Benzene, 1,1'-oxybis-, tetrapropylene derivs., sulfonated, sodium salts

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Benzene, 1,1'-oxybis-, tetrapropylene derivs., sulfonated, sodium salts

EC Number:

601-601-6

Cas Number:

119345-04-9

Molecular formula:

C24H32O7S2Na2

IUPAC Name:

Benzene, 1,1'-oxybis-, tetrapropylene derivs., sulfonated, sodium salts

Details on test material:

Major components: monoalkyl and dialkyl diphenyl ether sulfate

Specific details on test material used for the study:

Identification: Benzene 1,1ʹ-oxybis-, tetrapropylene derivs., sulfonated, sodium salts
CAS No.: 119345-04-9
Lot No.: CAC09162020
Expiration/Retest Date: 22 Apr 2023
Purity: 93.24%
Correction Factor: -
Storage Conditions: 18°C to 24°C
Provided by: Sponsor
- = not applicable.

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Details on test animals or test system and environmental conditions:

Receipt:
On 05 Nov 2021, time-mated female Crl:CD(SD) rats were received from Charles River Laboratories, Inc., Raleigh, NC on Gestation Day 2, 3, or 4. The animals were approximately 11–13 weeks old and weighed between 215 and 299 g at the initiation of exposure.

Justification for Test System and Number of Animals:
At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models that do not use live animals currently do not exist.
The Crl:CD(SD) rat is recognized as appropriate for developmental toxicity studies. Charles River Ashland has historical data on the background incidence of fetal malformations and developmental variations in the Crl:CD(SD) rat. This animal model has been proven to be susceptible to the effects of developmental toxicants.
The number of animals selected for this study was based on the US EPA Health Effects Test Guidelines OPPTS 870.3700, Prenatal Development Toxicity Study, Aug 1998 and the OECD Guidelines for the Testing of Chemicals: Guideline 414, Prenatal Developmental Toxicity Study, Jan 2018, which recommend evaluation of approximately 20 females with implantation sites at necropsy. Given the possibility of nongravid animals, unexpected deaths, or treatment-related moribundity and/or mortality, this was an appropriate number of animals to obtain a sample size of 20 at termination.

Animal Identification:
Each animal was identified using a subcutaneously implanted electronic identification chip.

Quarantine:
After receipt at the Testing Facility, the Crl:CD(SD) rats were acclimated prior to the initiation of dosing.

Selection, Assignment, and Disposition of Animals:
Animals were assigned to groups by a stratified randomization scheme designed to achieve similar group mean body weights.
The disposition of all animals was documented in the Study Records.

Housing:
Housing: Single/Individual:
Caging: Solid-bottom cages containing appropriate bedding material (Bed-OCobs® or other suitable material).
Cage Identification: Individual (color-coded) cage cards were affixed to each cage and displayed at least the animal number(s), group number, dietary concentration, study number, and sex of the animal.
Housing set-up was as specified in the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). Cages were arranged on the racks in group order. Where possible, control group animals were housed on a separate rack from the test substance-exposed animals.

Environmental Conditions:
The targeted conditions for animal room environment was as follows:
Temperature: 68°F to 77°F (20°C to 25°C)
Humidity: 30% to 70%
Light Cycle: 12 hours light and 12 hours dark.

Food:
Diet: PMI Nutrition International, LLC Certified Rodent LabDiet® 5002.
The aforementioned basal diet was offered to the control group throughout the study and was used to prepare the test diets. Test and basal diets were offered during the treatment period.
Type: Meal
Frequency: Ad libitum.
Analysis: Results of analysis for nutritional components and environmental contaminants were provided by the supplier and are on file at the Testing Facility. It was considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

Water:
Type: Municipal tap water, treated by reverse osmosis and ultraviolet
irradiation.
Frequency/Ration: Ad libitum, via an automatic watering system.
Analysis: Periodic analysis of the water was performed, and results of these analyses are on file at the Testing Facility. It was considered that there were no known contaminants in the water that could interfere with the outcome of the study.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

Test Substance Inventory and Disposition:
Test materials (e.g., test substance and basal diet) were received by the Testing Facility for distribution as needed. Records of the receipt, distribution, storage, and disposition of test materials (including empty containers of Sponsor-provided materials) were maintained. All unused bulk test substance was retained for subsequent studies.

Dose Formulation and Analysis:
Preparation of Formulations:
The test substance was added to PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 on a weight/weight basis and per Charles River SOPs. Each lot of diet utilized was identified and recorded.

Preparation Details:
Dose Formulation: Basal Diet/Carrier
Frequency of Preparation: Approximately weekly
Storage Conditions Set to Maintain: Set to maintain -20°C

Dose Formulation: Test Substance
Frequency of Preparation: Approximately weekly
Storage Conditions Set to Maintain: Set to maintain -20°C

Any residual volumes from each dosing occasion were discarded.

Preparation Details:
Diet formulations were prepared at appropriate concentrations to meet the target dietary level requirements.

Administration of Test Materials:
Animals were exposed to the test substance continuously in the diet during Gestation Days 6–20 (for exceptions, see Appendix 1). The test substance was administered as a constant concentration (ppm) in the diet for the entire study.

Justification of Route and Dose Levels:
The oral (dietary) route of exposure was selected because this is a potential route of exposure for humans. Historically, this route has been used extensively for studies of this nature.
The oral route (gavage or diet) for rat reproductive and developmental studies was specified by ECHA in the compliance final decision, and under EU REACH, the oral route is the preferred route of exposure for study conduct. The dietary route of exposure was selected because it is consistent with the existing repeat-dose dataset of the test substance.
The dietary levels for the current study were selected by the Sponsor based on the results of 2 previous studies, a 14-day toxicity study (Millard, 2021, 00410109) and dose range-finding prenatal developmental toxicity study (Millard, 2022, 00410110).
In the previous 14-day toxicity study, the test substance was offered at a constant concentration in the diet to male and female rats (3/sex/group) at concentrations of 5000, 10,000, or 15,000 ppm. Lower mean body weights and food consumption were noted for males at 15,000 ppm throughout the study, while these parameters for females were comparable to controls throughout the study.
In the previous dose range-finding prenatal developmental toxicity study, the test substance was offered at a constant concentration in the diet to time-mated female rats (5/group) at concentrations of 5000, 10,000, and 15,000 ppm during Gestation Days 6–20 (equivalent to 322 and 708 mg/kg/day for 5000 and 10,000 ppm, respectively). Due to body weight loss (31.0% from controls on Gestation Day 18) and reduced food consumption (≤ 13.4 g/day during Gestation Days 6–18), a dietary concentration of 15,000 ppm was considered to have exceeded the maximum tolerated dose (MTD). As a result, 1 female in this group was found dead and the remaining females were subsequently euthanized on Gestation Day 18 due to excessive toxicity/early group termination. At 10,000 ppm, a decreased (61%) mean body weight gain, with corresponding reduced (36%) food consumption, were noted when the entire exposure period (Gestation Days 6–21) was evaluated compared to the control group. In addition, a higher (13.3%) mean litter proportion of postimplantation loss was noted at 10,000 ppm compared to controls, with a corresponding lower (17.9%) mean number of live fetuses noted in this group compared to controls, corresponding to the lower (30%) mean gravid uterine weight. No remarkable clinical observations or gross pathological observations were noted, and mean body weights, body weight gains, food consumption, organ weights, pregnancy rates, number of corpora lutea, number of implantation, pre- or post-implantation loss, resorptions, and litter size were similar to controls at 5000 ppm. Therefore, due to the excessive decrease in body weight gain and food consumption at 10,000 ppm, concentrations of 850, 2500, and 7500 ppm were chosen for the current study.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sample Collection and Analysis:
Diet formulation samples were collected for analysis. All samples to be analyzed were transferred to the Analytical Chemistry Department at the Testing Facility for same day analysis, where possible or stored for analysis within known formulation stability period.

Analytical Method:
Analyses described below were performed by liquid chromatography-mass spectrometry(LC-MS) using a validated analytical procedure (Patel, 2022, 00410105).

Concentration and Homogeneity Analysis:
Storage Conditions: Set to maintain a target temperature of -20°C
Acceptance Criteria: For concentration: Mean sample concentration within 70% to 120% of theoretical concentration.
For homogeneity: Relative standard deviation (RSD) of concentrations of ≤ 15% for each group.

Stability Analysis:
Test substance formulations have been previously shown to be stable over the range of concentrations used on this study for at least 28 hours of room temperature storage and at least 10 days of frozen (-20°C) storage (Patel, 2022, 00410105). Therefore, stability of test substance formulations was not assessed on this study.

Details on mating procedure:

Animals are time-mated female Crl:CD(SD) rats.

Duration of treatment / exposure:

Rat dams were exposed to Benzene 1,1ʹ-oxybis-, tetrapropylene derivs., sulfonated salts continuously via the diet during Gestation Days 6–20.

Frequency of treatment:

Rat dams were exposed to Benzene 1,1ʹ-oxybis-, tetrapropylene derivs., sulfonated salts continuously via the diet during Gestation Days 6–20.

Duration of test:

Rat dams were exposed to Benzene 1,1ʹ-oxybis-, tetrapropylene derivs., sulfonated salts continuously via the diet during Gestation Days 6–20.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

850 ppm

Doses / concentrations 2

Dose / conc.:

2 500 ppm

Doses / concentrations 3

Dose / conc.:

7 500 ppm

No. of animals per sex per dose:

25 females per dose group.

Control animals:

yes, plain diet

Examinations

Maternal examinations:

Mortality (All animals): At least twice daily (morning and afternoon), beginning upon arrival through termination/release.
Detailed Clinical Observations (All animals): Once daily, beginning with the day of animal arrival and continuing through (and including) the day of euthanasia.
Individual Body Weights (All animals): Gestation Days 0 (by supplier) and 5–21 (daily).
Food Consumption (All animals): Gestation Days 5–21.

Terminal Procedures:
Method of Euthanasia:
Animals euthanized at study termination were euthanized by carbon dioxide inhalation.

Unscheduled Deaths:
No animals died during the course of the study.

Scheduled Euthanasia:
Animals surviving until scheduled euthanasia were weighed and euthanized by carbon dioxide inhalation (including any animals that delivered).

Histology:
Tissue trimming was performed at the Testing Facility. Maternal thyroid glands from all animals in all groups were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin.

Histopathology:
Histopathological evaluation was performed by a board-certified veterinary pathologist. Maternal thyroid glands were examined microscopically from all females in all groups.

Ovaries and uterine content:

Laparohysterectomy and Macroscopic Examination – Gestation Day 21:
Laparohysterectomies and macroscopic examinations were performed blind to treatment group. All surviving females were euthanized on Gestation Day 21. The cranial, thoracic, abdominal, and pelvic cavities were opened and the contents examined. The uterus of each dam was excised and its adnexa trimmed. Corpora lutea were also counted and recorded. Gravid uterine weights were obtained and recorded. The uterus of each dam was opened and the number of viable and nonviable fetuses, early and late resorptions, and total number of implantation sites were recorded, and the placentae were examined. The individual uterine distribution was documented using the following procedure: all implantation sites, including early and late resorptions, were numbered in consecutive fashion beginning with the left distal uterine horn, noting the position of the cervix and continuing from the proximal to the distal right uterine horn. Uteri which appear nongravid by macroscopic examination were opened and placed in a 10% ammonium sulfide solution for detection of early implantation loss (Salewski, 1964). For all animals, the liver, kidneys, and thyroid gland were excised, weighed (thyroid post-fixation), and preserved in 10% neutral-buffered formalin for possible future histopathologic examination. Macroscopic lesions were also collected and preserved in 10% neutral-buffered formalin for possible future histopathologic examination. Representative sections of corresponding organs from a sufficient number of controls were retained for comparison, if possible. The carcasses were discarded.

Blood sampling:

Thyroid Hormone Sample Processing:
Samples were allowed to clot at ambient temperature at ambient temperature for a minimum of 30 minutes before centrifugation. The samples were centrifuged and the resultant serum was separated and transferred to duplicate uniquely labelled polypropylene tubes. Approximately 150 µL of the resultant serum was placed in a tube and was used for T3 and T4 hormone analysis. Approximately 100 µL was placed in a second tube and was used for thyroid stimulating hormone (TSH) analysis. Samples were stored in a freezer set to maintain a target of -70°C. For T3 and T4 analysis, the samples to be analyzed were transferred to the Bioanalytical Chemistry Department. For TSH analysis, the samples to be analyzed were transferred to the Immunotoxicology Department.

Thyroid Hormone Sample Analysis:
For total T3 and T4 analysis, hormone samples were analyzed using validated ultra high performance liquid chromatography with dual mass spectroscopy (UHPLCMS/MS) assays (Lucarell, 2017, 99764).
Analysis of serum samples to determine TSH concentrations was conducted using a validated Luminex Bead Based (TSH) assay (Castagnier, 2017, 3600258; Peachee, 2020, 00099827).

Fetal examinations:

Fetal Examinations:
Fetal examinations were conducted without knowledge of treatment group. External, internal, and skeletal fetal findings were recorded as either developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or occur at high incidence, representing slight deviations from normal), malformations (those structural anomalies that alter general body conformity, disrupt or interfere
with normal body function, or may be incompatible with life), or incidental (minor changes in coloration, mechanical damage to specimen, etc.). Representative photographs of all malformations, as appropriate, were included in the Study Records. Corresponding low magnification photographs, depicting both the malformed fetus and a comparison control fetus or normal littermate, were also included in the Study Records as needed and as appropriate for
comparison, when possible.

External:
Each viable fetus was examined in detail, sexed, weighed, tagged, and euthanized by a subcutaneous injection of sodium pentobarbital in the scapular region. Following euthanasia, anogenital distance was measured for all viable fetuses. The absolute and normalized (relative to the cube root of fetal body weight) values were reported. The crown-rump length of late resorptions (advanced degree of autolysis) was measured, the degree of autolysis recorded, a gross external examination performed (if possible), and the tissue was discarded.

Visceral (Internal):
The sex of all fetuses was confirmed by internal examination. Approximately one-half of the fetuses in each litter were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe (1984). This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development (Woo and Hoar, 1972). The heads from these fetuses were removed and placed in Harrison’s fixative for subsequent processing and soft-tissue examination using the Wilson sectioning technique (Wilson, 1965). Following examination, the carcasses and cephalic slices were discarded.

Skeletal:
The remaining fetuses (approximately one-half from each litter, excluding any carcasses without heads) were eviscerated and fixed in 100% ethyl alcohol. Following fixation in alcohol, fetuses were stained with Alizarin Red S (Dawson, 1926) and Alcian Blue (Inouye, 1976). The skeletal examination was made following this procedure.

Statistics:

STATISTICAL ANALYSIS:
Numerical data and clinical and necropsy observations data were summarized by sex and
occasion or by litter. See "Any other information on materials and methods incl. tables" section for details on statistical analysis.

Indices:

The following parental indices and litter calculations were included, where applicable:
Pre-Implantation Loss = No. of corpora lutea – no. of implants/No. of corpora lutea x 100
Post-Implantation Loss = No. of implants – no. of live fetuses/No. of implants x 100
Sex Ratio (% males) = No. male fetuses/Total no. of fetuses x 100
Litter % of Fetuses with Abnormalities = No. of fetuses in litter with a given finding/No. of fetuses in litter examined x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

No test substance-related clinical observations were noted at the detailed examinations at any
dietary concentration. Observations noted in the test substance-exposed groups were noted infrequently, similarly in the control group, and/or in a manner that was not
concentration-related.

Mortality:

no mortality observed

Description (incidence):

All females in the control, 850, 2500, and 7500 ppm groups survived to the scheduled necropsy,
including 2 females in the 7500 ppm group (Nos. 4503 and 4516) that delivered on Gestation
Day 21. With the exception of 2 and 1 females in the 850 and 2500 ppm groups, respectively, all
females were gravid.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A lower (32.7%) mean body weight gain was noted in the 7500 ppm group when the entire
exposure period (Gestation Days 6–21) was evaluated compared to the control group, resulting in
a mean absolute body weight on Gestation Day 21 that was 11.4% lower than the control group;
the differences were statistically significant. Additionally, a statistically significantly lower mean
gravid uterine weight, adjusted body weight, and adjusted body weight gain were noted in the
7500 ppm group were noted compared to the control group. These effects were considered test
substance-related and adverse.
Mean maternal body weights, body weight gains, gravid uterine weights, adjusted body weights,
and adjusted body weight gains in the 850 and 2500 ppm groups were unaffected by test
substance exposure. Statistically significantly lower mean body weight gains were noted in both
test substance exposed groups on Gestation Day 19–20 compared to the control group; however,
these differences were transient, not noted in a concentration-related manner, and had no effect
on mean absolute body weights in either group. Other differences from the control group were
slight and not statistically significant.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In the 7500 ppm group, statistically significantly lower mean food consumption (18.2%),
evaluated as g/animal/day, and food utilization (18.0%) were noted when the entire exposure
period (Gestation Days 6–21) was evaluated compared to the control group, which corresponded
with the lower mean absolute body weights and body weight gains noted in this group. These
differences were considered test substance-related and adverse.
Mean maternal food consumption and food utilization in the 850 and 2500 ppm groups were
unaffected by test substance exposure. Any statistically significant differences from the control
group were transient in nature and had no effect on mean body weight gains, and therefore were
not attributed to test substance exposure. Other differences from the control group were slight
and not statistically significant.

Endocrine findings:

effects observed, treatment-related

Description (incidence and severity):

In the 2500 and 7500 ppm groups, concentration dependent, statistically significantly lower
(22.9% and 42.5%, respectively) mean T3 concentrations were noted compared to the concurrent
control group. Additionally, at 7500 ppm, the mean T3 concentration was below the minimum
mean value in the Charles River Ashland historical control data (version 2021.02). The effects on
mean T3 concentrations in these groups were considered test substance-related, but not adverse,
based on the lack of corresponding macroscopic and microscopic findings in the thyroid as well
as lack of concomitant, compensatory changes in T4 and TSH levels.
Mean T3, T4, and TSH concentrations in the 850 ppm group were unaffected by test substance
exposure. Differences from the control group were slight and not statistically significant.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Statistically significantly lower mean absolute kidney (8.0%) and liver (15.3%) weights were
noted in the 7500 ppm group compared to the control group. The lower mean absolute organ
weights corresponded with the lower mean terminal body weight noted in this group; thus, these
changes were not considered to be directly related to test substance exposure. In addition, lower
(11.3% to 14.0%; not statistically significant) mean absolute thyroid weights were noted for all
test substance-exposed groups compared to the control group. There were no histologic
correlates for these differences and the differences were not statistically significant; thus, these
changes were considered to be related to test substance exposure.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test substance-related macroscopic findings were noted. The macroscopic findings observed
were considered incidental, of the nature commonly observed in this strain and age of rats,
and/or were of similar incidence in control and exposed animals, and therefore, were considered
unrelated to exposure to the test substance.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No test substance-related microscopic findings were noted in the thyroid glands. The
microscopic findings observed were considered incidental, of the nature commonly observed in
this strain and age of rats, and/or were of similar incidence and severity in control and exposed
animals, and therefore, were considered unrelated to exposure of the test substance.

Maternal developmental toxicity

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Mean numbers of corpora lutea and implantation sites and the mean litter proportions of
pre-implantation loss were similar across all groups.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

2 500 ppm

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Lower (11.8% to 12.0%) mean fetal weights (male, female and combined sex) were noted in
7500 ppm group compared to the control group. The differences were statistically significant and
individual mean values were below the respective minimum mean values in the Charles River
Ashland historical control data. Therefore, the fetal weight effects in this group were considered
test substance-related and adverse.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Intrauterine survival at 7500 ppm was unaffected by test substance exposure. Intrauterine growth and survival and anogenital distances were unaffected by test substance exposure at dietary concentrations of 850 and 2500 ppm.

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significantly lower mean sexes combined fetal weight was noted at 2500 ppm (5.864 g) compared to the concurrent control group; however, the value was within the range of values in the Charles River Ashland historical control data, and therefore this difference was attributed to biological variation. Other differences from the control group were slight and not statistically significant.

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

Mean absolute and normalized anogenital distances in the 7500 ppm group were also unaffected by test substance exposure. Differences from the control group were slight and not statistically significant.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No external malformations were noted for fetuses in the test substance-exposed groups. External
malformations were limited to a short tail and absent anus for Fetus No. 1524-8 in the control
group.
No external developmental variations were noted for fetuses in this study.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No skeletal malformations were noted for fetuses in the test substance-exposed groups. Skeletal
malformations were limited to absent, branched, and fused ribs and absent caudal, lumbar, sacral,
and thoracic vertebrae for Fetus No. 1524-8 in the control group.
No test substance-related skeletal developmental variations were noted. Findings observed in the
test substance-exposed groups were noted infrequently, similarly in the control group, were not
observed in a concentration-related manner, the differences in the mean litter proportions were
not statistically significant compared to the concurrent control group, and/or were within the
ranges of the Charles River Ashland historical control data.

Visceral malformations:

no effects observed

Description (incidence and severity):

No visceral malformations were noted for fetuses in this study.
No test substance-related visceral developmental variations were noted. Findings observed in the
test substance-exposed groups were noted infrequently, similarly in the control group, were not
noted in a concentration-related manner, the differences in the mean litter proportions were not
statistically significant compared to the concurrent control group, and/or were within the range of
values in the Charles River Ashland historical control data.

Details on embryotoxic / teratogenic effects:

Fetal Morphological Data:
The numbers of fetuses (litters) available for morphological evaluation were 316(25), 288(23),
314(24), and 310(25) in the control, 850, 2500, and 7500 ppm groups, respectively.
Malformations were observed in 1(1) fetus (litter) in the control group and were considered
spontaneous in origin.

Summary of External, Visceral, and Skeletal Examinations:
The numbers of fetuses (litters) available for morphological evaluation were 316(25), 288(23),
314(24), and 310(25) in the control, 850, 2500, and 7500 ppm groups, respectively.
Malformations were observed in 1(1) fetus (litter) in the control group and were considered
spontaneous in origin. When the total malformations and developmental variations were
evaluated on a proportional basis, no statistically significant differences from the control group
were noted. Fetal malformations and developmental variations, when observed in the test
substance-exposed groups, occurred infrequently or at a frequency similar to that in the control
group, did not occur in a concentration-related manner, and/or were within the Charles River
Ashland historical control data ranges. Based on these data, no fetal malformations or
developmental variations were attributed to the test substance.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

2 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

Dietary Formulation Analyses:

The analyzed dietary formulations contained 73.6% to 95.0% of the test substance which was within the protocol-specified range of target concentrations (70% to 120%) and were homogeneous. The test substance was not detected in the analyzed basal diet that was offered to the control group (Group 1).
Results of the analyses of dietary formulations are summarized below.

Results of Homogeneity Analyses:

	Group 2 (850 ppm)	Group 4 (7500 ppm)
Homogeneity Assessment of the 04 Nov 2021 Formulations
Mean Concentration (mg/mL)	625	6149
RSD (%)	9.07	6.84
Mean % of Target	73.6	82.0

 

Results of Concentration Analyses:

	Mean Concentration, ppm (% of Target)
Date of Preparation	Group 2 (850 ppm)	Group 3 (2500 ppm)	Group 4 (7500 ppm)
04 Nov 2021	625 (73.6)	2057 (82.3)	6149 (82.0)
18 Nov 2021	744 (87.5)	2375 (95.0)	6458 (86.1)

Test Substance Consumption:

Mean compound consumptions (mg/kg/day) were based on theoretical dietary concentrations of the test substance and are presented below.

Mean Calculated Test Substance Consumption (mg/kg/day)

Theoretical Dietary Concentration (ppm)^a	Target Dose Levels (mg/kg/day)	Mean Test Substance Consumption (mg/kg/day)
850	55	57
2500	165	170
7500	490	443

^a Test substance was presented as a fixed dietary admix concentration without adjustment for body weight. Achieved dose levels (mg/kg/day) were calculated at the conclusion of the study based on recorded body weight and food consumption values for each animal.

Applicant's summary and conclusion

Conclusions:

Based on adverse effects on maternal body weights, body weight gains, and food consumption, and adverse effects on fetal body weights noted in the 7500 ppm group, a dietary concentration of 2500 ppm was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity and prenatal developmental toxicity of Benzene 1,1ʹ-oxybis-, tetrapropylene derivs., sodium salts administered via the diet to time-mated Crl:CD(SD) rats.

Executive summary:

The objectives of this study were to determine whether the test substance, Benzene 1,1ʹ-oxybis-, tetrapropylene derivs., sodium salts, could affect rat development after maternal dietary exposure from implantation to 1 day prior to expected parturition at the exposure levels tested and to determine a no-observed-adverse-effect level (NOAEL) for maternal and developmental toxicity
in rats.

Rat dams were exposed to Benzene 1,1ʹ-oxybis-, tetrapropylene derivs., sulfonated salts continuously via the diet during Gestation Days 6–20.
The following parameters and end points were evaluated in this study: mortality, clinical signs, body weights, body weight gains, gravid uterine weights, food consumption, food utilization, thyroid hormones, organ weights, macroscopic and microscopic examinations, intrauterine growth and survival, anogenital distances, and fetal morphology.
The estimated achieved intake of test substance in mg/kg body weight/day was calculated using the nominal concentration of the test substance in the diet and is summarized in the text table below.

Mean Calculated Test Substance Consumption (mg/kg/day)

Theoretical Dietary Concentration (ppm)^a	Target Dose Levels (mg/kg/day)	Mean Test Substance Consumption (mg/kg/day)
850	55	57
2500	165	170
7500	490	443

^a Test substance was presented as a fixed dietary admix concentration without adjustment for body weight. Achieved dose levels (mg/kg/day) were calculated at the conclusion of the study based on recorded body weight and food consumption values for each animal.

The analyzed dietary formulations were within the protocol-specified range of target concentrations and were homogeneous.
All females survived to the scheduled necropsy on Gestation Day 21, including 2 females in the 7500 ppm group that delivered on that day. No test substance-related clinical observations were noted at the daily examinations at any dietary concentration.
In the 7500 ppm group, lower mean body weight gains, with corresponding lower mean food consumption and food utilization, were noted when the entire exposure period (Gestation Days 6–21) was evaluated compared to the control group. As a result, mean absolute body weight in this group was 11.4% lower than the control group on Gestation Day 21. Furthermore, a lower mean gravid uterine weight, adjusted body weight, and adjusted body weight gain were
noted at 7500 ppm compared to the control group. The effects on body weight and food consumption noted in the 7500 ppm group were considered test substance-related and adverse.
Mean maternal body weights, body weight gains, gravid uterine weights, adjusted body weights, adjusted body weight gains, and food consumption in the 850 and 2500 ppm groups were unaffected by test substance exposure.
Concentration dependent, lower mean T3 concentrations were noted for females in the 2500 and 7500 ppm groups compared to the concurrent control group; the value in the 7500 ppm group was also below the minimum mean value in the Charles River Ashland historical control data (version 2021.02). Mean T3 concentration in the 850 ppm group, and mean T4 and TSH
concentrations in all test substance-exposed groups were unaffected by test substance exposure.
The effects on mean T3 concentrations in these groups were considered test substance-related, but not adverse, based on the lack of corresponding macroscopic and microscopic findings in the thyroid as well as lack of concomitant, compensatory changes in T4 and TSH levels.
No test substance-related effects on organ weights or macroscopic and microscopic observations were noted at any dietary concentration.
In the 7500 ppm group, lower (11.8% to 12.0%) mean fetal body weights (male, female, and sexes combined) were noted compared to the concurrent control group; the values were also below the respective minimum mean values in the Charles River Ashland historical control data.
Therefore, the fetal body weight effects at 7500 ppm were considered test substance-related and adverse. Intrauterine growth in the 850 and 2500 ppm groups, and intrauterine survival and mean anogenital distances in the 850, 2500, and 7500 ppm groups were unaffected by test substance exposure. No test substance-related effects on fetal morphology were note at any dietary
concentration.
In conclusion, based on adverse effects on maternal body weights, body weight gains, and food consumption, and adverse effects on fetal body weights noted in the 7500 ppm group, a dietary concentration of 2500 ppm was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity and prenatal developmental toxicity of Benzene 1,1ʹ-oxybis-, tetrapropylene
derivs., sodium salts administered via the diet to time-mated Crl:CD(SD) rats.