Reaction mass of divinylbenzene and ethylstyrene

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

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:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of divinylbenzene and ethylstyrene

EC Number:

910-757-7

Cas Number:

N/A

Molecular formula:

Divinylbenzene: C10H10 Ethylstyrene: C10H12

IUPAC Name:

Reaction mass of divinylbenzene and ethylstyrene

Specific details on test material used for the study:

Purity: 98.3%

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, NC
- Age at study initiation (Gestation Day 0): 11 to 13 weeks
- Weight at study initiation (Gestation Day 0): 229 to 289 g
- Animal Identification: Each animal was identified using a subcutaneously implanted electronic identification chip
- Housing: Individual; Solid bottom cages containing appropriate bedding material (Bed-O-Cobs® 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, dose level, study number, and sex of the animal
- Animal Enrichment: For enrichment, animals were provided items such as treats, a gnawing device, and/or nesting material, except when interrupted by study procedures/activities
- Diet (e.g. ad libitum): Ad libitum; PMI Nutrition International, LLC Certified Rodent LabDiet® 5002; Results of analysis for nutritional components and environmental contaminants is provided by the supplier; It was considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
- Water (e.g. ad libitum): Ad libitum, via an automatic watering system. Water bottles were provided, if required; Municipal tap water, treated by reverse osmosis and ultraviolet irradiation Periodic analyses of the water are performed; It was considered that there were no known contaminants in the water that could interfere with the outcome of the study.
- Acclimation period: Arrival to GD6; GD0 - GD6; Time-mated rats were received by Charles River on Gestation Days 1, 2, 3 and 4. The day on which confirmation of mating was made was designated Gestation Day 0.
- Animal Disposition: Animals were assigned to groups by a stratified randomization scheme designed to achieve similar group mean body weights.

JUSTIFICATION FOR TEST SYSTEM & NUMBER OF ANIMALS
-Test System: The Crl:CD(SD) rat has been recognized to be appropriate for developmental toxicity studies. Charles River has historical data on the background incidence of fetal malformations and developmental variations in this species. This animal model has been proven to be susceptible to the effects of developmental toxicants.
- Number of Animals: The number of animals is 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, 25 females/group is an appropriate number of animals to obtain a sample size of 20 at termination.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 68°F to 77°F (20°C to 25°C)
- Humidity (%): 30% to 70%
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark

IN-LIFE DATES: From: 19 Jul 2021 To: 06 Aug 2021

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% methylcellulose (400 cps) and 0.1% Cremophor EL in deionized water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Dose formulations were divided into aliquots where required to allow them to be dispensed on each dosing occasion. Dosing formulations were prepared (approximately weekly) at appropriate concentrations to meet dose level requirements. The prepared formulations were not adjusted for purity. Storage conditions were set to maintain a target temperature of 5°C

VEHICLE: 0.5% methylcellulose (400 cps) and 0.1% Cremophor EL in deionized water
- Concentration in vehicle: 0.5% methylcellulose (400cps); 0.1% Cremophor EL
- Amount of vehicle (if gavage): 10 mL/kg
- Storage Conditions: Set to maintain a target temperature of 5°C

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Doses were analyzed via high performance liquid chromatography with ultraviolet absorbance detection (HPLC-UV).

The analyzed dosing formulations contained 77.1% to 105.9% of the test substance which was within the protocol-specified range of target concentrations for suspensions (85% to 115%) and were homogeneous, with the following exceptions. On the first day of formulation sample analysis (16 Jul 2021), Group 2 and 3 formulations failed to meet protocol specified acceptability criteria for concentration. However, further investigations of formulations prepared on 19 Jul 2021 revealed that additional stirring time during formulation preparation resulted in concentrations that met acceptance criteria. Furthermore, analysis of stored aliquots from the 16 Jul 2021 formulations, for Groups 2 and 3, following increased stir time, met protocol specified acceptance criteria for concentration acceptability thereby confirming that the initial low results was due to inadequate stirring of the bulk formulations. For all subsequent formulations during the study, the stir time was increased to 2 hours before sample collection, analysis and preparation of aliquots for dose administration. All subsequent formulations met protocol-specified acceptance criteria for concentration acceptability.

Details on mating procedure:

Time-mated rats were received by Charles River on Gestation Day 1, 2, 3 or 4. The day on which confirmation of mating was designated Gestation Day 0.

Duration of treatment / exposure:

GD 6 - 20

Frequency of treatment:

Single daily dose

Duration of test:

Terminal sacrifice on GD 21

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

350 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

650 mg/kg bw/day

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The oral route of exposure was selected because this a possible route of human exposure. In a GLP-compliant study conducted according to OECD Test Guideline 422, at doses of 30, 100, 300, and 1000 mg test substance/kg/day, via oral gavage, clear evidence of maternal and developmental toxicity was evidenced at the highest dose (1000 mg/kg/day) as reduced maternal body weights and increased fetal toxicity. At 100 and 300 mg/kg/day, maternal effects were minimal, and there were no effects on embryo/fetal or pup survival or pup body weights. The results obtained for the test substance were consistent with other acute and repeated-dose studies and were hence considered to be representative of the different grades of the test substance.
In a previous dose range-finding prenatal developmental toxicity study conducted at Charles River, the test substance was administered via oral gavage to pregnant female rats at dose of 500, 600, 700 and 800 mg/kg/day. Test substance related clinical signs, maternal body weight losses and reduced food consumption, and a significant reduction in mean fetal weights, without concurrent effects on external fetal morphology were noted at 800 mg/kg/day. At lower doses, similar effects were observed, however, with much lower severity.
Based on these data, dosage levels of 100, 350 and 650 mg/kg/day were selected for the current study. It is anticipated that the high dose will show some maternal effects but not produce an incidence of fatalities that would prevent a meaningful evaluation of test substance related effects. The lower dosage levels were selected to identify potential dose response relationships.

- Rationale for animal assignment (if not random): Animals were assigned to groups by a stratified randomization scheme designed to achieve similar group mean body weights.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily; conducted prior to dosing on days of scheduled dosing; approximately 1 hr post-dose

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Gestation Days: 5, 6, 9, 12, 15, 18, 21

MORTALITY: Yes
-Time schedule: At least twice daily (morning and afternoon), beginning upon arrival through termination/release; (except on days of receipt and necropsy where frequency was at least once daily)

BODY WEIGHT: Yes
- Time schedule for examinations: Gestation Days: 0 (by supplier), 5, 6, 9, 12, 15, 18, and 21 (not collected from animals found dead)

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

POST-MORTEM EXAMINATIONS: Yes
- Method of Euthanasia: Animals euthanized for humane reasons or at study termination were euthanized by carbon dioxide inhalation.
- Unscheduled Deaths: If necessary for humane reasons, animals were euthanized as per Testing Facility SOPs. These animals underwent necropsy, and specified tissues were retained. The number and location of implantation sites, corpora lutea, and viable fetuses were recorded. Recognizable fetuses were examined externally, euthanized (if necessary), and preserved in 10% neutral buffered formalin.
- Scheduled Euthanasia: Main study animals surviving until scheduled euthanasia were weighed and euthanized by carbon dioxide inhalation.
- Sacrifice on gestation day # 21
- Organs examined: Liver and thyroid gland were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for animals euthanized in poor condition or in extremis. Representative samples of the liver and thyroid gland (gross lesions when possible) were collected from all animals and preserved in 10% neutral buffered formalin. Representative sections of corresponding organs from a sufficient number of controls were retained for comparison, if possible. The thyroid and liver from all animals in all groups were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin. The thyroid and liver were examined microscopically from all females in all groups.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Blood sampling:

Thyroid Sample Hormone Collection:
- Method/Route of Collection: Jugular vein using the hand-held restraint method
- Target Volume: Approximately 1.0 mL/time point collected
- Special Requirements: Collected prior to noon on each day of collection, around the same time each day, and within a 2-hour window on each collection day, to reduce variability due to normal diurnal variations in physiological levels of thyroid hormones. Blood collections were performed in animal ante room, the necropsy laboratory, or as far away from live animals as possible to minimize stress-induced hormone fluctuations.
Thyroid Hormone Sample Processing: Samples were allowed to clot at ambient temperature for a minimum of 30 minutes before centrifugation. All samples were centrifuged (approximately 3000 rpm [approximately 2056xg] for approximately 10 min) at approximately 4°C. The maximum amount of serum was recovered and transferred into 2 new, uniquely labeled polypropylene tubes. Approximately 150 µL of the resultant serum was placed in 1 tube to be used for total T3 and T4 hormone analysis and approximately 100 µL was placed in a separate tube for TSH analysis. Samples were stored in a freezer set to maintain a target of -70°C.

Thyroid Hormone Sample Analysis: Samples to determine total T3 and T4 concentrations were analyzed using a validated UHPLC/MS/MS assay. Samples for TSH determination were analyzed using a validated Luminex Bead-Based (TSH) assay.

Fetal examinations:

FETAL EXAMINATIONS: Fetal examinations were conducted without knowledge of treatment group. External, internal, and skeletal fetal findings were recorded as either developmental variation (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 examinations: Yes: [all per litter]
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.

- Soft tissue examinations: Yes: [approximately half per litter]
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. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development. Following examination, the carcasses and cephalic slices were discarded.

- Skeletal examinations: Yes: [approximately half per litter]
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 macerated in potassium hydroxide and stained with Alizarin Red S and Alcian Blue. The skeletal examination was made following this procedure.

- Head examinations: Yes: [approximately half per litter]
The heads from these fetuses examined for visceral alterations were removed and placed in Harrison’s fixative for subsequent processing and soft-tissue examination using the Wilson sectioning technique.

Statistics:

Numerical data and clinical and necropsy observations data were summarized by sex and occasion or by litter. Body Weight Gains were calculated between each scheduled interval as well as between GD 6-21. Food Consumption was calculated for each corresponding body weight interval.

Means, standard deviations, percentages, numbers, and/or incidences were reported, as appropriate by dataset. Data from nongravid females were excluded from calculation of means and from comparative statistics.

All statistical tests were conducted at the 5% significance level. All pairwise comparisons (vs. control) were conducted using two sided tests and were reported at the 1% and 5% levels, unless otherwise noted. Body weight, body weight gains, food consumption, thyroid hormone data, gravid uterine weight and adjusted maternal body weights, organ weights, litter means, and anogenital distance were analyzed by the parametric/non-parametric methods listed below. Ovarian and uterine content and litter % of fetuses with gross/external/visceral/skeletal abnormalities were analyzed by the non-parametric methods listed below.

Parametric/Non-Parametric: Levene’s test was used to assess the homogeneity of group variances. The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively.

Non-Parametric: The groups were compared using an overall Kruskal-Wallis test. If the overall Kruskal-Wallis test was found to be significant, then the above pairwise comparisons were conducted using Dunn’s test.

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. of male fetuses/Total No. of fetuses) x 100

- Litter % of Fet. with Abnor. = (No. of fet. in litter with a finding/No. of fet. in litter examine) x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no significant test substance-related clinical observations noted at any dose level. Observations noted in the test substance-treated groups, including fur staining or thin fur cover on various body surfaces, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female in the 650 mg/kg/day group was euthanized in extremis on Gestation Day 12 following body weight losses (26.4% of Gestation Day 6 body weight), and severely reduced food consumption (≤ 1 g/animal/day during Gestation Days 6-12) and clinical observations of hunched posture (Gestation Days 9 and 12), erected fur (Gestation Day 12), decreased activity (Gestation Days 9 and 10), and thin (Gestation Days 9 and 12). At necropsy, there were no gross observations, and the female was gravid. Other tissues were not examined microscopically given the lack of gross findings. A microscopic cause of moribundity and subsequent euthanasia could not be determined. All other animals survived to scheduled euthanasia.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related, statistically significant mean maternal body weight losses were noted in the 650 mg/kg/day group immediately following the initiation of treatment (Gestation Day 6-9). Thereafter, mean body weight gains were generally comparable to or slightly lower than the control group throughout the remainder of the treatment period (Gestation Days 9-21). Consequently, mean body weight gains were significantly lower than the control group when the overall treatment period (Gestation Days 6-21) was evaluated and mean absolute body weights in the 650 mg/kg/day group were 8.8% lower than the control group at scheduled termination on Gestation Day 21; differences from the control group attained statistical significance generally throughout the treatment period. Test substance-related lower mean gravid uterine weight, adjusted body weight, and adjusted body weight gain were noted for females in this group; the differences in adjusted body weight and adjusted body weight gain were statistically significant. Mean maternal body weights, body weight gains, adjusted body weights, adjusted body weight gains, and gravid uterine weights in the 100 and 350 mg/kg/day groups were unaffected by test substance administration. 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):

Mean maternal food consumption, evaluated as g/animal/day, in the 650 mg/kg/day group was statistically significantly lower than the control group following the initiation of dose administration (Gestation Days 6-9). This was considered test substance-related and corresponded to the mean body weight loss noted for these females. Thereafter, mean food consumption in this group was generally comparable to or slightly lower than the control group throughout the remainder of the dosing period (Gestation Days 9-21). Consequently, mean food consumption was significantly lower than the control group when the overall treatment period (Gestation Days 6-21) was evaluated.
Mean maternal food consumption, evaluated as g/animal/day, in the 100 and 350 mg/kg/day groups was unaffected by test substance administration. Mean food consumption in the 350 mg/kg/day group was statistically significantly lower than the control group during Gestation Days 6-9; however, this was transient and did not affect mean body weight gains. Other differences from the control group were slight and not statistically significant.

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related changes in serum T4; however, serum T3 levels were 9.2%, 9.0%, and 15.5% lower than the control group in the 100, 350, and 650 mg/kg/day groups. Correspondingly, TSH levels were 54.4% and 49.8% higher than the control group in the 350 and 650 mg/kg/day groups. The lower T3 levels are consistent with microsomal enzyme induction due to hypertrophy in the liver. Given the lack of a clear dose response, and the lack of any differences in T4 levels, these differences were also considered adaptive and non-adverse.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related changes were observed in the liver and thyroid in the 650 mg/kg/day group. Mean absolute liver and thyroid/parathyroid weights were slightly elevated in the 650 mg/kg/day group when compared to the control group. The percent differences for liver and thyroid/parathyroid in all test substance-treated groups from the control group did not show a clear dose-response. However, there was a test substance-related statistically significantly lower mean body weight in the 650 mg/kg/day group. Both liver and thyroid weights show a linear relationship to body weight and should mirror changes in body weight, therefore, liver and thyroid weights relative to body weight are the best endpoint and slight elevations in absolute values would be toxicologically relevant. The rats were pregnant at the time of necropsy, so calculating organ weights relative to body weight would have had too much variability. The liver and thyroid/parathyroid weight elevations in the 650 mg/kg/day group in the face of body weight loss with correlating hepatocellular hypertrophy (liver) and follicular cell hypertrophy (thyroid) were considered test substance related.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test substance-related gross findings were noted. The gross findings observed were considered incidental, of the nature commonly observed in this strain and age of rat and, therefore, were considered unrelated to administration of the test substance.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related findings were observed in the liver of the 350 and 650 mg/kg/day groups and the thyroid of 100, 350, and 650 mg/kg/day groups.

The liver finding of centrilobular hepatocellular hypertrophy was noted in the 350 and 650 mg/kg/day group with a dose-response in incidence and severity. Minimal (350 and 650 mg/kg/day) to mild (650 mg/kg/day) hypertrophy was characterized by enlargement of hepatocytes with glassy, eosinophilic cytoplasm that was confined to the centrilobular area in minimal hypertrophy and extended into the midzonal area in mild hypertrophy. Hypertrophy correlated with higher absolute liver weight in the 650 mg/kg/day group.

In the thyroid, follicular cell hypertrophy was noted in the 100 (minimal to mild), 350 (minimal to moderate), and 650 (minimal to mild) mg/kg/day groups. Minimal to mild changes were characterized by enlargement of thyroid follicular cells with cytoplasmic vacuolation with cellular enlargement greater in mild findings. Moderate follicular cell hypertrophy was noted in a single female in the 350 mg/kg/day group with enlarged, vacuolated follicular cells that often-showed piling. Follicular cell hypertrophy correlated with higher absolute thyroid/parathyroid weight in the 650 mg/kg/day group.

Hypertrophy in the liver and/or thyroid gland was consistent with microsomal enzyme induction and was considered an adaptive response and non-adverse given the severity of changes and lack of test substance-related hepatocellular injury.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Other microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of the test substance.

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

350 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean fetal weights (male/female/combined) in the 650 mg/kg/day group were 6.476% to 6.740% lower than the control group; differences were statistically significant. Mean values were below the ranges of values in the historical control data and were hence considered substance related but non-adverse, based on the lower magnitude of the difference versus controls.

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

Intrauterine survival in the 650 mg/kg/day group was unaffected by test substance administration. A slightly higher (not statistically significant) mean litter proportion of post implantation loss was noted for females in this group; however, the difference was partially attributed to a single female with 80% post implantation loss and the mean value was within the range of values in the historical control data.

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

The numbers of fetuses (litters) available for morphological evaluation were 297(25), 299(24), 294(25), and 248(23) in the control, 100, 350, and 650 mg/kg/day groups, respectively.

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

Mean anogenital distance in the 650 mg/kg/day group was comparable to the control group.

Changes in postnatal survival:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related external malformations were noted at any dose level. Omphalocele (portion of intestine protruded with remnants of a membranous sac) was noted for one fetus in the 100 mg/kg/day group. However, this was only noted for a single fetus and was not noted in a dose-related manner.
No external developmental variations were observed in fetuses in this study.

Skeletal malformations:

no effects observed

Description (incidence and severity):

No test substance-related skeletal developmental variations were noted. Findings observed in the test substance-treated groups were noted infrequently, similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the historical control data.

Visceral malformations:

no effects observed

Description (incidence and severity):

No test substance-related visceral developmental variations were noted. Findings observed in the test substance-treated groups were noted infrequently, similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the historical control data.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

650 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Summary of Maternal Performance and Mortality
Sex: Female   Days(s) Relative to Mating (Litter: A)		0 mg/kg/day   Group 1	100 mg/kg/day   Group 2	350 mg/kg/day   Group 3	650 mg/kg/day   Group 4
Group Size - Females		25	25	25	25
Number of Females Pregnant	N+ve	25	24	25	24
	%	100.0	96.0	100.0	96.0
Females with Live Fetuses	N+ve	25	24	25	24
	%	100.0	100.0	100.0	100.0
Female with Resporptions	N+ve	8	9	13	9
	%	32.0	37.5	52.0	37.5
Female with all Nonviable	N+ve	0	0	0	0
	%	0.0	0.0	0.0	0.0
Placenta exam Normal	N+ve	25	23	25	23
	%	100.0	95.8	100.0	100.0
Terminal Euthanasia	N+ve	25	25	25	24
	%	100.0	100.0	100.0	96.0
Unscheduled Death/Euthanasia	N+ve	0	0	0	1
	%	0.0	0.0	0.0	4.0
Found Dead	N+ve	0	0	0	0
	%	0.0	0.0	0.0	0.0
Unscheduled Euthanasia	N+ve	0	0	0	1
	%	0.0	0.0	0.0	4.0
Aborted	N+ve	0	0	0	0
	%	0.0	0.0	0.0	0.0
Delivered	N+ve	0	0	0	0
	%	0.0	0.0	0.0	0.0

 

Summary of Ovarian and Uterine Examinations and Litter Observations
Sex: Female   Days(s) Relative to Mating (Litter: A)		0 mg/kg/day   Group 1	100 mg/kg/day   Group 2	350 mg/kg/day   Group 3	650 mg/kg/day   Group 4
Females with Live Fetuses	N+ve	25	24	25	23
	%	100.0	100.0	100.0	100.0
Number of Corpora Lutea [k]	Mean	15.5	15.7	14.5	15.0
	SD	2.7	2.5	2.8	2.9
	N	25	24	25	23
	%Diff	-	1.5	-6.2	-3.4
Number of Implantations [k]	Mean	12.3	13.3	12.6	11.6
	SD	2.4	2.2	2.3	3.3
	N	25	24	25	23
	%Diff	-	8.2	1.9	-5.8
Pre-implantation Loss (%) [k]	Mean	18.69	14.14	11.69*	22.81
	SD	17.10	12.39	15.57	15.94
	N	25	24	25	23
	%Diff	-	-24.38	-37.49	22.03
Total Number of Resorptions [k]	Mean	0.4	0.9	0.8	0.8
	SD	0.7	1.6	0.8	1.3
	N	25	24	25	23
	%Diff	-	98.9	72.7	87.7
Number of Early Resorptions [k]	Mean	0.4	0.9	0.7	0.8
	SD	0.7	1.6	0.8	1.3
	N	25	24	25	23
	%Diff	-	98.9	63.6	87.7
Number of Late Resorptions [k]	Mean	0.0	0.0	0.0	0.0
	SD	0.0	0.0	0.2	0.0
	N	25	24	25	23
	%Diff	-	-	-	-
Total Number of Fetuses [k]	Mean	11.9	12.5	11.8	10.8
	SD	2.3	2.6	2.3	3.8
	N	25	24	25	23
	%Diff	-	4.9	-0.7	-9.2
Number of Live Fetuses [k]	Mean	11.9	12.5	11.8	10.8
	SD	2.3	2.6	2.3	3.8
	N	25	24	25	23
	%Diff	-	4.9	-0.7	-9.2
Number of Live Male Fetuses [k]	Mean	5.8	6.4	6.4	5.3
	SD	2.2	2.5	2.5	2.4
	N	25	24	25	23
	%Diff	-	11.4	11.1	-8.7
Number of Live Female Fetuses [k]	Mean	6.1	6.0	5.4	5.5
	SD	2.0	2.6	2.4	2.6
	N	25	24	25	23
	%Diff	-	-1.3	-11.8	-9.8
Number of Dead Fetuses [k]	Mean	0.0	0.0	0.0	0.0
	SD	0.0	0.0	0.0	0.0
	N	25	24	25	23
	%Diff	-	-	-	-
Post-implantation Loss (5) [k]	Mean	3.29	7.10	6.01	9.42
	SD	5.24	14.19	6.47	18.50
	N	25	24	25	23
	%Diff	-	115.96	82.77	186.69
Live Male Fetus/Litter (5) [k]	Mean	48.24	51.33	54.26	47.50
	SD	14.20	17.57	18.23	18.79
	N	25	24	25	23
	%Diff	-	6.39	12.47	-1.53
Mean Fetal Weight all (g) [G]	Mean	5.920	5.958	5.863	5.537*
	SD	0.257	0.380	0.395	0.493
	N	25	24	25	23
	%Diff	-	0.647	-0.970	-6.476
Mean Fetal Weight males (g) [G]	Mean	6.099	6.151	6.003	5.700*
	SD	0.258	0.369	0.475	0.567
	N	25	24	25	22
	%Diff	-	0.861	-1.564	-6.541
Mean Fetal Weight females (g) [G1]	Mean	5.756	5.775	5.699	5.368**
	SD	0.273	0.409	0.391	0.468
	N	25	24	25	23
	%Diff	-	0.321	-0.987	-6.740
Mean Fetal AGD males (mm) [G]	Mean	2.32	2.32	2.21	2.22
	SD	0.17	0.15	0.16	0.17
	N	25	24	25	22
	%Diff	-	0.26	-4.41	-4.14
Mean Fetal AGD females (mm) [G]	Mean	0.89	0.92	0.87	0.90
	SD	0.08	0.11	0.14	0.11
	N	25	24	25	23
	%Diff	-	4.08	-2.25	1.23
Mean Normalized Fetal AGD m [G]	Mean	1.268	1.269	1.220	1.245
	SD	0.092	0.082	0.082	0.083
	N	25	24	25	22
Mean Normalized Fetal AGD f [G]	Mean	0.494	0.515	0.485	0.512
	SD	0.041	0.067	0.074	0.058
	N	25	24	25	23
[k] - Kruskal-Wallis & Dunn: * = p ≤ 0.05 [G] - Kruskal-Wallis & Dunn: * = p ≤ 0.05 [G1] - Anova & Dunnett: ** = p ≤ 0.05

 

Summary of Thyroid Hormone Values - Terminal Euthanasia (Gestation Day 21)
Group	1	2	3	4
Dose (mg/kg/day)	0	100	350	650
T3 (pg/mL)
Mean	318.1	288.9	289.2	268.7
SD	99.3	94.3	63.3	94.0
N	25	24	25	23
% Difference	-	-9.2%	-9.0%	-15.5%
T4 (pg/mL)
Mean	15166.8	14503.8	16066.8	14647.4
SD	4547.8	5100.4	4212.7	5909.7
N	25	24	25	25
% Difference	-	-4.4%	+5.9%	-3.4%
TSH (pg/mL)
Mean	1692.1	1772.5	2613.1	2536.3
SD	851.9	1011.3	1866.3	1907.5
N	21	22	20	20
% Difference	-	+4.8%	+54.4%	+49.9%
None statistically significant versus control. Bold - Test substance-related

Applicant's summary and conclusion

Conclusions:

Based on the effects on body weight, body weight gain, and food consumption at 650 mg/kg/day, a dosage level of 350 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity when the test substance was administered orally by gavage to time-mated Crl:CD(SD) rats. Due to the absence of adverse effects at any dose level, a dose level of 650 mg/kg/day, the highest dose level evaluated, was considered to be the NOAEL for embryo/fetal development.

Executive summary:

The objective of this study was to determine the potential of the test substance to induce developmental toxicity after maternal exposure (via oral gavage) from implantation to one day prior to expected parturition, to characterize maternal toxicity at the exposure levels tested and to determine a no-observed-adverse-effect level (NOAEL) for maternal and developmental toxicity.

The study design was as follows:

Experimental Design

Group No.	Test Substance	Dose Level^a (mg/kg bw/d)	Dos Volume^b (mL/kg)	Dose Concentration (mg/mL)	Number of Females
1	Vehicle	0	10	0	25
2	DVB-HP	100	10	10	25
3	DVB-HP	350	10	35	25
4	DVB-HP	650	10	65	25

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

^b Based on the most recent body weight measurement

 

Animals were dosed via oral gavage once daily 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, thyroid hormones, organ weights, macroscopic and microscopic examinations, intrauterine growth and survival, fetal anogenital distance, and fetal morphology.

 

The analyzed dosing formulations were within the protocol-specified range of target concentrations for suspensions and were homogeneous.

 

One female in the 650 mg/kg/day group was euthanized in extremis on Gestation Day 12 following body weight losses and severely reduced food consumption and clinical observations of hunched posture, erected fur, decreased activity, and thin. A microscopic cause of moribundity and subsequent euthanasia could not be determined. None of the other females in the 650 mg/kg/day group were found dead or euthanized in extremis or noted with similar clinical findings. However, several females in this group (17 of 24 gravid females) lost weight following the initiation of treatment (Gestation Days 6-9), therefore, a relationship of to test substance administration cannot be ruled out. All other animals survived to scheduled euthanasia.

 

With the exception of the aforementioned clinical findings for the female euthanized in extremis in the 650 mg/kg/day group, there were no significant test substance-related clinical observations noted at any dose level.

 

A test substance-related initial mean body weight loss, with corresponding lower mean food consumption, was noted for females in the 650 mg/kg/day group during Gestation Days 6-9. Thereafter, mean body weight gains and food consumption in the 650 mg/kg/day group were generally comparable to or slightly lower than the control group throughout the remainder of the treatment period (Gestation Days 9-21). Consequently, mean body weight in this group was 8.8% lower than the control group on Gestation Day 21. Mean gravid uterine weight, adjusted body weight, and adjusted body weight gain in the 650 mg/kg/day group were lower than the control group. No test substance-related effects were noted on mean body weight, body weight gain, food consumption, gravid uterine weight, adjusted body weight, and adjusted body weight gain in the 100 and 350 mg/kg/day groups when compared to controls.

 

No test substance-related maternal gross findings were noted at any dose level.

 

Test substance-related higher absolute liver and thyroid/parathyroid weights were noted in the 650 mg/kg/day group when compared to the control group. These results correlated to the liver finding of centrilobular hepatocellular hypertrophy in the 350 and 650 mg/kg/day groups and the thyroid finding of follicular cell hypertrophy in the 100, 350, and 650 mg/kg/day groups. While there were no changes in maternal T4 at any dosage level, lower serum T3 levels were noted in the 100, 350 and 650 mg/kg/day groups, with correspondingly higher mean TSH levels in the 350 and 650 mg/kg/day groups. Hypertrophy in the liver and/or thyroid gland was consistent with microsomal enzyme induction and lower T3 levels and these findings were considered nonadverse and adaptive given the severity of changes and lack of test substance-related hepatocellular injury. No other test substance-related organ weight effects or microscopic finding were noted at any dose level.

 

Test substance-related lower (6.476% to 6.740%) mean fetal weights (male/female/combined) were noted in the 650 mg/kg/day group. These effects were considered nonadverse based on the lower magnitude of the difference versus controls. No other test substance-related effects were noted on intrauterine growth and survival or anogenital distance at any dose level.

 

No test substance-related fetal malformations or variations were noted for fetuses at any dose level.

 

Based on the effects on maternal body weight, body weight gain, and food consumption at 650 mg/kg/day, a dosage level of 350 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity when the test substance was administered orally by gavage to time-mated Crl:CD(SD) rats. Due to the absence of adverse effects at any dose level, a dose level of 650 mg/kg/day, the highest dose level evaluated, was considered to be the NOAEL for embryo/fetal development.