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

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / 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

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

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

Specific details on test material used for the study:

Identification: Benzene 1,1’-oxybis-, tetrapropylene derivs., sulfonated, sodium salts
Alternate Identification: -
CAS No.: 119345-04-9
Batch/Lot No.: CAC09162020
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 species / strain selection:

Receipt:
On 03 Aug 2021 and 17 Aug 2021, female and male Crl:CD(SD) rats, respectively, were received from Charles River Laboratories, Inc., Raleigh, NC. The animals were approximately 11 weeks old and weighed between 277 and 368 g (males) or 201 and 286 g (females) 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 reproduction studies. Charles River Ashland has reproductive historical control 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. The number of animals selected for this study was based on the OECD Guideline for the Testing of Chemicals: Guideline 421, Reproduction/Development Toxicity Screening Test, 29 Jul 2016 (as modified), which recommends that evaluation of each group be initiated with at least 10 males and 12–13 females per group. Females are evaluated for estrous cyclicity during the pretest period and any females that fail to exhibit normal 4–5 day estrous cycles (e.g., EDDDE),
during the pretest period, are excluded from the study, therefore, the extra females are included to yield at least 10 females per group. Given the possibility of nongravid animals, unexpected deaths, total litter losses, or treatment-related moribundity and/or mortality, this was an appropriate number of animals to obtain a sample size of 8 at termination.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal Identification:
Each animal was identified using a subcutaneously implanted electronic identification chip. Offspring were identified by tattoo markings applied to the digits after parturition and by microchip after weaning.

Environmental Acclimation:
After receipt at the Testing Facility, the Crl:CD(SD) rats were acclimated prior to initiation of dosing. The testes were palpated at least once for all males during acclimation.

Selection, Assignment, and Disposition of Animals:
Animals were assigned to groups by a stratified randomization scheme designed to achieve similar group mean body weights. Males and females were randomized separately. Animals at
extremes of body weight range, or not exhibiting normal, 4- to 5-day estrous cycles were not assigned to groups.
To reduce variability among the F1 litters, 10 pups/litter of equal sex distribution, if possible, were randomly selected on PND 4. Standardization of litter size was not performed on litters with fewer than 10 pups. The remaining offspring were euthanized by an intraperitoneal injection of sodium pentobarbital (following thyroid hormone blood collection for those selected) and discarded.
The disposition of all animals was documented in the Study Records.

Housing:
Group housed (2–3 animals of the same sex and same dosing group together) during the premating period.
During cohabitation, the females were paired (1:1) with a male for mating (home cage of the male).
Single/Individual following positive signs of mating or the end of the breeding period.
All offspring selected to constitute the F1 generation were group housed (2–3 animals of the same sex and same dosing group together) from weaning until euthanasia.
Caging: Solid-bottom cages containing heat-treated aspen bedding material (Northeastern Products Corp.).
Cage Identification: Individual (color-coded) cage cards were affixed to each cage and displayed at least the animal number(s), cage number, 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-treated
animals.

Environmental Conditions:
The targeted conditions for animal room environment were as follows:
Temperature: 68°F to 78°F (20°C to 26°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 exposure period.
Type: Meal
Frequency: Ad libitum
Analysis: Results of analysis for nutritional components and environmental contaminants are provided by the supplier and are on file at the Testing Facility. It is considered that there are 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. Water bottles were provided, if required.
Analysis: Periodic analysis of the water is performed, and results of these analyses are on file at the Testing Facility. It is considered that there are no known contaminants in the water that could interfere with the outcome of the study.

Veterinary Care:
Veterinary care was available throughout the course of the study, and animals were examined by the veterinary staff as warranted by clinical signs or other changes. All veterinary examinations and recommended therapeutic treatments, if any, were documented in the study records and reviewed by the Study Director.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

Diet Formulation:
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:
Diet formulations were prepared at appropriate concentrations to meet dose level requirements.

Administration of Test Materials:
Animals were exposed to the test substance continuously via the diet. Males were exposed for 14 days prior to mating and continuing through the day of euthanasia (for a minimum of 56 days). Females were exposed for 14 days prior to mating and continuing throughout mating, gestation, and lactation until euthanasia. F1 offspring were potentially exposed in utero and through nursing during lactation. Offspring selected to constitute the F1 generation were directly exposed beginning at weaning (PND 21) and continuing until euthanasia (PND 35).
The test substance was administered as a constant concentration (ppm) in the diet to F0 males for the entire study and to F0 females during the premating, mating, and gestation periods. During lactation for F0 females, test substance dietary concentrations were adjusted based on calculated corrected body weights and historical control food consumption data to compensate for the higher caloric demand on maternal animals during this period in order to adhere to target doses.
Likewise, during PND 21 to euthanasia for the F1 generation, the test substance concentrations
were adjusted based on calculated corrected body weights and historical control food consumption data.

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 was consistent with the existing repeat-dose dataset of the test material.
A 13-week dietary toxicity study was previously conducted in rats at concentrations of 0, 100, 300, 1000, 3000, or 10,000 ppm (The Dow Chemical Company, 1957). The no-observed-effect level (NOEL) was determined to be 3000 ppm in the diet (male/female) based on histopathological changes in the liver of male and female rats given 10,000 ppm Benzene 1,1’-oxybis-, tetrapropylene derivs., sulfonated, sodium salts in the diet. In addition, female rats at the 10,000 ppm dose level showed a retardation of growth, and an increase in the average weight of the liver and kidneys.
A 2-year chronic combined repeated dose and carcinogenicity study was conducted in rats at concentrations of 300, 1000, 3000, or 10,000 ppm (The Dow Chemical Company, 1963). On a body weight basis, the test substance was consumed in the amounts of 15, 50, 150, or 500 mg/kg/day by the rats on the respected levels. The NOEL was determined to be 3000 ppm based on growth depression observed in rats given 10,000 ppm Benzene 1,1’-oxybis-, tetrapropylene derivs., sulfonated, sodium salts in the diet.
The concentrations for the current study were determined from the results of a previous 14-day toxicity study (Millard, 2022, 00410109). In the previous study, male and female rats (3/sex/group) were administered the test substance in diet for 14 consecutive days at concentrations of 0, 5000 10,000, or 15,000 ppm. In that study, there were no remarkable clinical observations noted at the daily examinations. Lower body weight gains were noted for males at 10,000 and 15,000 ppm during Study Days 0–14 compared to controls, resulting in body weights that were 4.1% to 10.0% lower, respectively on Study Day 14. Female body weights and body weight gains at all concentrations were similar to controls. Males given 10,000 and 15,000 ppm had decreased feed consumption for the first two days of treatment compared to controls, but feed consumption was similar to controls for the remainder of the study. Feed consumption for all female treated groups was similar to controls. Therefore, a concentration of 15,000 ppm was selected for the high dose in the current study as the limit dose, with concentrations of 7500 and 3500 ppm selected as the low- and mid-dose levels to establish a potential dose response and no observed-adverse-effect level (NOAEL).

Details on mating procedure:

Following a minimum of 14 days of exposure, 1 female was cohabited with 1 male rat of the same treatment group, avoiding sibling mating (Charles River Laboratories will supply non-litter mates),
in a solid-bottom cage for mating (home cage of the male). A maximum of 14 days was allowed for mating. If no evidence of mating was obtained after 14 days, the animals were separated without further opportunity for mating, and the female was placed in a solid-bottom cage containing bedding material.
Daily vaginal lavages performed beginning during the pretest period and continuing through the
2-week premating and mating treatment periods untilevidence of mating was observed.

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.
Dose analysis results were verified prior to diet administration at each sampling interval.
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, Draft, 00410105).

Concentration and Homogeneity Analysis:
Storage Conditions: Temperature set to maintain 18°C to 24°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, Draft, 00410105). Therefore, stability of test substance formulations was not assessed on this study.

Duration of treatment / exposure:

Animals were exposed to the test substance continuously via the diet. Males were exposed for 14 days prior to mating and continuing through the day of euthanasia (for a minimum of 56 days). Females were exposed for 14 days prior to mating and continuing throughout mating, gestation, and lactation until euthanasia (Lactation Day 21). F1 offspring were potentially exposed in utero and through nursing during lactation. Offspring selected to constitute the F1 generation were directly exposed beginning at weaning (Postnatal Day [PND] 21) and continuing until euthanasia (PND 35).

Frequency of treatment:

Animals were exposed to the test substance continuously via the diet.

Details on study schedule:

Breeding:
Following a minimum of 14 days of exposure, 1 female was cohabited with 1 male rat of the same treatment group, avoiding sibling mating (Charles River Laboratories will supply non-litter mates), in a solid-bottom cage for mating (home cage of the male). A maximum of 14 days was allowed for mating. If no evidence of mating was obtained after 14 days, the animals were separated without further opportunity for mating, and the female was placed in a solid-bottom cage containing bedding material.
Detection of mating was confirmed by evidence of a copulatory plug in the vagina or by evidence of sperm in a vaginal lavage. After confirmation of mating, the female was returned to an individual solid-bottom cage, and the day was designated as day 0 of gestation.

Standardization of Litter Size:
To reduce variability among the litters, 10 pups from each litter, of equal sex distribution (if possible), were randomlyselected on PND 4.

Selection of F1 Generation:
Prior to PND 21, 1 F1 pups per sex per litter from all available litters were selected.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

15 000 ppm

Doses / concentrations 2

Dose / conc.:

7 500 ppm

Doses / concentrations 3

Dose / conc.:

3 500 ppm

Doses / concentrations 4

Dose / conc.:

0 ppm

No. of animals per sex per dose:

10/sex/dose group

Control animals:

yes, plain diet

Details on study design:

The concentrations for the current study were determined from the results of a previous 14-day toxicity study (Millard, 2022, 00410109). In the previous study, male and female rats (3/sex/group) were administered the test substance in diet for 14 consecutive days at concentrations of 0, 5000 10,000, or 15,000 ppm. In that study, there were no remarkable clinical observations noted at the daily examinations. Lower body weight gains were noted for males at 10,000 and 15,000 ppm during Study Days 0–14 compared to controls, resulting in body weights that were 4.1% to 10.0% lower, respectively on Study Day 14. Female body weights and body weight gains at all concentrations were similar to controls. Males given 10,000 and 15,000 ppm had decreased feed consumption for the first two days of treatment compared to controls, but feed consumption was similar to controls for the remainder of the study. Feed consumption for all female treated groups was similar to controls. Therefore, a concentration of 15,000 ppm was selected for the high dose in the current study as the limit dose, with concentrations of 7500 and 3500 ppm selected as the low- and mid-dose levels to establish a potential dose response and no observed-adverse-effect level (NOAEL).

Determination of Achieved Dosage Levels:
The estimated achieved intake of test substance in mg/kg body weight/day was calculated
separately for each cage for each period of food consumption recorded, using the following
formula:
Individually Housed:
Achieved Dose (mg/kg/day) =
((FF-FL)/NDays / ((BWS + BWE) / 2)) x CONC
Where FF = Food Fed (g)
FL = Food Left (g)
NDays = Number of days between FF and FL (days)
BWS = Body weight for first animal at start of interval (g)
BWE = Body weight of first animal at end of interval (g)
CONC = Nominal concentration of test substance in diet (ppm)

Socially Housed:
Achieved Dose (mg/kg/day) =
(((FF-FL)/NDays / (((BWS1 x AD1) / Ndays) + ((BWS2 x AD2) / NDays) + …) + ((BWE1 +
BWE2 + …) / 2)) x CONC
Where FF = Food Fed (g)
FL = Food Left (g)
NDays = Number of days between FF and FL (days)
AD1 = Number of days first animal was in cage during interval (days)
AD2 = Number of days second animal was in cage during interval (days)
BWS1 = Body weight for first animal at start of interval (g)
BWS2 = Body weight for second animal at start of interval (g)
BWE1 = Body weight of first animal at end of interval (g)
BWE2 = Body weight of second animal at end of interval (g)
CONC = Nominal concentration of test substance in diet (ppm)
It was assumed the food consumption was the same over the body weight period as was
calculated during the food consumption period.
Data were presented as a cage mean for each period of food consumption and as an overall
achieved dose, which was the arithmetic mean of all calculations during the treatment period for
each group.
An overall estimated achieved dose for each group was calculated by averaging the group means
for each period.

Examinations

Parental animals: Observations and examinations:

In-life Procedures, Observations, and Measurements:
F0 Generation:
Mortality - At least twice daily (morning and afternoon) beginning upon arrival through termination.
Detailed Clinical Observation (DCO) - Weekly, beginning on the first day of test diet administration for males, and beginning at the start of estrous lavages for females, through evidence of mating.
Gestation Days 0, 4, 7, 11, 14, and 20.
Lactation Days 1, 4, 7, 10, 14, 17, and 21.
Cage-Side Observations - Once daily (except on days where DCO's were performed).
Individual Body Weights Males- Study Days 0, 1, 3, and 7, and weekly thereafter through euthanasia. Body weights were also recorded on the day of euthanasia.
Individual Body Weights Females - Study Days 0, 1, 3, and 7, and weekly thereafter until
evidence of copulation was observed.
Gestation Days 0, 4, 7, 11, 14, 17, and 20.
Lactation Days 1, 4, 7, 10, 14, 17, and 21.
Food Consumption - Daily, beginning on the first day of test or control diet administration, until euthanasia.
Thyroid Hormone Analysis - Blood samples for thyroid hormone analyses were collected at termination for all F0 males and females. Samples were collected prior to noon to reduce variability due to normal diurnal variations in physiological levels of thyroid hormones. Blood samples were maintained at room temperature and allowed to clot. Serum was isolated in
a refrigerated centrifuge and stored in a freezer set to maintain a target of -70°C. Blood samples were analyzed for Triiodothyronine (T3), Thyroxine (Total T4) and Thyroid Stimulating Hormone (TSH).

Oestrous cyclicity (parental animals):

Estrous - Daily vaginal lavages performed beginning during the pretest period and continuing through the 2-week premating and mating treatment periods until evidence of mating was observed.

Litter observations:

Parturition, Lactation, and Littering - Females were observed for dystocia (prolonged or
difficult labor), or other difficulties, and any findings were recorded. When parturition was judged to be complete, the sex of each pup was determined, pups were examined for gross malformations, and the number of stillbirths and live pups were recorded. Any
changes or abnormalities in nesting and nursing behavior were recorded.

F1 Litter Parameters:
Mortality - All pups were observed daily for general appearance and behavior and survival from birth through euthanasia.
Detailed Clinical Observations - PND 1, 4, 7, 10, 13, 17, 21, and twice weekly thereafter until euthanasia.
Cage-Side Observations - Once daily beginning on PND 21. Cage side observations need
not be conducted on days that the detailed clinical observations are performed.
Pup Sex - PND 0 or 1, 4, 13, and 21.
Individual Pup Body Weights - PND 1, 4 (before culling), 7, 10, 13, 17, 21 and daily thereafter until euthanasia.
Food Consumption - Daily, beginning on PND 21 through euthanasia.
Anogenital Distance - PND 1.
Assessment of Areolae/Nipple Anlagen Retention Males - PND 13.

Thyroid Hormone Analysis - Blood samples for thyroid hormone analyses were collected at the time of culling, on PND 4from 2 culled pups/litter; samples were pooled regardless of sex. Additional culled pups may have been bled to obtain the required blood volume, if necessary. However, based on litter size, if fewer than 2 pups are available for culling, blood was collected from the available pups only. Blood samples were also collected at termination on PND 21 and PND 35 from 1 pup/sex/litter (samples analyzed separately, by sex). Blood samples were maintained at room temperature and allowed to clot for a minimum of 30 minutes. Serum was isolated in a refrigerated centrifuge and stored in a freezer set to maintain a target of -70°C.
Blood samples were analyzed for Triiodothyronine (T3), Thyroxine (Total T4) and Thyroid Stimulating Hormone (TSH).

Postmortem examinations (parental animals):

Unscheduled Deaths:
Due to excessive toxicity, Group 4 animals were euthanized as per Testing Facility SOPs. These
animals underwent a complete necropsy examination, which included evaluation of the carcass;
all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic,
abdominal, and pelvic cavities with their associated organs and tissues. The number and location
of implantation sites or scars were recorded for females. No organs were weighed, and no tissues
were collected. Viable fetuses were euthanized by a subcutaneous injection of sodium
pentobarbital in the scapular region.

Scheduled Euthanasia:
All surviving animals, including females that failed to deliver or with total litter loss, were
euthanized by carbon dioxide inhalation.

Necropsy:
F0 animals were subjected to a complete necropsy examination, which included examination of
the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal
cord, and the thoracic, abdominal, and pelvic cavities, with their associated organs and tissues.
The numbers of implantation sites and former implantation sites were recorded for females that
delivered or had macroscopic evidence of implantation. The number of unaccounted-for sites
was calculated for each female by subtracting the number of pups born from the number of
former implantation sites observed. The numbers of corpora lutea were recorded for females with
total litter loss between Lactation Days 0–4 and for females with evidence of macroscopic
implantations. Uteri of females without macroscopic evidence of implantation were opened and placed in 10% ammonium sulfide solution for detection of early implantation loss (Salewski, 1964).

Organ Weights:
The following organs were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together, unless otherwise indicated. Organ to body weight
ratio (using the terminal body weight) and organ to brain weight ratios were calculated.
Adrenal glands
Brain
Epididymidesa
Heart
Kidneys
Liver
Ovaries with oviducts
Pituitary gland
Prostate gland
Seminal vesicles
Spleen
Testesa
Thymus gland
Thyroids with parathyroids (after fixation)

Tissue Collection and Preservation:
Representative samples of the tissues identified below were collected from all animals and preserved in 10% neutral buffered formalin, unless otherwise indicated.
Adrenal glands (2)
Brain (forebrain, midbrain, hindbrain)
Coagulating glands (2)
Kidneys (2)
Liver (sections of 2 lobes)
Mammary gland
Ovaries and oviducts (2)
Pituitary gland
Prostate gland
Seminal vesicles (2)
Testes with epididymides (2)
Thyroids (with parathyroids, if present [2])
Uterus with cervix and vagina. Uterus not retained if placed in 10% ammonium sulfide solution.
Vas deferens (2)
All gross lesions

Histology:
Tissues collected, except adrenal glands, from all F0 animals at the scheduled necropsy were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin. In addition, PAS staining was used for the testes and epididymides.

Histopathology:
Pathological evaluation was performed by a board-certified veterinary pathologist. Tissues
collected, with the exception of adrenal glands, for microscopic examination were evaluated from all F0 animals in Groups 1 and 3 at the scheduled necropsy. Gross lesions, ovaries, and uterus with cervix were examined from all groups at the scheduled necropsy.

Postmortem examinations (offspring):

F1 Litters: Terminal procedures for PND 21 offspring
Unscheduled Deaths:
A necropsy was conducted for animals that died on study, and specified tissues were saved.
Intact offspring that were found dead, during PND 0–4, were necropsied using a fresh dissection
technique, which included examination of the heart and major vessels (Stuckhardt and Poppe,
1984). Findings were recorded as developmental variations or malformations, as appropriate. A
gross necropsy was performed on any pup found dead after PND 4.

Scheduled Euthanasia:
On PND 21, all non-selected pups were euthanized by carbon dioxide inhalation or
exsanguination following thyroid blood collection.

Necropsy:
On PND 21, 1 pup/sex/litter was subjected to a complete necropsy examination, with emphasis
on developmental morphology and organs of the reproductive system. All other non-selected pups were discarded without examination.

Organ Weights:
The organs collected (identified below) were weighed at necropsy from 1 pup/sex/litter at the scheduled euthanasia. Organ to body weight ratios (using the terminal body weight) were calculated.
Kidney (2)
Liver
Thyroid (with parathyroids, if present)

Tissue Collection and Preservation:
Representative specimens with malformations from offspring found dead were preserved in
10% neutral buffered formalin.
Representative samples of the tissues collected and gross lesions were collected from 1 pup/sex/litter at the scheduled euthanasia and preserved in 10% neutral buffered formalin.

F1 Generation: Terminal procedures for PND 35 offspring
Unscheduled Deaths:
No animals died during the course of the F1 generation dosing period.

Scheduled Euthanasia:
On PND 35, all F1 male and female adults were euthanized by intraperitoneal injection of sodium
pentobarbital or exsanguination following thyroid hormone blood collections.

Necropsy:
On PND 35, all F1 males and females were subjected to a complete necropsy examination, which
included evaluation of the carcass; all external surfaces and orifices; cranial cavity and external
surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs
and tissues.

Organ Weights:
The organs identified below were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together, unless otherwise indicated. Organ to body weight
ratios (using the terminal body weight) were calculated.
Adrenal glands
Epididymidesa
Kidneys
Liver
Ovaries
Testes
Thyroids with parathyroids
Uterus (with oviducts and cervix)

Tissue Collection and Preservation:
Representative samples of the tissues identified below were collected from all animals
and preserved in 10% neutral buffered formalin, unless otherwise indicated.
Adrenal glands
Epididymides
Kidney
Liver
Ovaries
Testes
Thyroids with parathyroids
Uterus (with oviducts and cervix)
Gross lesions

Statistics:

See "Materials and Methods" section for details on statistical analysis.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

No test substance-related clinical observations were noted for F0 males and females in the 3500 and 7500 ppm dose concentrations. Clinical observations noted in the test substance-exposed groups occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not exposure-related. See "Details on Results (P0)" section for details on the 15,000 ppm dose group observations.

Mortality:

no mortality observed

Description (incidence):

All F0 males and females in the 3500 and 7500 ppm dose groups survived to the scheduled necropsies. See "Details on Results (P0)" section for details on the 15,000 ppm dose group mortality.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males:
At 7500 ppm, lower mean body weight gains were noted for F0 males during the premating period (Study Days 0–14) and when the entire generation (Study Days 0–56) was evaluated compared to the control group, resulting in mean absolute body weights that were 7.9% to 11.1% lower during Study Days 14–56; differences were statistically significant. These body weight effects at 7500 ppm were considered test substance-related and adverse.
Mean body weights and body weight gains in the 3500 ppm group F0 males were unaffected by test substance exposure throughout the study. None of the differences from the control group were statistically significant.

Females:
Weekly:
Mean body weights and body weight gains in the 3500 and 7500 ppm group F0 females were unaffected by test substance exposure during the premating period (Study Days 0-14). None of the differences from the control group were statistically significant.

Gestation:
At 7500 ppm, a lower mean body weight gain was noted for F0 females throughout gestation compared to the control group; differences were statistically significant during Gestation Day 14–20 and when the entire gestation period (Gestation Days 0–20) was evaluated. These deficits in body weight gain resulted in mean absolute body weight in this group that was 16.3% (statistically significant) lower than the control group on Gestation Day 20. These effects on mean body weight during gestation were considered test substance-related, and partiallyattributed to the lower number of pups born in this group.
In the 3500 ppm group, a slightly lower (not statistically significant) mean body weight gain was noted when the entire gestation period (Gestation Days 0–20) was evaluated compared to the control group. As a result, mean absolute body weight on Gestation Day 20 in this group was 7.9% lower (statistically significant) than the control group. These effects on mean body weight during gestation were primarily attributed to the lower number of pups born in this group.

Lactation:
Mean absolute body weight for F0 females in the 7500 ppm group was 7.0% lower (not statistically significant) than the control group on Lactation Day 1, a carryover effect from gestation. A higher (statistically significant) mean body weight gain was noted in this group when the entire lactation period (Lactation Day 1–21) was evaluated, resulting in mean absolute body weight on Lactation Day 21 that was comparable (0.9% difference) to the control group.
Mean body weights and body weight gains for F0 females in the 3500 ppm group were unaffected by test substance exposure during lactation. None of the differences from the control group were statistically significant.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males:
Mean food consumption, evaluated as g/animal/day, in the 3500 and 7500 ppm group F0 males was unaffected by test substance exposure during the premating (Study Days 0–14) and postmating (Study Days 28–56) periods. Lower (16.7%) mean food consumption was noted in the 7500 ppm group during Study Days 0–7 compared to the control group; the differences during Study Days 0–1 and 1–3 were statistically significant. These effects on food consumption corresponded to lower mean body weight gains or body weight loss noted in this group during these intervals, and were likely due to palatability of the test substance in the diet following the initiation of exposure, as food consumption values during Study Days 7–14 were comparable to the control group. Any other statistically significant differences were sporadic, transient, and did not impact the overall evaluation intervals.

Females:
Weekly:
Mean food consumption, evaluated as g/animal/day, in the 3500 and 7500 ppm group F0 females was unaffected by test substance exposure during the premating period (Study Days 0–14). Any statistically significant differences were transient and did not impact the overall interval.

Gestation:
Mean maternal food consumption, evaluated as g/animal/day, in the 3500 and 7500 ppm group F0 females was unaffected by test substance exposure during gestation (Gestation Days 0–20).
Any statistically significant differences were transient and did not impact the overall interval.

Lactation:
Mean maternal food consumption, evaluated as g/animal/day, in the 3500 and 7500 ppm group F0 females was unaffected by test substance exposure during lactation (Lactation Days 1-21). None of the differences from the control group were statistically significant.

Endocrine findings:

no effects observed

Description (incidence and severity):

Thyroid Hormone Analysis:
There were no test substance-related effects on thyroid hormone values (T3, T4, and TSH) in the F0 males at 3500 and 7500 ppm. Differences from the control group were not statistically significant, considered to be the result of normal biological variation, and were not considered to be of toxicological significance. As no effect was noted for F0 males, F0 females were not analyzed for thyroid hormone levels.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No test substance-related microscopic findings were noted for F0 males and males at the
scheduled necropsies. 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 treated animals and, therefore, were considered unrelated to exposure to
the test substance.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Statistically significantly lower mean numbers of implantation sites and pups born were noted in
the 3500 and 7500 ppm groups compared to the control group. These effects were considered test substance-related and adverse. Mean numbers of unaccounted-for sites in these groups were higher (not statistically significant) compared to the control group, which was indicative of
postimplantation loss; the increase in the 7500 ppm group was driven by 1 dam (No. 2428) with
16 unaccounted-for sites.

Reproductive function / performance (P0)

Reproductive performance:

no effects observed

Description (incidence and severity):

No test substance-related effects on reproductive performance were observed at 3500 and
7500 ppm. No statistically significant differences were noted. All F0 males in the control, 3500,
and 7500 ppm groups sired a litter and all F0 females in these same groups were gravid.
The mean numbers of days between pairing and coitus in the 3500 and 7500 ppm groups were
similar to the control group value. The mean lengths of estrous cycles in these groups were also
similar to the control group value. None of these differences were statistically significant.

Gestation Length and Parturition:
Mean gestation lengths in the 3500 and 7500 ppm groups were similar to those in the control group. No statistically significant differences were noted. No signs of dystocia were noted in these groups.

Details on results (P0)

In the 15,000 ppm group, mean body weight losses and lower mean body weight gains, with corresponding reduced mean food consumption, were noted for F0 males and females following approximately 3 weeks of test substance exposure. As a result, mean absolute body weights for F0 males and females in this group were up to 27.5% and 21.8% lower, respectively, than the control group during this period and a dietary concentration of 15,000 ppm was considered to have exceeded the maximum tolerated dose. This group of animals was subsequently terminated and necropsied on 23 Sep 2021 (Study Day 23 [males] or Gestation Day 4, 5, 6, 7, or 8 [females]), thus precluding evaluation of any other endpoints on the study. No test substance-related macroscopic findings were observed in the 15,000 ppm group at necropsy.

One 7500 ppm group F0 female failed to deliver and 1 experienced total litter loss. Additionally, one 3500 ppm group F0 female experienced total litter loss. These 3 animals were euthanized in accordance with the protocol. The cause for failed delivery/litter loss was not identified.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

3 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: Based on effects on body weight for F0 males and females at 7500 ppm and excessive toxicity (early group termination) noted at 15,000 ppm.

2

Dose descriptor:

NOAEL

Effect level:

7 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: 7500 ppm was considered to be the no-observed-adverse-effect level (NOAEL) for F0 reproductive toxicity.

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

In the 7500 ppm group, 5 pups from 1 litter, and an additional pup from a separate litter, were noted with missing eyes (bilateral). Anophthalmia is present in the Sprague Dawley rat historical control data for embryo/fetal development studies, and is the ninth most common external malformation in the current HCD (range of 0.00–0.500% per litter, with a mean of 0.014%). The general physical condition (defined as the occurrence and severity of clinical observations) of the remaining F1 pups in this study was unaffected by test substance exposure. Four (1), 3(3), and 3(2) pups (litters) in the control, 3500, and 7500 ppm groups, respectively, were found dead. Zero (0), 1(1), and 2(2) pups (litters) in the same respective groups were missing and presumed to have been cannibalized.
With the exception of the previously mentioned missing eyes for pups in the 7500 ppm group, no clinical observations were noted for F1 males and females at any concentration.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Four (1), 3(3), and 3(2) pups (litters) in the control, 3500, and 7500 ppm groups, respectively, were found dead. No internal findings that could be attributed to parental test substance exposure were noted at the necropsies of pups that were found dead. Aside from the presence or absence
of milk in the stomach, internal findings were limited to lobular dysgenesis of the lung in one pup in the 3500 ppm group. No other internal findings were noted.
All F1 males and females assigned to the F1 generation survived to the scheduled necropsy on
PND 35.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean F1 pup birth weights (PND 1) in the 3500 and 7500 ppm groups were comparable to the control group. Mean male and female pup body weights and body weight changes during PND 1–21 in the 3500 and 7500 ppm groups were unaffected by parental exposure to the test substance. Any statistically significant differences from the control group were transient.
Mean body weights and body weight gains in the 3500 and 7500 ppm group F1 males and females were unaffected by test substance exposure throughout the generation (PND 21–35).
Differences from the control group were slight and not statistically significant.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Mean food consumption, evaluated as g/animal/day, in the 3500 and 7500 ppm group F1 males
and females were unaffected by test substance exposure throughout the generation (PND 21-35).
Differences from the control group were slight and not statistically significant.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

The mean anogenital distances (absolute and relative to the cube root of pup body weight) in the 3500 and 7500 ppm groups were unaffected by parental administration of the test substance. Differences from the control group were slight and not statistically significant.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Areolae/nipple anlagen in the F1 male pups was unaffected by parental administration of the test substance when evaluated on PND 13. No areolas/nipples were present for any F1 male pups.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on organ weights in the F1 males and females at any
concentration on PND 21. Differences from the control group were considered to be the result of
normal biological variation and were not considered to be of toxicological significance.
There were no test substance-related effects on organ weights in the F1 males and females at any concentration on PND 35. Differences from the control group were considered to be the result of normal biological variation and were not considered to be of toxicological significance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No internal findings that could be attributed to parental test substance administration were noted
at the necropsy of pups euthanized on PND 21. Internal findings included a dilated renal pelvis
in one pup in the 3500 ppm group and dilation of the brain in one pup in the 7500 ppm group. No other internal findings were noted.
No test substance-related macroscopic findings were noted for F1 males and females at the scheduled necropsy on PND 35. The 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 treated animals and, therefore, were considered unrelated to exposure to the test substance.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

In the 3500 and 7500 ppm groups, the mean number of pups born (8.5 and 7.6 pups/litter, respectively) and live litter sizes on PND 0 (8.4 and 7.3 pups/litter, respectively) were statistically significantly lower than the control group (13.4 and 13.3 pups/litter, respectively). In addition, lower (not statistically significant) mean postnatal survival was noted in these same groups (86.3% and 84.7% per litter, respectively) during birth to PND 4 compared to the control group (97.9% per litter), and attributed to 1 dam in each group with total litter loss on Lactation Day 0. The aforementioned differences were considered test substance-related and adverse. Mean postnatal survival in these groups was unaffected by the test substance during PND 4–21.

Details on results (F1)

Thyroid Hormone Analysis (PND 21):
There were no test substance-related effects on thyroid hormone values (T3, T4, and TSH) in the F1 males and females at any concentration on PND 21. Differences from the control group were not statistically significant, considered to be the result of normal biological variation, and were not considered to be of toxicological significance. As no effect was noted for F1 males and females on PND 21, samples collected on PND 4 and 35 were not analyzed for thyroid hormone levels.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

7 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: Based on the lack of any evidence of toxicity, the NOAEL for F1 systemic toxicity was considered to be 7500 ppm.

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

Analyses of Diet Admix Formulations:

The analyzed dietary formulations contained 72.6% to 91.0% 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 administered to the control group (Group 1).

 

Test Substance Consumption (F0 Generation):

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^a )

(Target dose Levels of 250, 500, and 1000 mg/kg/day)

Theoretical Dietary Concentration (ppm)	Mean Test Substance Consumption (mg/kg/day)
	Males		Females
	Prior to Mating	After Mating	Prior to Mating	Gestation	Lactation^a
3500	243	193	228	258	231
7500	479	446	449	506	503
15,000	477	N/A	391	N/A	N/A

N/A = not applicable. Group was terminated due to excessive toxicity shortly following the initiation of the breeding period, thus precluding calculation of test substance consumption thereafter.

^a Dietary test substance concentrations were adjusted for lactating females to accommodate the higher caloric demands of milk production and to prevent overdosing of dams.

Test Substance Consumption (F1 Generation):

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)

Target Dietary Concentration (ppm)^a	Mean Test Substance Consumption (mg/kg/day)
	Males (PND 21–35)	Females (PND 21–35)
3500	203	209
7500	427	483

^a Dietary concentrations of the test substance were adjusted for F1 males and females to accommodate the higher caloric demands of growth and to prevent overdosing of the animals.

 

Applicant's summary and conclusion

Conclusions:

Under the conditions of this OECD 421 screening study, a concentration of 7500 ppm (equivalent to 446 to 506 mg/kg/day for males and females) was considered to be the no-observed-adverse-effect level (NOAEL) for F0 reproductive toxicity of Benzene 1,1’-oxybis-,tetrapropylene derivs., sulfonated, sodium salts when administered continually in the diet to Crl:CD(SD) rats. The NOAEL for F0 systemic toxicity was considered to be 3500 ppm (equivalent to 193 to 258 mg/kg/day for males and females), based on effects on body weight for F0 males and females at 7500 ppm and excessive toxicity (early group termination) noted at 15,000 ppm. The NOAEL for F1 neonatal toxicity was unable to be determined based on the lower numbers of pups born and lower postnatal survival from birth to PND 4 in both treated groups (3500 and 7500 ppm); some of these effects were driven by a single dam/group and need to be followed up in the definitive OECD 443 study. Based on the lack of any evidence of toxicity, the NOAEL for F1 systemic toxicity was considered to be 7500 ppm (equivalent to 427 and 483 mg/kg/day for males and females, respectively).

Executive summary:

The objective of this study was to provide information on the potential adverse effects of the test substance, Benzene, 1,1’-oxybis-, tetrapropylene derivs., sulfonated, sodium salts, on male and female reproduction within the scope of a screening OECD 421 study following dietary exposure in rats. This encompassed gonadal function, mating behavior, conception, parturition, and lactation of the parental generation and the development of offspring from conception through day 35 of postnatal life.

The study design was as follows:

Text Table 1: Experimental Design

Group No.	Test Material	Dietary Concentration^a (ppm)	Number of Animals
			Male	Female
1	Vehicle Control	0	10	10
2	Test Substance	3500	10	10
3	Test Substance	7500	10	10
4	Test Substance	15,000	10	10

^a Not corrected for salt, purity, and water content.

Animals were exposed to the test substance continuously via the diet. Males were exposed for 14 days prior to mating and continuing through the day of euthanasia (for a minimum of 56 days). Females were exposed for 14 days prior to mating and continuing throughout mating, gestation, and lactation until euthanasia (Lactation Day 21). F1 offspring were potentially exposed in utero and through nursing during lactation. Offspring selected to constitute the
F1 generation were directly exposed beginning at weaning (Postnatal Day [PND] 21) and continuing until euthanasia (PND 35).
Dietary test substance concentrations were adjusted based on historical control food consumption and body weight data for lactating females in order to compensate for the higher caloric demands of milk production. Dietary test substance concentrations were also adjusted for F1 pups beginning on PND 21, based on historical control food consumption and body weight data for age-matched animals in order to compensate for rapid growth rates and higher food efficiency during this period.
The analyzed dietary formulations were within the protocol-specified range of target concentrations and were homogeneous.
The following parameters and end points were evaluated in this study: mortality, clinical signs, body weights, body weight gains, food consumption, estrous cycles, reproductive performance, parturition, litter viability and survival, anogenital distance, areolae/nipple anlagen retention, thyroid hormones, macroscopic findings, organ weights, and microscopic examinations, including evaluations of the thyroid gland.

Mean calculated test substance consumption for males and females in the F0 and F1 generations are presented below:

Text Table 2: Mean Calculated F₀ Test Substance Consumption (mg/kg/day) (Target Dose Levels of 250, 500, and 1000 mg/kg/day)

Theoretical Dietary Concentration (ppm)	Mean Test Substance Consumption (mg/kg/day)
	Males		Females
	Prior to Mating	After Mating	Prior to Mating	Gestation	Lactation
F0 Generation
3500	243	193	228	258	231
7500	479	446	449	506	503
15,000	477	N/A	391	N/A	N/A
F1 Generation
Target Dietary Concentration (ppm)	Males (PND 21–35) (Post Weaning)		Females (PND 21–35) (Post Weaning)
3500	203		209
7500	427		483
15,000	N/A		N/A
N/A = not applicable. Group was terminated due to excessive toxicity shortly following the initiation of the breeding period, thus precluding calculation of test substance consumption thereafter.

In the 15,000 ppm group, mean body weight losses and lower mean body weight gains, with correspondingly reduced mean food consumption, were noted for F₀ males and females beginning immediately following initiation of dietary administration and continuing through approximately 3 weeks of test substance exposure. As a result, mean absolute body weights for F₀ males and females in this group were up to 27.5% and 21.8% lower, respectively, than the control group during this period. Therefore, a concentration of 15,000 ppm was considered to have exceeded the maximum tolerated dose, and the group was subsequently terminated and necropsied on 23 Sep 2021 (Study Day 23 [males] or Gestation Day 4, 5, 6, 7, or 8 [females]), thus precluding any further evaluations. No test substance-related macroscopic findings were observed in the 15,000 ppm group at necropsy.

In the 3500 and 7500 ppm groups, there were no test substance-related effects on F₀ male or female survival, clinical observations, estrous cyclicity, reproductive parameters, thyroid hormone levels, macroscopic and microscopic findings, or organ weights, including evaluations of the thyroid gland.

Test substance-related lower mean body weight gains, compared to the control group, were noted for F₀ males in the 7500 ppm group during the premating period (Study Days 0–14) and when the entire generation (Study Days 0–56) was evaluated, resulting in mean absolute body weights that were 7.9% to 11.1% lower than the control group during Study Days 14–56. These effects were considered adverse. Mean body weights and body weight gains in the 3500 ppm group F₀ males were unaffected by test substance exposure.

In F₀ females, mean body weights, body weight gains, and food consumption in the 3500 and 7500 ppm groups were unaffected by test substance exposure during the premating period. Lower mean body weight gains, without correlating effects on mean food consumption, were noted in the 3500 and 7500 ppm groups throughout gestation (Days 0–20) compared to the control group, resulting in mean body weights on Gestation Day 20 that were 7.9% and 16.3% lower, respectively, than the control group. The effects on mean body weight during gestation at 7500 ppm were considered test substance-related, and partially attributed to the lower number of pups born in this group, while the lower number of pups born was considered the primary factor in the 3500 ppm group. During lactation (Days 1–21), a higher mean body weight gain was noted in the 7500 ppm group compared to the control group, resulting in a mean absolute body weight on Lactation Day 21 that was comparable to the control group; mean food consumption in this group was comparable to the control group throughout lactation. Mean body weights, body weight gains, and food consumption for F₀ females in the 3500 ppm group were unaffected by test substance exposure during lactation.

Lower mean numbers of implantation sites, numbers of pups born, and live litter sizes on PND 0, and higher mean numbers of unaccounted-for sites (indicative of postimplantation loss) were noted at 3500 and 7500 ppm compared to the control group. In addition, lower mean postnatal survival was noted in these same groups during birth to PND 4 compared to the control group, which were attributed to 1 dam in each group with total litter loss on Lactation Day 0. These differences were considered test substance-related and adverse. Mean postnatal survival in these groups was unaffected by the test substance during PND 4–21. No other test substance-related findings were noted in the F₁ pups prior to weaning. In the 7500 ppm group, 6(2) pups (litters) were noted with missing eyes bilaterally; however, anophthalmia is a common external malformation in the current OECD 414 historical control data. Mean body weights, body weight gains, anogenital distances (absolute and relative to cube root of bodyweight), areolae/nipple anlagen, thyroid hormone levels on PND 21, macroscopic findings, and organ weights in F₁ pups were unaffected by parental exposure to the test substance in the 3500 and 7500 ppm groups.

F₁ survival, clinical observations (with the exception of the previously mentioned missing eyes), body weights, body weight gains, food consumption, macroscopic findings, and organ weights were unaffected by direct exposure to the test substance at 3500 and 7500 ppm during PND 21-35.

In conclusion, under the conditions of this OECD 421 screening study, a concentration of 7500 ppm (equivalent to 446 to 506 mg/kg/day for males and females) was considered to be the no-observed-adverse-effect level (NOAEL) for F₀ reproductive toxicity of Benzene 1,1’-oxybis-, tetrapropylene derivs., sulfonated, sodium salts when administered continually in the diet to Crl:CD(SD) rats. The NOAEL for F₀ systemic toxicity was considered to be 3500 ppm (equivalent to 193 to 258 mg/kg/day for males and females), based on effects on body weight for F₀ males and females at 7500 ppm and excessive toxicity (early group termination) noted at 15,000 ppm. The NOAEL for F₁ neonatal toxicity was unable to be determined based on the lower numbers of pups born and lower postnatal survival from birth to PND 4 in both treated groups (3500 and 7500 ppm); some of these effects were driven by a single dam/group and need to be followed up in the definitive OECD 443 study. Based on the lack of any evidence of toxicity, the NOAEL for F₁ systemic toxicity was considered to be 7500 ppm (equivalent to 427 and 483 mg/kg/day for males and females, respectively).