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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22Jan2020 to 13Oct2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
June 2018
Deviations:
yes
Remarks:
The OECD414 Guideline requires that histological examination of the thyroid should be conducted for all animals. In this study, only the thyroids of all Group 1 and 4 animals were evaluated
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2'-[(1-methylethylidene)bis[(2,6-dibromo-4,1-phenylene)oxymethylene]]bisoxirane
EC Number:
221-346-0
EC Name:
2,2'-[(1-methylethylidene)bis[(2,6-dibromo-4,1-phenylene)oxymethylene]]bisoxirane
Cas Number:
3072-84-2
Molecular formula:
C21H20Br4O4
IUPAC Name:
2,2'-{propane-2,2-diylbis[(2,6-dibromo-4,1-phenylene)oxymethylene]}dioxirane
Constituent 2
Chemical structure
Reference substance name:
3,3'-((propane-2,2-diylbis(2,6-dibromo-4,1-phenylene))bis(oxy))bis(1-(2,6-dibromo-4-(2-(3,5-dibromo-4-oxiran-2-ylmethoxy)phenyl)propan-2-yl)phenoxy)propan-2-ol
Molecular formula:
C57H52Br12O10
IUPAC Name:
3,3'-((propane-2,2-diylbis(2,6-dibromo-4,1-phenylene))bis(oxy))bis(1-(2,6-dibromo-4-(2-(3,5-dibromo-4-oxiran-2-ylmethoxy)phenyl)propan-2-yl)phenoxy)propan-2-ol
impurity 1
Reference substance name:
n/
Molecular formula:
C93H84Br20O16
IUPAC Name:
n/
impurity 2
Chemical structure
Reference substance name:
1,3-bis(2,6-dibromo-4-(2-(3,5-dibromo-4-(oxiran-2-ylmethoxy)phenyl)propan-2-yl)phenoxy)propan-2-ol
Molecular formula:
C39H36Br8O7
IUPAC Name:
1,3-bis(2,6-dibromo-4-(2-(3,5-dibromo-4-(oxiran-2-ylmethoxy)phenyl)propan-2-yl)phenoxy)propan-2-ol
Test material form:
solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: Olin Corporation
lot/batch number of test material: F289J6O241
- Purity: 100% (Test Substance used as provided as it is a multicomponent mixture of TBBA-GE monomer/oligomers)
approximately 58% monomer, 2% dimer, 30% trimer, and 7% pentimer

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stable when stored refrigerated protected from light
- Stability and homogeneity: Dietary preparations were demonstrated to be homogenious and stable for 3 weeks when stored frozen and thereafter for at least 10 days at room tmemperature under conditions of this feeding study

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): test material was ground to fine powder using a mortar and pestle prior to addition to powdered diet for preparation of the test diets

FORM AS APPLIED IN THE TEST (if different from that of starting material)
Test substance (ground solid) was homogenoiusly mixed into powdered diet.





Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
Rats arrived on Day 0 or Day 1 post-coitum (Day 0 post-coitum is defined as the day of successful mating). They were 11-15 weeks old and weighed between 175 and 290 g at the initiation of administration.

Animal husbandry:
Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained.
A 12 hour light/12 hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.
Free access to prepared powder diets, except during designated procedures. During the acclimatization period, animals had free access to standard powder rodent diet
Municipal tap water was freely available to each animal via water bottles.

Administration / exposure

Route of administration:
oral: feed
Details on exposure:
Dietary exposure
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of prepared diets were sampled twice during the study to demonstrate concentration (all groups) and homogeneity (low and high dose group).
Samples were analyzed inaccordance with a validated method utilizing an Acquity UPLC /UPLC TUV detector system.
Details on mating procedure:
Time-mated pregnant rats were obtained on Day 0 and 1 post-coitum from a commercial vendor.
Duration of treatment / exposure:
The test item was administered to the appropriate animals by inclusion in the diet ad libitum from Day 6 to Day 21 post-coitum, inclusive.
Frequency of treatment:
Daily
Duration of test:
The test item was administered to the appropriate animals by inclusion in the diet ad libitum from Day 6 to Day 21 post-coitum, inclusive.
Doses / concentrationsopen allclose all
Dose / conc.:
1 350 ppm (nominal)
Remarks:
109 mg/kg mean intake
Dose / conc.:
6 750 ppm (nominal)
Remarks:
548 mg/kg mean intake
Dose / conc.:
13 500 ppm (nominal)
Remarks:
1075 mg/kg mean intake
No. of animals per sex per dose:
22 animals per group
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: based on dose-renge finder study with graduated dose levels to provide dose response
- Rationale for animal assignment (if not random): At arrival, animals were randomly assigned to groups
- Fasting period before blood sampling for (rat) dam thyroid hormones: Animals were not fasted
- Time of day for (rat) dam blood sampling: between 7 and 9 am.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Daily morbidity/mortality checks performed twice a day.

DETAILED CLINICAL OBSERVATIONS: Clinical observations were performed at least once daily, beginning on Day 2 post-coitum and
lasting up to the day prior to necropsy

BODY WEIGHT: Animals were weighed individually on Days 2, 6, 9, 12, 15, 18 and 21 post-coitum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes


POST-MORTEM EXAMINATIONS: Yes / No / No data
- Sacrifice on gestation day # 21
- Organs examined: All animals (including females with that delivered on the day of scheduled necropsy) were
subjected to an external, thoracic and abdominal examination, with special attention being
paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and
fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution).
No organs (except for the gravid uterus and thyroid gland) were weighed

Ovaries and uterine content:
Each ovary and uterine horn of all animals were dissected and examined as quickly as
possible to determine:
• The number of corpora lutea.
• The weight of the uterus (not for animals that delivered on the day of scheduled necropsy
and for Female No. 37).
• The number of implantation sites.
• The number and distribution of live and dead fetuses.
• The number and distribution of early and late resorptions.
• The sex of each fetus based on the anogenital distance.
Blood sampling:
- Serum: Yes
- Volume collected : 1.0 ml
- Other:
Fetal examinations:
- External examinations: Yes: [all per litter)
- Soft tissue examinations: Yes: [ half per litter)
- Skeletal examinations: Yes: [all per litter / half per litter / #? per litter ] / No / No data
- Head examinations: Yes: [half per litter]
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics: number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.

Parametric
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).

Non-Parametric
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).
Mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and sex distribution were compared using the Mann Whitney test.
Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. As the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.

Incidence
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using a two-sided Fisher’s exact test at the 5% significance level if the overall test was significant.
No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and postimplantation loss.

Historical control data:
See attached PDF (Historical Control Data)

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
On Day 9 post-coitum, a lower body weight gain (3%) compared to concurrent controls (5%)was observed for animals treated at 13500 ppm, though not statistically significant. A body weight gain comparable to control animals was observed in the periods thereafter.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 13500 ppm, lower food consumption (absolute (16% lower then control) and relative (13% lower then control)) was observed during the Period “Day 6 to 9 post-coitum”. This effect was observed in the first period after the test item-containing diet was provided to the animals and was accompanied with a small reduction in mean body weight gain on Day 9 post-coitum. However, both food consumption and body weight gain recovered to normal values in the periods thereafter and did not result in clinical signs. Therefore, it was considered that the lower food consumption and body weight gain was an effect of the palatability of the diet, rather than being an effect of toxicity of the test item.
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
Endocrine findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Thyroid weights and thyroid-to-body weight ratios of treated animals were considered to be comparable to those of control animals.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic observations in the thyroid glands of animals treated at 1000 mg/kg/day.
The recorded microscopic findings in the control and high dose groups were within the range of background pathology encountered in rats of this age and strain
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic observations in the thyroid glands of animals treated at 1000 mg/kg/day.
The recorded microscopic findings in the control and high dose groups were within the range of background pathology encountered in rats of this age and strain
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic observations in the thyroid glands of animals treated at 1000 mg/kg/day.
The recorded microscopic findings in the control and high dose groups were within the range of background pathology encountered in rats of this age and strain

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
Female No. 46 (6750 ppm) had a litter with dead fetuses only. For this female, it was noted that she might be in delivery on the day of scheduled necropsy and macroscopic findings included emaciating, ruptured uterus and abdominal cavity containing yellowish, watery-clear fluid. In addition, this female was noted with a low food consumption on Days 18-21 post coitum. Due to the single occurrence observed at the mid dose level, this was considered to be unrelated to treatment with the test item.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOEL
Effect level:
1 075 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
food consumption and compound intake
Key result
Dose descriptor:
NOAEL
Effect level:
13 500 ppm
Based on:
test mat.
Remarks:
highest dose group
Basis for effect level:
other: No adverse effects at any dose level
Remarks on result:
not determinable due to absence of adverse toxic effects

Results (fetuses)

Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Anogenital distance of all rodent fetuses:
not examined
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Remarks:
No treatment related effects upto and including highest dose
Effect level:
> 1 075 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effect observed

Overall developmental toxicity

Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
1 075 mg/kg bw/day (actual dose received)
Treatment related:
yes

Applicant's summary and conclusion

Conclusions:
The results of this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for 2,2’,6,6’-Tetrabromo-4,4’-isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane (TBBA-GE) was established as being 13500 ppm (corresponding to an actual test item intake of 1075 mg/kg/day).
Executive summary:

The objectives of this study were to determine the potential of 2,2’,6,6’-Tetrabromo-4,4’-isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane (TBBA-GE) to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given via diet to time-mated female Wistar Han rats from Day 6 to 21 post-coitum, inclusive.

The dose levels in this study were selected to be 0, 1350, 6750, 13500 ppm (estimated dose equivalent of 100, 500 and 1000 mg test item//kg body weight, respectively), based on the results of the dose range finder Chemical analyses of dietary preparations were conducted twice during the study to assess accuracy and homogeneity.

The following parameters and end points were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, test item intake, thyroid hormone levels (triiodothyronine (Total T3), thyroxine (Total T4), thyroid-stimulating hormone (TSH)), gross necropsy findings, organ weights (thyroid gland), uterine contents, histopathologic examination (thyroid gland), corpora lutea, implantation sites and pre- and postimplantation loss.

In addition, the following parameters were determined for the F1-generation: the number of live and dead fetuses, fetal body weights, sex ratio, anogenital distance, external, visceral and skeletal malformations and developmental variations.

Test item containing diets prepared for the use from Day 6 post-coitum onwards were considered inhomogeneous at 1350 and 13500 ppm (criteria of acceptance: i.e. coefficient of variation (CV) ≤10%, whereas in the present study diets at 1350 and 13500 ppm had a CV of 26% and 15%, respectively). Despite this inhomogeneity, the accuracy of the diet was well within specification at all dose levels. Moreover, averages of top, middle and bottom samples at each dose level were at or above the target concentration. Additionally, with the exception of a single Group 2 sample, all analyzed diet samples were at or above target concentrations and all Group 4 individual samples were within 20% of target concentration.

Based on the results of the first diet analysis, measures were taken to increase homogeneity of the diet. A sieving step was added to the diet preparation, after mortaring the test item and new diet was prepared and given to the animals from Day 13-16 onwards (depending on mating date).

Diet analysis of the newly prepared diet revealed that homogeneity was remarkedly improved and within specification (CV of 5.5% and 2.1% at 1350 and 13500 ppm, respectively), whereas accuracy of the diet was still within specified criteria (94 - 97% of targeted concentration).

Taken together, although the animals received an inhomogeneous diet for the majority of the treatment period (used between Day 6 and 13-16 post-coitum, depending on mating date), this inhomogeneity had no impact on the overall test item intake and hence on the outcome of the study. Based on the average food consumption during the study, the average test item intake at the highest dose level was even slightly above the aimed 1000 mg test item/kg body weight/day (average between 1055-1146 mg test item/kg body weight/day). Given the diet accuracies at or above the target concentration, it was considered that all animals of the high dose group were exposed to at least 1000 mg test item/kg body weight/day during the entire study. 

No test item-related mortality occurred during the study period.

In total, three females (No. 58 at 6750 ppm and Nos. 67 and 70 at 13500 ppm) were sacrificed when they started to deliver their litters on the day of scheduled necropsy.

At 13500 ppm, lower food consumption (absolute and relative) was observed during the Period “Day 6 to 9 post-coitum”. This effect was observed in the first period after the test item-containing diet was provided to the animals and was accompanied with a small reduction in mean body weight gain on Day 9 post-coitum. However, both food consumption and body weight gain recovered to normal values in the periods thereafter and did not result in clinical signs. Therefore, it was considered that this lower food consumption and body weight gain was an effect of the palatability of the diet, rather than being an effect of toxicity of the test item.

No maternal toxicity was observedin the 1350, 6750 and 13500 ppm groups.

No developmental toxicity was observedin the 1350, 6750 and 13500 ppm groups.

In conclusion, based on the results in this prenatal developmental toxicity study a maternal and developmental No Observed Adverse Effect Level (NOAEL) for 2,2’,6,6’-Tetrabromo-4,4’-isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane (TBBA-GE) of 13500 ppm (corresponding to an actual test item intake of 1075 mg/kg/day) was established.