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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:
02Sept2020 to awaiting final report
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
June 2018
Deviations:
yes
Remarks:
• Body weights of Day 0 post-coitum (i.e. the day of mating) reported were collected non GLP by the Supplier
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:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
The females arrived on Day 2 post-coitum (Day 0 post-coitum is defined as the day of successful mating). They were 17-19 weeks old and weighed between 2962 and 4127 g at the initiation of administration.

Animal husbandry:
Target temperatures of 17 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.

Prepared powder diet for rabbits was provided ad libitum throughout the study, except during designated procedures. In addition, pressed hay was provided during the study period.
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility.
It was considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

Municipal tap water was freely available to each animal via water bottles.

Administration / exposure

Route of administration:
oral: feed
Details on exposure:
The test item was administered to the appropriate animals by inclusion in the diet from Day 7 to Day 29 post-coitum, inclusive.
The amount of test item incorporated in the diet was kept at a constant level in terms of ppm, throughout the study period. After termination, the actual test item intake was estimated based on the body weight and food consumption values.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of prepared diet was sampled twice during the first week of 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 from Day 7 to Day 29 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.:
2 600 ppm (nominal)
Remarks:
85 (70 - 102) mg/kg mean intake
Dose / conc.:
7 800 ppm (nominal)
Remarks:
241 (198 - 278) mg/kg mean intake
Dose / conc.:
28 500 ppm (nominal)
Remarks:
897 (706 - 1119) 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-range 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
The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of administration.

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 7 post-coitum and lasting up to the day prior to necropsy

BODY WEIGHT: Animals were individually weighed on Days 7, 9, 12, 15, 18, 21, 24, 27 and 29 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 day 29 post-coitum
- 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: No
-
Fetal examinations:
- External examinations: Yes: [all per litter)
- Soft tissue examinations: Yes: [ all per litter)
- Skeletal examinations: Yes: [ all per litter ]
- 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
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
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:
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

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:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
28 500 ppm (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
changes in number of pregnant
changes in pregnancy duration
dead fetuses
early or late resorptions
effects on pregnancy duration
food consumption and compound intake
gross pathology
maternal abnormalities
mortality
necropsy findings
number of abortions
pre and post implantation loss
total litter losses by resorption

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:
effects observed, treatment-related
Description (incidence and severity):
An increased mean litter incidence of the variation “retrocaval ureter” was observed in fetuses of dams treated at 28500 ppm. This variation was observed in 1.5%, 1.5%, 0.8% and 6.0% per litter in the control, 2600, 7800 and 28500 ppm groups, respectively. The incidence at the high dose was above the maximum historical control value (4.1% per litter) , but did not reach statistical significance. As this incidence was increased at the high dose level, a test item-related effect could not be excluded. It should be noted that this variation is the most occurring variation in the historical control data. However this concerns a variation with no detrimental effects on development, it was considered non-adverse.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Remarks:
No treatment related effects upto and including highest dose
Effect level:
> 28 500 ppm (nominal)
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.:
28 500 ppm (nominal)
Treatment related:
yes

Applicant's summary and conclusion

Conclusions:
In conclusion, based on the results in this prenatal developmental toxicity study in New Zealand White rabbits, the maternal and developmental No Observed Adverse Effect Levels (NOAELs) for 2,2’,6,6’-Tetrabromo-4,4’-isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane (TBBA-GE) were established as being at least 28500 ppm.
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 New Zealand White rabbits from Days 7 to 29 post‑coitum, inclusive. In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated.

The dose levels in this study were selected to be 0, 2600, 7800, 28500 ppm (anticipated dose levels of 100, 300 and 1000 mg/kg bw/day), based on the results of the Dose Range Finder. The study design was as follows:


Experimental Design

Group No.

Test Item Identification

Dose Level

(ppm)#

Number of Females

1

-

0a

22

2

TBBA-GE

2600

22

3

7800

22

4

28500

22

a  Standard powder diet for rabbits. # Corresponding to anticipated dose levels of 100, 300 and 1000 mg/kg bw/day.

The following parameters and endpoints were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, test item intake, macroscopic findings, (gravid) uterine weight and uterine contents.

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

Dietary analyses conducted twice during the study confirmed that diets containing test item were prepared accurately and homogenously.

During the course of this study, one control female was sacrificed on Day 25 post‑coitum as it started to deliver its offspring. As this concerned a control animal,this was considered to be of spontaneous origin.

No maternal toxicity was observed in the 2600, 7800 and 28500 ppm groups.

No developmental toxicity was observedin the 2600, 7800 and 28500 ppm groups.

In conclusion, based on the results in this prenatal developmental toxicity study in New Zealand White rabbits, the maternal and developmental No Observed Adverse Effect Levels (NOAELs) for 2,2’,6,6’-Tetrabromo-4,4’-isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane (TBBA-GE) were established as being at least 28500 ppm (corresponding to an actual mean test item intake of 897 mg/kg bw/day).

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