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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 August 2020 to 06 January 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
August 1998
Deviations:
yes
Remarks:
with no impact on experimental results or study integrity (see below)
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
January 2018
Deviations:
yes
Remarks:
with no impact on experimental results or study integrity (see below)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Fatty acids, tall-oil, reaction products with boric acid (H3BO3) and diethanolamine
EC Number:
294-785-9
EC Name:
Fatty acids, tall-oil, reaction products with boric acid (H3BO3) and diethanolamine
Cas Number:
91770-03-5
IUPAC Name:
Fatty acids, tall-oil, reaction products with boric acid (H3BO3) and diethanolamine
Test material form:
liquid
Details on test material:
- Physical state/appearance: dark brown liquid
- Storage conditions: room temperature in the dark over silica gel

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Details on test animals or test system and environmental conditions:
TEST SYSTEM
- 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 number of animals was based on the United States 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 recommended evaluation of approximately 20 females with implantation sites at necropsy. Given the possibility of nongravid animals, unexpected deaths, or treatment-related moribundity and/or mortality, this was an appropriate number of animals to obtain a sample size of 20 at termination.
- Animal identification: Each animal was identified using a subcutaneously implanted electronic identification chip (BMDS system). Microchips were implanted following receipt.
- Quarantine: After receipt at the Testing Facility, the Crl:CD(SD) rats were acclimated prior to the initiation of dosing.
- Selection, assignment, replacement and disposition of animals: Animals were assigned to groups by a stratified randomization scheme designed to achieve similar group mean body weights. Before the initiation of dosing, any assigned animals considered unsuitable for use in the study were replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions. The disposition of all animals was documented in the study records.

HUSBANDRY
- Housing: Animals were individually housed in solid-bottom cages containing appropriate bedding material. Individual (colour-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. Housing set-up was as specified in the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). Where possible, control group animals were housed on a separate rack from the test substance-treated animals.
- Animal enrichment: Animals were provided items such as treats, a gnawing device, and/or nesting material, except when interrupted by study procedures/activities.
- Environmental conditions: Target temperatures of 68 °F to 77 °F (20 °C to 25 °C); target relative humidity 30 % to 70 %; 12-hour light/12-hour dark cycle.
- Water: Municipal tap water treated by reverse osmosis and ultraviolet irradiation was available ad libitum via an automatic watering system. Water bottles were provided if required. Periodic analysis of the water was performed, and results of these analyses held on file at the testing facility. It was considered that there were no known contaminants in the water that could interfere with the outcome of the study.
- 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: gavage
Vehicle:
arachis oil
Remarks:
Peanut oil NF (Lot 2JA0064; expiry date 31 August 2021; storage conditions 18 to 24 °C protected from light)
Details on exposure:
DOSE FORMULATION AND ANALYSIS
- Preparation of formulations: Dose formulations were divided into aliquots where required to allow them to be dispensed on each dosing occasion. The neat test substance was closed and returned to a desiccator immediately after each use to minimize exposure to air or moisture. Any residual volumes from each dosing occasion were discarded unless otherwise requested by the Study Director.
- Preparation details: Dosing formulations (see table below) were prepared at appropriate concentrations to meet dose level requirements. The prepared formulations were not adjusted for purity. Any procedures not covered by SOPs required for formulation were approved by the Study Director and included in the Study Records. Any residual volumes from each dosing occasion were discarded unless otherwise requested by the Study Director.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
SAMPLE COLLECTION AND ANALYSIS
- Dose formulation samples were collected for analysis as indicated in the table below.
- Dose analysis results were verified prior to dose administration at each sampling interval, if possible.
- 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. Care was taken to minimize exposure of the test substance in the dosing formulations to air or moisture prior to analysis.

ANALYTICAL METHOD
- Analyses were performed by a high-performance liquid chromatography method with ultraviolet detection using a validated analytical procedure. The full experimental procedure was presented in Appendix 1 of the full study report and a summary is given in the table below.
- Concentration acceptance criteria: mean sample concentration within 100 % ± 15 % of theoretical concentration.
- Homogeneity acceptance criteria: relative standard deviation (RSD) of concentrations of ≤ 10 % for each group.
Details on mating procedure:
Female animals were received pre-mated.
Duration of treatment / exposure:
Gestation Days 6 to 20
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25 females per dose level
Control animals:
yes, concurrent vehicle
Details on study design:
EXPERIMENTAL DESIGN
- Experimental design is summarised in Table 6 (below).
- Administration of test materials: The test substance and vehicle were administered as a single daily oral gavage dose during Gestation Days 6 through 20. All animals were dosed at approximately the same time each day. The plastic feeding tube was thoroughly wiped of residual test substance prior to each dose.
- Justification of route and dose levels: The route of administration was oral (gavage) because this is a potential route of exposure for humans. The dose levels were selected based on data from a dose range-finding study of test item using oral dose levels of 50, 250, 500, and 1000 mg/kg/day. Based on results from the study, a high dosage level of 1000 mg/kg/day was selected. This high-dosage level was not expected to produce an incidence of test substance-related fatalities that would prevent a meaningful evaluation. Lower dosage levels of 50 and 250 mg/kg/day of test item were selected to evaluate any dose-related trends.

COMPUTERIZED SYSTEMS
- Critical computerized systems used in the study were listed in the full study report or presented in the appropriate phase report. All computerized systems used in the conduct of this study have been validated (with the exception of Microsoft Office); when a particular system has not satisfied all requirements, appropriate administrative and procedural controls were implemented to assure the quality and integrity of data.

Examinations

Maternal examinations:
IN-LIFE PROCEDURES, OBSERVATIONS AND MEASUREMENTS
- General in-life assessments are summarised in Table 7 (below).

THYROID HORMONE SAMPLE COLLECTION
- Blood samples for thyroid hormone analysis were collected as described in Table 8 (below).

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

THYROID HORMONE SAMPLE ANALYSIS
- For total T3 and T4 analysis, hormone samples were analyzed using validated ultra high performance liquid chromatography with dual mass spectroscopy (UHPLC/MS/MS) assays.
- Analysis of serum samples to determine TSH concentrations was conducted using a validated Luminex Bead Based (TSH) assay.

TERMINAL PROCEDURES
- Terminal procedures are summarised in Table 9 (below).
- All animals survived until scheduled euthanasia and were weighed before being euthanised by carbon dioxide inhalation.

ORGAN WEIGHTS
- The organs identified in Table 10 (below) were weighed at necropsy for all scheduled euthanasia animals.
- Representative samples of the tissues identified in Table 11 (below) were collected from all animals and preserved in 10% neutral buffered formalin for possible histopathologic examination.

HISTOLOGY
- Tissue trimming was performed at the testing facility. Maternal thyroids and livers from all animals in all groups were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin.

HISTOPATHOLOGY
- Histopathological evaluation was performed by a board-certified veterinary pathologist. Thyroids and livers for all animals in the control and high-dose groups (Groups 1 and 4) were subjected to microscopic examination.
Ovaries and uterine content:
LAPAROHYSTERECTOMY AND MACROSCOPIC EXAMINATION – Gestation Day 21 (embryo/fetal development phase)
- Laparohysterectomies and macroscopic examinations were performed blind to treatment group.
- All surviving females were euthanized and subjected to a gross necropsy. The thoracic, abdominal, and pelvic cavities were opened and the contents examined. Additionally, special attention was paid to the esophagus, stomach, and intestines for signs of irritation. The uterus of each dam was excised and its adnexa trimmed. Corpora lutea were also counted and recorded. Gravid uterine weights were obtained and recorded. The uterus of each dam was opened and the number of viable and nonviable fetuses, early and late resorptions, and total number of implantation sites were recorded, and the placentae were examined.
- The individual uterine distribution was documented using the following procedure: all implantation sites, including early and late resorptions, were numbered in consecutive fashion beginning with the left distal uterine horn, noting the position of the cervix and continuing from the proximal to the distal right uterine horn. Uteri which appear nongravid by macroscopic examination were opened and placed in a 10% ammonium sulfide solution for detection of early implantation loss (Salewski, 1964).
- Gross lesions were also collected and preserved in 10% neutral-buffered formalin for possible future histopathologic examination. Representative sections of corresponding organs from a sufficient number of controls were retained for comparison, if possible. The carcasses were discarded.
Fetal examinations:
FETAL EXAMINATIONS
- Fetal examinations were conducted without knowledge of treatment group.
- External, internal, and skeletal fetal findings were recorded as either developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or occur at high incidence, representing slight deviations from normal), malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life), or incidental (minor changes in coloration, mechanical damage to specimen, etc.).
- Representative photographs of all malformations, as appropriate, were included in the study records. Corresponding low magnification photographs, depicting both the malformed fetus and a comparison control fetus or normal littermate, were also included in the study records as needed and as appropriate for comparison, when possible.

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

VISCERAL (INTERNAL)
- The sex of all fetuses was confirmed by internal examination.
- Approximately one-half of the fetuses in each litter were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe (1984). This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development (Woo and Hoar, 1972). The heads from these fetuses were removed and placed in Harrison’s fixative for subsequent processing and soft-tissue examination using the Wilson sectioning technique (Wilson, 1965). Following examination, the carcasses and cephalic slices were discarded.
- The remaining fetuses (approximately one-half from each litter, excluding any carcasses without heads) were eviscerated and fixed in 100% ethyl alcohol for subsequent examination of skeletons.
SKELETAL
- Following fixation in alcohol, each eviscerated fetus was macerated in potassium hydroxide and stained with Alizarin Red S (Dawson, 1926) and Alcian Blue (Inouye, 1976).
- The skeletal examination was made following this procedure.
Statistics:
See below
Indices:
See below

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- Test substance-related clinical observations included abnormal breathing for 2 and 3 females in the 250 and 1000 mg/kg/day groups, respectively, at the daily examinations and/or 1 to 2 hours post-dosing and increased incidences of red fur staining and/or wet fur around the mouth and/or muzzle in the same groups at 1–2 hours postdosing; these findings occurred sporadically throughout the study and were considered non-adverse. Additionally, the abnormal breathing sounds were considered likely related to aspiration of the test substance and not the result of a systemic response to test substance administration. Other observations noted in the test substance-treated groups occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.
- All females were determined to be gravid with the exception of Female No. 4525 in the 1000 mg/kg/day group.
Mortality:
no mortality observed
Description (incidence):
- There were no test substance-related effects on survival. All females in the control, 50, 250, and 1000 mg/kg/day groups survived to the scheduled necropsy on Gestation Day 21.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Test substance-related effects on body weight parameters were noted in the 1000 mg/kg/day group. Statistically significantly lower mean body weight gains were noted in this group following initiation of dosing (Gestation Days 6 to 9) compared to the control group. Mean body weight gains in the 1000 mg/kg/day group were generally comparable to the control group during Gestation Days 9 to 12 and 12 to 15 and lower (statistically significant) during Gestation Days 15 to 21 and when the entire treatment period (Gestation Days 6 to 21) was evaluated. As a result, mean absolute body weights in this group were 3.6 % to 6.8 % lower than the control group during Gestation Days 9 to 21; differences were generally statistically significant. Lower mean corrected body weight and body weight gain were also noted in the 1000 mg/kg/day group compared to the control group; differences were statistically significant. Due to the magnitude in difference from the control group, the test substance-related effects on body weight and body weight gains at 1000 mg/kg/day were considered adverse.
- Mean maternal body weights, body weight gains, corrected body weights, corrected body weight gains, and gravid uterine weights in the 50 and 250 mg/kg/day groups were unaffected by test substance administration. Any statistically significantly differences between these groups and the control group were noted in a manner that was not dose-responsive, transient, and/or did not impact mean absolute body weights.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- In the 1000 mg/kg/day group, mean food consumption, evaluated as g/animal/day, was statistically significantly lower than the control group during Gestation Days 6 to 9, 9 to 12, 15 to 21, and when the overall treatment period (Gestation Days 6 to 21) was evaluated. Mean food consumption in this group was comparable to the control group during Gestation Days 12 to 15. The decrements in mean food consumption in the 1000 mg/kg/day group corresponded with the adverse effects on mean body weight and body weight gain and were considered test substance-related and adverse.
- Mean maternal food consumption in the 50 and 250 mg/kg/day groups was unaffected by test substance administration; differences from the control group were slight and not statistically significant.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
- No test substance-related liver or thyroid gland weight changes were noted. The organ weight differences observed were considered incidental and unrelated to administration of the test item.
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/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to administration of the test item.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
- No test substance-related microscopic findings on the maternal livers or thyroid glands were noted. The 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 item.

Maternal developmental toxicity

Other effects:
effects observed, treatment-related
Description (incidence and severity):
OVARIAN AND UTERINE EXAMINATIONS
- Mean fetal body weights (males, females, and combined) in the 1000 mg/kg/day group were statistically significantly lower (6.194 %, 6.712 %, and 6.134 %, respectively) than the concurrent control group and below the minimum mean values in historical control data. The effects on fetal body weight were considered test substance-related and adverse. Intrauterine growth at 50 and 250 mg/kg/day was unaffected by test substance administration.
- Intrauterine survival at 50, 250, and 1000 mg/kg/day were unaffected by test substance administration. Parameters evaluated included mean litter proportions of post-implantation loss, mean number and percentage of live fetuses, and fetal sex ratios. In addition, mean anogenital distances (absolute and relative to the cube root of the fetal weight) were unaffected by test substance administration at all dosage levels. Differences from the control group were slight and not statistically significant.
- Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- No test substance-related external developmental malformations were observed in fetuses in this study. Gastroschisis or omphalocele was noted for 6 fetuses from a single litter (No. 2508) in the 50 mg/kg/day group and consisted of portions of the intestine, stomach, liver, and/or spleen protruding. These malformations were not considered to be test substance-related because they occurred in a single litter in the low-dose group.
- No external developmental variations were observed in fetuses in this study.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- Skeletal malformations were noted for 1(1), 2(1), and 1(1) fetuses (litters) in the control, 50, and 1000 mg/kg/day groups, respectively. Fetus No. 4521-07 in the 1000 mg/kg/day group was noted with absent vertebra (only 5 lumbar vertebra). In the 50 mg/kg/day group, Fetus Nos. 2508-07 and 2508-14 were noted with sternoschisis. Both of these fetuses also had an omphalocele noted at the visceral examination. These malformations were not considered test substance-related because they occurred in single fetuses/litters and/or were noted similarly in the control group. In the control group, Fetus No. 1516-08 was noted with sternoschisis.
- No test substance-related skeletal developmental variations were noted. Significantly higher incidence of wavy ribs and short supernumerary cervical ribs were noted at 1000 mg/kg/day and significantly lower incidence of short supernumerary thoracolumbar ribs were noted at 50 and 1000 mg/kg/day relative to the concurrent control group. These rib findings were within range of historical control data and as such were not considered test substance-related. Additional 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 developmental historical control data.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- No visceral developmental malformations were observed in fetuses in this study.
- No test substance-related visceral developmental variations were noted. Variations observed in the test substance-treated groups were noted infrequently, similarly in the control group, and/or were not observed in a dose-related manner.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
FETAL MORPHOLOGICAL DATA
- The numbers of fetuses (litters) available for morphological evaluation were 299(25), 275(25), 289(25), and 274(24) in the control, 50, 250, and 1000 mg/kg/day groups, respectively.
- Malformations were observed in 1(1), 8(2), 0(0), and 1(1) fetuses (litters) in these same respective dose groups and were considered spontaneous in origin.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
maternal dose
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes

Fetal abnormalities

Key result
Abnormalities:
effects observed, non-treatment-related

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

ANALYSES OF DOSING FORMULATIONS

- The analyzed dosing formulations contained 93.8% to 109% of the test substance which was within the protocol-specified range of target concentrations for suspensions (85% to 115%) and were homogeneous. The test substance was not detected in the analyzed vehicle formulation that was administered to the control group (Group 1).

- Results of the analyses of dosing formulations are summarized in Tables 15 and 16 (below).

Table 15 – Results of homogeneity analysis

Homogeneity Assessment of the

13 Aug 2020 formulations

Group 2 (12.5 mg/L)

Group 4 (250 mg/L)

Mean Concentration (mg/mL)

13.6

263

RSD (%)

1.8

3.3

Mean Concentration % of Target

109

105

 

Table 16 – Results of concentration analysis

Date of preparation

Group 2 (12.5 mg/L)

Group 3 (62.5 mg/L)

Group 4 (250 mg/L)

13 Aug 2020

13.6 mg/mL (109 % of target)

64.8 mg/mL (104 % of target)

263 mg/mL (105 % of target)

27 Aug 2020

12.5 mg/mL (100 % of target)

58.6 mg/mL (93.8 % of target)

254 mg/mL (102 % of target)

THYROID HORMONE ANALYSIS                 

- In the 1000 mg/kg/day group, test substance-related lower total maternal T3 hormone levels were noted relative to the concurrent control group and historical control minimum values. However, in the absence of effects on T4 and TSH levels and without corresponding microscopic findings on the thyroid gland or effects on thyroid weights, these effects were considered non-adverse.

- The total T3, and T4, and TSH hormone levels were unaffected by test item administration at 50 and 250 mg/kg/day.

Applicant's summary and conclusion

Conclusions:
Lower mean maternal body weights with corresponding effects on mean food consumption and lower mean fetal body weights were noted at 1000 mg/kg/day. Based on these results, a dosage level of 250 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity and prenatal developmental toxicity when test item was administered orally by gavage to time-mated Crl:CD(SD) rats.
Executive summary:

GUIDELINE

The design of the study was based on United States EPA Guideline OPPTS 870.3700 and OECD Test Guideline 414. The objectives of the study were to determine the potential of the test substance to induce developmental toxicity after maternal exposure from implantation to 1 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.

 

METHODS

Female Crl:CD(SD) rats (25 per group) were dosed via oral gavage at 0, 50, 250 or 1000 mg/kg bw/day once daily during Gestation Days 6 to 20. Parameters and end points evaluated during the study were clinical signs, body weights, body weight gains, gravid uterine weights, food consumption, thyroid hormone assessments, gross necropsy findings, organ weights, histopathologic examinations, intrauterine growth and survival, anogenital distance and fetal morphology.

 

RESULTS

All maternal animals survived to scheduled necropsy on Gestation Day 21. Test substance-related clinical observations included abnormal breathing, red fur staining and wet fur around the mouth and/or muzzle at the daily examinations and/or 1–2 hours postdosing in the 250 and 1000 mg/kg/day groups. These clinical observations were considered non-adverse due to their sporadic occurrence.

 

Test substance-related maternal effects in the 1000 mg/kg/day group, included lower mean body weight gains with correspondingly lower mean food consumption when the entire dosing period was evaluated (Gestation Days 6 to 21), resulting in a mean absolute body weight that was 6.8 % lower than the control group on Gestation Day 21. In addition, lower mean corrected body weight and corrected body weight gain were noted in this group compared to the control group. Maternal food consumption in the 1000 mg/kg/day group was lower than the control group generally throughout the entire treatment period. Due to the magnitude of these differences from the control group, the test substance-related effects on mean maternal body weight, body weight gains, and food consumption were considered adverse. Mean body weights, body weight gains, food consumption, corrected body weights, corrected body weight gains, and gravid uterine weights in the 50 and 250 mg/kg/day groups were unaffected by test substance administration.

 

In the 1000 mg/kg/day group, lower maternal T3 hormone levels were noted relative to the control group in the absence of effects on TSH and T4 levels. Due to the magnitude of the difference from the control group, the effect on T3 levels was considered test substance-related. However, due to the lack of corresponding microscopic findings or effects on the thyroid gland weight, the lower mean T3 level was not considered adverse. Thyroid hormone levels (T3, T4, and TSH) were unaffected by test item administration at 50 and 250 mg/kg/day.

 

No remarkable maternal macroscopic or microscopic (thyroid gland and liver) findings were noted at any dosage level at the scheduled necropsy. There were no test substance-related effects on mean thyroid gland or liver weights. Test substance-related lower (up to 6.712%) mean fetal body weights (males, females, and combined) were noted in the 1000 mg/kg/day group compared to the control group and correlated with lower mean gravid uterine weight and adverse maternal toxicity in this group.

 

Due to the magnitude of difference from the controls, the test substance-related effects on fetal weights were considered adverse. There were no test substance-related effects on intrauterine growth at 50 and 250 mg/kg/day or intrauterine survival, anogenital distance, and fetal morphology (external, visceral, and skeletal) at 50, 250, and 1000 mg/kg/day.

 

CONCLUSION

Lower mean maternal body weights with corresponding effects on mean food consumption and lower mean fetal body weights were noted at 1000 mg/kg/day. Based on these results, a dosage level of 250 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity and prenatal developmental toxicity when test item was administered orally by gavage to time-mated Crl:CD(SD) rats.