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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
toxicity to reproduction
Remarks:
other: screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
9th October 1991 to 7th December 1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study following GLP. Klimisch code reduced to 2 as this is read across.
Justification for type of information:
Category and read across justification attached in section 13 and in appendix of CSR

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1993

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
Certain organ weights not recorded - not considered to affect the validity of the test. For details, see postmortem effects.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name and of test material: Phenol, dodecyl-, sulfurized, carbonates, calcium salts, overbased (alternate name: Phenol, tetrapropenyl-, sulfurized, carbonates, calcium salts, overbased)
- CAS number of test material: 68784-26-9 (alternate CASRN 122384-87-6)
Testing was performed on a commercial sample of this material. Typical composition of this UVCB material as distributed in commerce is the same as identified in Section 1.2 of IUCLID. Residual calcium carbonate (overbasing) of the commercial substance is typically 12 wt%.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
peanut oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of test article for each group was drawn into a syringe. A specified amount of vehicle was placed in a storage container. The test article was added to the vehicle while mixing with a laboratory mixer until the test material had been incorporated. The preparation was then stirred (magnetic stir bar) throughout the sampling, dispensation and dosing procedures, and periods of non-use. Storage containers were wrapped in foil for additional light protection. Test article preparations were formulated weekly; the prepared suspensions for each dose group were stirred at least 30 minutes prior to sampling.

Details on mating procedure:
- M/F ratio per cage: After a minimum of 28 days of test article administration, one female was cohabited with one male from the same dose group in the home cage of the male. Animals were at least 11 weeks of age.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in a vaginal smear or by appearance of a vaginal copulatory plug. The day mating was confirmed was designated gestation day 0.
- If no evidence of copulation was noted after 10 days, a given female was placed with another male in the same treatment group for an additional 5 days; this second male had previously shown evidence of successful mating.
- After successful mating each pregnant female was caged (how): Females were transferred to plastic maternity cages containing nesting material (Bed-O'Cobs; The Andersons, Industrial Products Division, Maumee, Ohio 43537) upon confirmation of mating or after 15 days of mating, when no evidence of copulation was apparent.



Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Peanut oil dosing solutions were prepared weekly, and their test material concentrations, homogeneity and stability verified by chemical analysis.
Duration of treatment / exposure:
28 days prior to mating; dosing was continued for a total minimum of 70 days in the parental (F0) males, while F0 females continued to receive the test article through day 4 of lactation.
Frequency of treatment:
Daily
Details on study schedule:
No data
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 200 and 1000 mg/kg b.wt./day administered in a dose volume of 5 mL/kg b.wt./day
Basis:
actual ingested
No. of animals per sex per dose:
There were 12 rats/sex/group for the F0 generation. For the control 12 rats/sex received peanut oil vehicle only.
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
Animals were observed twice daily (morning and afternoon) for mortality or moribundity. Detailed clinical observations were recorded twice daily,
before and one hour after dosing. Individual body weight and food consumption were recorded pre-study, weekly during exposure and at
termination, except for females during the gestation and lactation period when shorter intervals were used and food consumption was not recorded during mating.
Litter observations:
At completion of parturition, litters were examined for viability, sex and gross malformations. Litters were observed daily until lactation day 4. Pups
were given a detailed physical examination and weighed on lactation days 1 and 4
Postmortem examinations (parental animals):
Complete necropsy examinations were performed on parental animals. Seven major organs were weighed and 41 organs / tissues were preserved for all parental animals. Tissues with macroscopic findings from all groups were examined for microscopic pathology. In addition, microscopic examination of the following tissues were made for all
parental animals in the control and high-dose groups: brain, pituitary, adrenals, heart, kidney, liver, spleen, epididymides, prostate, seminal vesicles, testes, and ovaries.

All guideline recommendations were exceeded, except for the following: heart, epididymides, spleen, and thymus weights were not measured.
Microscopic observations were not obtained for the following tissues: spinal cord, stomach, small and large intestines, thymus, thyroid, trachea,
lungs, uterus, urinary bladder, lymph nodes, peripheral nerve and bone marrow.
Postmortem examinations (offspring):
Necropsy of pups emphasized developmental morphology and abnormal tissues were preserved. All pup skeletons were processed for future skeletal examinations and skulls were examined for hyoid bone development.
Statistics:
Body weight, food consumption, organ weight, litter weight and gestation interval data were analyzed by ANOVA with a Dunnett’s post-hoc test if
significance was observed with ANOVA. Male and female mating and fertility indices, and litter data were analyzed with the Chi-square test with Yate’s correction factor. Histopathology data of the control and high dose groups were compared with the Kolmogorov-Smirnov test.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed

Details on results (P0)

Parental data and F1 (toxic response/effects with NOAEL value): Decreased body weight gain in males after 8-10 weeks treatment and in pregnant
females during the last two weeks of gestation at 1000 mg/kg b.wt./day. Increased food consumption without commensurate body weight gain in
males and females at 1000 mg/kg b.wt./day. NOAEL for all effects in males and females was 200 mg/kg b.wt./day.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

Decreased live litter size and prenatal toxicity (decreased gestation body weight); embryotoxicity (pre-implantation loss) at 1000 mg/kg b.wt./day. Developmental toxicity NOAEL in offspring was 200 mg/kg b.wt./day

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: decreased gestation body weight

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Body weights and body weight gains for high-dose males were significantly lower than controls during the treatment period from weeks 2 to 10 (p=<0.05 - p=<0.01). Maternal body weight gain in the high-dose group was reduced during the second and third weeks of gestation (p=<0.05 - p=<0.01).

Food consumption for high-dose males was significantly higher than controls during weeks 7-10 (p=<0.01). Maternal food consumption in the high-dose group was significantly elevated during the last week of gestation (p=<0.05) and was significantly lower (p=<0.01) than controls during lactation. Implantation sites/dam for the high-dose group was significantly lower than concurrent control (p=<0.01). There were no other statistically significant differences of toxicological significance.

Survival: All animals survived to termination except for one low-dose male that died due to a dosing error.

Clinical observations: Material and/or staining around the mouth of high dose animals were the only treatment-related signs and were seen only at the 1-hour post-dose observation time. Onset of these signs occurred within 1-2 weeks and persisted until the end of the exposure period.

Body weights and food consumption: Mean body weights for high-dose males were significantly lower (p=<0.01) than controls during the treatment period from weeks 3 to 10. Mean body weight gain for high-dose males was significantly lower than controls by 15-21% starting week 2 (p=<0.05) through week 10 (p=<0.01). Food consumption (g/kg b.wt./day) for high-dose males was significantly higher (10-14%) than controls during weeks 7 -10 (p=<0.01). Female body weights and food consumption were not affected during the 4-week pre-mating exposure period. During gestation, however, maternal body weights were affected in the high-dose group. Mean body weight gain was reduced 13% (p=<0.05) and 29% (p=<0.01) during the second and third weeks of gestation, respectively, while food consumption was elevated 4-11% (p=<0.05, gestation days 14-20). On lactation days 1 and 4, maternal body weights for the high-dose group were lower than controls but the differences were not statistically significant. Maternal food consumption during lactation was significantly (p=<0.01) lower than controls by 24%.

Reproductive parameters: Male and female mating and fertility indices were not affected by treatment. The mean number of days until mating was comparable among all groups. The length of gestation was not affected by treatment.

Litter data: The number of corpora lutea/dam for the high-dose group was lower than concurrent controls (not statistically significant) and the range of historical controls. Implantation sites/dam for the high-dose group were significantly lower than concurrent controls (p=<0.01) and were below the range of historical controls. These two factors combined to result in significant (p=<0.01) pre-implantation loss of 30% and to significantly (p=<0.01) reduce the mean number of pups/litter in the high-dose group. Post-implantation loss was elevated for the high-dose group, but was not significantly different from control. There were no treatment-related differences of stillborn pups, viability index and sex ratio. There were no significant differences of male and female pup weights at days 1 and 4 of lactation. No pups were malformed.

Terminal organ and body weights: Brain, kidney, liver and testes organ/body weight ratios were significantly higher in high-dose males than controls, while absolute and organ/brain ratios were not different. Terminal body weights for high-dose males were significantly (p=<0.01) reduced 11% and there were no microscopic pathology findings for these tissues. Therefore the elevated organ/body weight ratios in high-dose males are not toxicologically significant. Mean adrenal and pituitary gland weights in high-dose males were 39% and 18% higher, respectively, than controls. Relative to body and brain weight, adrenal and pituitary gland weights were also elevated and all of these differences in male adrenal and pituitary gland weights were statistically significant (p=<0.05 - p=<0.01). There were no microscopic pathology findings for adrenal and pituitary gland in males, therefore the toxicological significance of consistently elevated weights for these organs is equivocal. Mean adrenal weights were equally (~16%) higher in both mid- and high-dose females, but the differences were not statistically significant. Relative to body and brain weight, the differences were statistically significant (p=<0.01 for organ/body, p=<0.05 for organ/brain) for both dose groups. Mean body weights at termination did not vary significantly for females. There were no microscopic pathology findings for adrenal gland in females and the adrenal weight differences between the two dose groups were not proportional to dose. Therefore the toxicological significance of elevated adrenal weight ratios in mid- and high-dose females are equivocal. There were no other dose-related differences of absolute or relative organ among males or females. Macroscopic and microscopic pathology: No treatment-related macroscopic or microscopic pathology was observed in parental animals or pups sacrificed at termination or found dead.

Applicant's summary and conclusion

Conclusions:
Based upon the results of this study, the apparent NOAEL (no observable adverse effect level) parental toxicity was considered to be 200 mg/kg/day. Equivocal prenatal toxicity was observed at a dose level of 200 mg/kg/day. The results of the present screen indicated that further toxicity testing for adverse reproductive effects should be considered.
Executive summary:

The potential reproductive toxicity related to the administration of the test material in the Sprague-Dawley Crl: CD®BR rat was evaluated in this combined repeated dose study conducted in accordance with GLP to OECD guideline 422. In the reproduction phase the test material was administered to 12 animals/sex/group for 28 days prior to mating; dosing was continued for a total minimum of 70 days in the parental (F0) males, while F0 females continued to receive the test article through day 4 of lactation. Dose levels were 50, 200 and 1000 mg/kg/day. A concurrent control group received the vehicle, peanut oil, on a comparable regimen. A dose volume of 5 ml/kg was used for all groups. Parameters common to all study phases included viability, clinical signs, body weights, food consumption and necropsy evaluations. Mating indices, neonatal parameters, organ weights and histopathology data were recorded in the reproductive phase.

The adverse effects observed in this study are summarized below:

* Salivation, clear material around the mouth, red or yellow staining around the mouth and/or red material around the nose were treatment-related clinical signs noted one-hour following dosing in males and females receiving 1000 mg/kg/day.

* Weekly group mean body weights and body weight gains in males were decreased at a dose level of 1000 mg/kg/day. This occurred during weeks 2-10 in the reproduction phase.

* Increased group mean adrenal gland and pituitary gland weights (absolute and relative to brain weights) were observed in males receiving 1000 mg/kg/day during the reproduction phase. No other organ weights (brain, liver, kidneys, testes or ovaries) were adversely affected.

* Decreased live litter size occurred at dose levels of 200 and 1000 mg/kg/day. The effect was pronounced at 1000 mg/kg/day. This was due to increased pre- and post-implantation loss. The prenatal mortality was also reflected in decreased gestation body weight data and subsequent lactation food consumption values (1000 mg/kg/day).

Based upon the results of this study, the apparent NOAEL (no observable adverse effect level) parental toxicity was considered to be 200 mg/kg/day. Equivocal prenatal toxicity was observed at a dose level of 200 mg/kg/day. The results of the present screen indicated that further toxicity testing for adverse reproductive effects should be considered.