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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
27 July 2020 to 23 February 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Remarks:
Dose-Range Finding Study does not have a Test Guideline

Data source

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

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
- Principle of test: Similar to OECD 422 study design to screen for Reproductive and Developmental Toxicity
- Short description of test conditions: Male and Female rats were dosed via dietary admix of test substance for at least 28 days, consecutively
- Parameters analysed / observed: Male and female reproductive performance such as gonadal function, mating behavior, conception, parturition and early postnatal development was evaluated.
No Observed Adverse Effect Levels (NOAELs) were evaluated for parental reproduction (up to and including implantation) and developmental (from implantation onwards) toxicity .
GLP compliance:
yes (incl. QA statement)
Limit test:
no

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: 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 homogeneous and stable for 3 weeks when stored frozen and thereafter for at least 10 days at room temperature 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 homogenously mixed into powdered diet.

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. The Testing laboratory has a rich general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Male and female Crl: WI(Han) rats were allowed to acclimate to the Test Facility toxicology accommodation for 8 days before the commencement of administration. At initiation of administration, males were 11-12 weeks old and weighed between 277 and 338 g and females were 12-13 weeks old and weighed between 202 and 251 g.

Animal husbandry:
Temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were targeted. Humidity excursions to as high as 87% was observed during the first 21 days of study.
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
Vehicle:
unchanged (no vehicle)
Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Diets were prepared for use at room temperature for a maximum of 8 days.

- Mixing appropriate amounts with (Type of food): The test item was mortared until it was a fine powder, sieved using a sieve of 500 µm and was mixed without the use of a vehicle, directly with some powder feed (premix) and subsequently mixed with the bulk of the diet. Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was used.

- Storage temperature of food: Diets were kept in the freezer (≤ 15°C) until use

Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Until evidence of mating upto 14 days.
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy: By evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug
- There were no unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually housed in Macrolon plastic cages (MIII type, height 18 cm).
Analytical verification of doses or concentrations:
no
Remarks:
Stability of dietary preparations in the range of concentrations was demonstrated in support of the 90-day study with this material
Duration of treatment / exposure:
Males were exposed for 29 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females were exposed for 51 to 56 days, i.e. 14 days prior to mating (to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 14 days after delivery, up to and including the day of scheduled necropsy. The female that failed to deliver was treated for 44 days.
Frequency of treatment:
Administered ad libitum to the appropriate animals by inclusion in the diet from Day 1 onwards for a minimum of 28 days.
Doses / concentrationsopen allclose all
Dose / conc.:
7 000 ppm (nominal)
Remarks:
From Day 1 through parturition
Dose / conc.:
14 000 ppm (nominal)
Remarks:
From Day 1 through parturition
Dose / conc.:
3 500 ppm (nominal)
Remarks:
Low dose during Lactation
Dose / conc.:
7 000 ppm (nominal)
Remarks:
High dose during Lactation
No. of animals per sex per dose:
10 males and 10 females per dose
Control animals:
no
Details on study design:
- Dose selection rationale: Dose levels were chosen to achieve the limit test dose level as high dose and low dose to provide dose response
- Rationale for animal assignment (if not random): Animals were randomly assigned to groups at arrival. Males and females were randomized separately.

Dose levels were adjusted for the lactation phase of the study due to anticipated higher food consumption. Low dose group from 7000 ppm reduced to 3500 ppm. High dose group from 14000 ppm reduced to 7000 ppm.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
Animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
Animals were weighed individually on the first day of administration, and weekly thereafter.
Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.
A terminal weight was recorded on the day of scheduled necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

Organ Weight: Epididymis and Testes

Necropsy: All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.
The numbers of former implantation sites were recorded for all paired females.

Fertility Index, Gestation Index and Duration, Parturition, Litter Size.


Oestrous cyclicity (parental animals):
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.
Daily vaginal lavage for all females during the first 14 days of treatment and during mating until evidence of copulation was observed.
On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrous.
Sperm parameters (parental animals):
Not examined
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes
- If yes, maximum of [8] pups/litter ([4]/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:

Litter size, Live Birth Index, Viability Index (live offspring on PND4 vs PND1), Lactation Index (live offspring on PND13 vs PND4), Clinical signs, Body Weight, Sex ratio, Macroscopic Observations.




Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals following completion of the mating period (a minimum of 28 days of administration).
- Female animals: All surviving animals on PND 14-16 except for females that failed to deliver but with evidence of mating were sacrificed on Post-coitum Day 27




HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
Pups that died before scheduled termination were examined externally and sexed (both externally and internally).
The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

On PND 4, the surplus pups were euthanized by decapitation. Sex was determined both externally and internally.

All remaining pups were euthanized on PND 14-16. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.
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.
Reproductive indices:
Mating Index, Fertility Index, Gestation Index and Duration, Live Birth Index,
Offspring viability indices:
Viability Index, Lactation Index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Diarrhoea was noted for all animals from Day 7 of treatment onwards and continued until Day 3 of the mating period for males and all females at 7000 ppm, and until the end of the
treatment period for males at 14000 ppm. All females at 14000 ppm exhibited diarrhoea from Day 7 until Day 3 of mating and only one female at 14000 ppm continued until the end of the
post-coitum period.

Hunched posture, piloerection and quick breathing were incidentally noted at both diet concentrations (quick breathing only at 7000 ppm) for a maximum duration of two weeks in
individual animals. As these clinical signs occurred at a low incidence these were considered not toxicologically relevant.

Other clinical signs occurred within the range of background findings to be expected for rats of this age and strain
which are housed and treated under the conditions in this study.
Dermal irritation (if dermal study):
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights and body weight gain of treated animals were considered not affected by treatment with the test item.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before or after correction for body weight was considered not affected by
treatment with the test item.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
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
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Oestrus cycles in the two weeks prior to mating, during mating, and on the day of necropsy were considered normal for this age and strain of rat.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
14 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
gross pathology
reproductive function (oestrous cycle)
reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
No treatment related clinical signs in pups was observed through 13-15 days of lactation
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
Viability index (number of live offspring on PND 4 before culling as percentage of number of
live offspring on PND 1) was not affected by treatment with the test item. Viability indices
were 98 and 100% for the 7000 and 14000 ppm groups, respectively.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights of pups were considered not to be affected by treatment with the test item.
Food consumption and compound intake (if feeding study):
not examined
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
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered not to be related to treatment with
the test item.
Histopathological findings:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no behavioral findings to suggest developmental neurotoxicity.

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

No developmental toxicity was observed up to the highest concentration tested (14000 ppm). No treatment-related changes were noted in any of the developmental parameters investigated
in this study (i.e. gestation index, duration of gestation, parturition, sex ratio, maternal care, litter size, viability and lactation indices and early postnatal pup development consisting of
mortality, clinical signs, body weight, and macroscopic examination).

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
14 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
clinical signs
mortality
body weight and weight gain
gross pathology
developmental neurotoxicity

Overall reproductive toxicity

Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
14 000 ppm (nominal)
Treatment related:
no

Applicant's summary and conclusion

Conclusions:
In conclusion, based on the results of this reproduction/developmental toxicity screening test, the following No Observed Adverse Effect Levels (NOAELs) of 2,2’,6,6’-Tetrabromo-4,4’-
isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane (TBBA-GE) were established:

Parental NOAEL: at least 14000 ppm, corresponding to an actual test article intake of 910 and 1039 mg/kg/day for males and females, respectively.

Reproduction NOAEL: at least 14000 ppm, corresponding to an actual test article intake of 910 and 1039 mg/kg/day for males and females, respectively.

Developmental NOAEL: at least 14000 ppm, corresponding to an actual test article intake of 910 and 1039 mg/kg/day for males and females, respectively.

Based on the results of this study in combination with the results of the 90-day study, 14000 ppm can be selected as highest dose level in the Extended One Generation Reproductive Toxicity study in rat.
Executive summary:

The objective of this study was to select dose levels for the extendend one generation reproductive toxicity study in rats.

The potential toxic effect of 2,2’,6,6’-Tetrabromo-4,4’-isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane (TBBA-GE) when given via diet for a minimum of 28 days to Wistar Han rats was determined, and the potential to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition and early postnatal development was evaluated. In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated.

The dose levels in this study were selected based on the results of the Dose Range Finder conducted for a OECD 414 study in rats with this material.

The following parameters and end points were evaluated in this study: mortality/ moribundity, clinical signs, body weight and food consumption, gross necropsy findings and organ weights. In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex and macroscopy).

No parental toxicity was observed up to the highest concentration tested (14000 ppm).

Diarrhoea was noted for all animals from Day 7 of treatment onwards and continued until Day 3 of the mating period for males at 7000 ppm and all females, and until the end of the treatment period for males at 14000 ppm. Except for one female at 14000 ppm, for which diarrhea continued until the end of the post-coitum period. As this was without an effect on body weight gain it was not considered adverse. No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. body weight, food consumption, macroscopic examination and organ weights).

Reproductive results

No reproduction toxicity was observed up to the highest concentration tested (14000 ppm).

No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time and number of implantations).

Developmental results

No developmental toxicity was observed up to the highest concentration tested (14000 ppm).

No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care, litter size and early postnatal pup development consisting of mortality, clinical signs, body weight, and macroscopic examination).

In conclusion, based on the results of this reproduction/developmental toxicity screening test, the following No Observed Adverse Effect Levels (NOAELs) of 2,2’,6,6’-Tetrabromo-4,4’-isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane (TBBA-GE) were established:

Parental NOAEL:                  at least 14000 ppm, corresponding to an actual test article intake of 910 and 1039 mg/kg/day for males and females, respectively.

Reproduction NOAEL:         at least 14000 ppm, corresponding to an actual test article intake of 910 and 1039 mg/kg/day for males and females, respectively.

Developmental NOAEL:      at least 14000 ppm, corresponding to an actual test article intake of 910 and 1039 mg/kg/day for males and females, respectively.

Based on the results of this study and in combination with the results of the 90-day study, 14000 ppm can be selected as highest dose level in the planned Extended One Generation Reproductive Toxicity study in rat.

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