2,2',6,6'-Tetrabromo-4,4'-isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 September 2020 - 13 April 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

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

Test material information

Constituent 1

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

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

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 / concentrations

Doses / concentrations 1

Dose / conc.:

2 600 ppm (nominal)

Remarks:

85 (70 - 102) mg/kg mean intake

Doses / concentrations 2

Dose / conc.:

7 800 ppm (nominal)

Remarks:

241 (198 - 278) mg/kg mean intake

Doses / concentrations 3

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	-	0^a	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).