident with decreases in body weight gains which was considered overtly toxic in the mallard study. Therefore, overall, there is a lack of convincing evidence for potential interaction with the estrogen pathway for mammals or wildlife.

For the androgen pathway, the Tier 1 in vitro assays were negative. In the male pubertal assay, the only effect observed in the absence of overt toxicity was a decrease in serum testosterone levels seen at the low dose (62.5 mg/kg/day). In the Hershberger assay, there was a dose-dependent decrease in androgen responsive tissue weights; ventral prostate and LABC weights were decreased at all doses as well as effects on seminal vesicles at the high dose (150 mg/kg/day); however the effects at the high dose were in the presence of overt toxicity (>10% decrease in body weight). No androgen-related effects were observed in the Part 158 studies. In the FSTRA, again, while gonadal histopathological alterations were observed in both controls and treatment groups, there were no effects observed in the male fish. Therefore, given the lack of responses in the in vitro assays, the male pubertal and Part 158 mammalian studies and the FSTRA, there is no convincing evidence of potential interaction with the androgen pathway in mammals and wildlife. Thyroid-related effects were observed in the male and female pubertal assays in the absence of overt toxicity, as well as in the amphibian metamorphosis assay (AMA). Increased follicular cell height and decreased colloidal area were observed in the male and female pubertal assays, while increased follicular cell hyperplasia and hypertrophy were observed in the AMA. Serum T4levels were also increased in the male and female pubertal assays. While the T4increase in the females was not statistically significant, it was of similar magnitude to the T4 increase in males. In the EDSP Tier 1 assays, there was evidence for potential interaction with the thyroid pathway in studies conducted with adult animals, but no such data exists for its effects in the young animals. For propargite, the point of departure (POD) of 4 mg/kg/day used for the current human health risk assessment is based on systemic toxicity characterized as increased mortality, decreases in body weight/body weight gain and food consumption seen at 19 mg/kg/day (LOAEL) in a chronic toxicity/carcinogenicity study. This POD is used for deriving the chronic reference dose (RfD) for chronic dietary risk assessment and calculating the Margins of Exposure (MOEs) for non-dietary risk assessments. In general, since the POD and the toxicity endpoints of concern are based on data obtained from adult animals, there usually would be a concern that the POD may not be protective of potential thyroid toxicity in the young. However in the case of propargite although no data exists for potential thyroid toxicity in the young, a comparative thyroid assay (CTA) is not recommended since:

1) the dose level (4 mg/kg/d) (or POD) driving the long term DNELs (oral Long Term systemic DNEL = ADI) is approximately 15-fold lower than the dose (62.5 mg/kg/day) that caused thyroid lesions in the EDSP Tier 1 pubertal assays;

2) no thyroid-related effects were seen in any of the mammalian toxicity studies e.g. Trutter (1991);

3) the EOGRTS going to higher doses would not be expected to yield a POD lower than the level currently used for risk assessment;

4) the DNELS (ADI) and the MOEs are protective of the thyroid effect seen in the pubertal assays. 

Therefore, mammalian higher Tier ED testing (e.g. EOGRTS) is NOT recommended for propargite since additional testing is not expected to impact the regulatory point of departures and endpoints for human health risk assessments.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to reproductive toxicity.

Additional information

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