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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
toxicity to reproduction
Remarks:
other: Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study, however spacing of doses is not adequate and historical control data is missing
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): TP-90B Rubber Chemical
- Analytical purity: 97% (v/v)

Test animals

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on mating procedure:
Pairings were monogamous. Vaginal smears were taken during pairing up to the day of positive identification of mating. Each cage was checked each morning for the presence of copulation plugs. The pairing combinations of animals which had not had positive identification of mating after 14 days of pairing were changed within each treatment group. Animals that did not have signs of mating during the initial pairing were placed with a second male that previously had positive signs of mating. The subsequent pairing was monitored for mating as described above for the first pairing. No more than 28 days was allowed for pairing.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
Exposure period: at least ca. 6 weeks:
For males, treatment commenced when the males were approximately 12 weeks old and continued for two weeks prior to pairing, during pairing until mating was confirmed, and up to and including the day before sacrifice.
For females, treatment commenced when they were approximately 12 weeks old and continued for two weeks prior to pairing through the mating and gestation periods, up to day 3 of lactation. Dams and offspring were sacrificed on Day 4 post-partum.
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
10, 100, 800 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
see information in IUCLID section 7.5.1. RTC (2004).
Oestrous cyclicity (parental animals):
Vaginal smears were taken daily in the morning, starting two weeks before pairing (first day of dosing) until a positive identification of mating was made. The vaginal smear data were examined to determine anomalies of the estrous cycle, and the pre-coital interval (i.e., the number of nights paired prior to the detection of mating.
Sperm parameters (parental animals):
Each cage was checked each morning for the presence of copulation plugs.
Litter observations:
The total litter size (live and dead) was counted as soon as possible after parturition. Live pups were identified individually within the litter by toe amputation, sexed and examined for external abnormalities. Live and dead pups were counted and weighed on days 1 and 4 post-partum, with the exception of one mid-dose female, where pups were weighed on days 2 and 5 post-partum. All litters were observed daily. All pups found dead were given a post-partum examination.
Postmortem examinations (parental animals):
- Organ weights P: The following organs from all adult animals in all groups were dissected free of fat and weighed: adrenals, brain, epididymides, heart, kidneys, liver, spleen, thymus, testes.

- Histopathology P: The tissues listed, as follows, were examined in all adult animals: corpora abnormalities (gross lesions), adrenals (paired), bone marrow (from the sternum), brain, caecum, coagulating glands (paired), colon, duodenum, epididymides (paired), esophagus, heart, ileum, jejunum, kidneys (paired), liver, lymph nodes (cervical), lymph nodes (mesenteric), lungs, ovaries with oviduct (paired), prostate, pituitary, rectum, sciatic nerve, seminal spinal column with spinal cord, spleen, stomach, thymus, thyroid, trachea, testes (paired), urinary bladder, uterus with cervix, vagina.
Statistics:
For continuous variables, the significance of the difference amongst group means was assessed by analysis of variance. Differences between the treated group and the control group were assessed by Dunnett's test using a pooled error of variance. The homogeneity of the data was assessed by Bartlett's Test before Dunnett's Test was performed. If the data were found to be inhomogeneous, a modified t-test (Cochran and Cox) was applied. Statistical analysis of histopathological findings was not carried out by means of the non-parametric Kolmogorov-Smirnov test.
The non-parametric Kruskal-Wallis analysis of variance was used for all other parameters. Intergroup differences between the control and the treated group were assessed by the non-parametric version of the Williams' test.

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

Body weight:
Body weight and body weight gain were unaffected by treatment in the males throughout the study and in females before pairing. Slight decreases, not statistically significant, in body weight and body weight gain were observed in the high dose females during the gestation period when compared to controls. No differences were noted in females of any treatment group during the post-partum period when compared to controls.

Food consumption:
Food consumption remained comparable between control and treated groups in both sexes before pairing. A statistically significant reduction in food consumption was noted in high dose females on day 7 post-coitum. A similar reduction (not statistically significant) was noted on day 14 post-coitum in the same group.

Description, severity, time of onset and duration of clinical signs:
Weekly physical examinations including detailed clinical signs with neurotoxicity assessment did not show any signs which could be correlated to the treatment with the test item in males or females in any group during the treatment period.

Fertility index:
Fertility index in the high-dose group was decreased compared to controls. A treatment-related effect on fertility was apparent in the high dose group. A total of fifteen females proved not to be pregnant at necropsy; three in the control group and in the mid-dose group, two in the low-dose group and seven in the high-dose group.

Pre-coital interval:
Pre-coital interval in tested rats was comparable to controls.

Duration of gestation:
Gestation periods in tested rats were comparable to controls.

Gestation index:
An increased incidence, not statistically significant, in the number and percentage of pre-birth loss was noted in high dose females when compared to controls.

Changes in lactation: not characterized

Changes in estrus cycles:
The estrus cycles of tested animals were comparable to controls.

Effects on sperm: not characterized

Hematological findings incidence and severity:
No toxicologically significant changes in hematology occurred in this study. Statistically significant decreases in prothrombin time were observed in the high dose male group. This change was considered incidental and not treatment-related since it was seen in only one sex, and the magnitude of the effect was low.
Statistically significant decreases in neutrophil percentage were observed in the high dose females. This change was considered incidental and not treatment-related since it was seen in one sex only and no other changes were seen in the white blood cell differential counts.

Clinical biochemistry findings incidence and severity:
No toxicologically significant change in any clinical chemistry parameters occurred at any dose level.
A statistically significant decrease in alkaline phosphatase level was observed in the high dose male group. Since this change was minimal, was decreased compared to the control group and occurred in only one sex, it was not considered toxicologically significant. A statistically significant decrease in aminotransferase level was noted in the low dose male group when compared to controls. This decrease was considered incidental to treatment since no dose response was evident.
Statistically significant decreases in urea were seen in the low and high dose males compared to controls. These changes were not considered treatment-related since the changes were decreased relative to controls and there was no evident dose response. Statistically significant changes in creatinine and inorganic phosphorus levels were observed in the high dose male group. These changes were not considered treatment-related since the values were decreased compared to controls, the magnitude of the change was small and they occurred only in one sex.
Urea level was also statistically significantly decreased in the low dose male group. This change was not considered treatment-related since the effect was decreased, there was no obvious graded dose response at the mid-and high dose groups and the effect occurred in only one sex.
No differences in clinical chemistry parameters were noted between control and treated females.

Mortality:
One mid-dose female was sacrificed, on day 0 post-partum, for humane reasons, since signs of difficult delivery were noted. No other animals died prior to study termination.

Gross pathology incidence and severity:
No macroscopic change was reported at necropsy in the examined organs/tissues of the animals killed at termination that could be considered related to the administration of the test item. The recorded changes were considered to be incidental or spontaneous in origin.

Number of implantations:
There were no effects on the mean number of implantation sites at any dose level.

Number of corpora lutea: not reported

Organ weight changes:
Treatment-related organ weight changes occurred in the liver of high dose males and females. Statistically significant increases in absolute and relative liver weights were observed in the high dose males when compared to controls. Increases in absolute (not statistically significant) and relative (statistically significant) liver weights were also noted in only the high dose females.
Statistically significant increases in absolute and relative kidney weights were noted in the mid-dose male group only.
Since the absolute and relative kidney weights of the high dose males were not affected (i.e., no apparent dose response), there was no corresponding increase in females, and no corresponding microscopic change in this tissue, the kidney weight changes were considered not related to treatment with the test item. Slight, but statistically significant increases in absolute and relative adrenal weights were observed in the high dose female group. This change was considered incidental and not treatment-related since there was no corresponding microscopic histopathologic change in this tissue and similar effects were not seen in the high dose male group.

Histopathology incidence and severity:
No microscopic changes clearly related to test substance administration were observed in male or female rats given 10 or 100 mk/kg bw/day of the test article.
Diffuse hepatocellular hypertrophy (enlarged hepatocytes in all areas of the lobules) occurred in male and female rats of the high dosage group and was considered to be treatment-related.
Focal germ-cell depletion and degeneration occurred in one testis (unilateral) of each of four different male rats of the high dosage group. Exfoliated spermatogenic cells were observed in the epididymis in three of these high dose rats and in one additional high dose rat without testicular changes. The significance of this finding was uncertain. Systemic toxicity usually does not affect only one of a paired organ.
All other microscopic changes were considered to have occurred spontaneously and to be unrelated to test article administration. These changes generally occurred at single, low, or similar frequencies among the groups and their type, incidence or severity was not influenced by compound administration.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios

Results: F1 generation

Details on results (F1)

Litter size and weights:
A decrease in the average number of pups per litter was observed at birth, and on day 4 post-partum in the high-dose group. Decreased litter weight and mean pup weight in the high-dose group (not statistically significant) were noted at birth and on day 4 post-partum.

Sex and sex ratios:
Sex ratios at birth and on day 4 post-partum did not show any differences between control and treated groups when calculated as the percentage of males.

Viability index:
Pup survival to day 4 was decreased in the high-dose-group. The number of females with live pups on day 4 post-partum was 6, 8, 6, and 3 in the control, low-, mid- and high-dose groups, respectively. A statistically significant increase in pup mortality (females and total) from day 0 to day 4 was noted in the high dose group. Similar results were also seen in male pups but the difference was not statistically significant.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 100 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: decreased fertility indices and increased pre-birth loss

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

The relevance of the observed effects (decreased fertility indices and increased pre-birth losses,

embryo-fetal toxicity as well as developmental effects of microdactyly and/or agenesis of digits (one litter) and

flexure of hind limbs (one litter)) with regard to the hazard assessment

(i.e., classification) is unclear due to the following concerns about the study:

 Low fertility rate for the control group which limits the predictive (i.e., statistical)

power of the study as well as its reliability

 Lack of a clear dose-response relationship (effects only in the high dose group);

Observed parental toxicity raises the concern that 800 mg/kg for the high dose level

may have been too high

 Inadequate sample size for evaluation of fetal malformations in the high dose

group

 Lack of the historical control data in the study report does not allow for

distinguishing between substance-related and spontaneous effects

 Some incorrect values stated in the final report call into question the QA process

for the study, the data collection software, and /or perhaps some of the results

Applicant's summary and conclusion