Thiophene, tetrahydro-, 1,1-dioxide, 3-(C9-11-isoalkyloxy) derivs., C10-rich

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 November 2016 to 25 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

minor deviation to planned post-mortem and pathology procedure with no impact on study results or integrity (see below)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

minor deviation to planned post-mortem and pathology procedure with no impact on study results or integrity (see below)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Thiophene, tetrahydro-, 1,1-dioxide, 3-(C9-11-isoalkyloxy) derivs., C10-rich

EC Number:

800-172-4

Cas Number:

398141-87-2

Molecular formula:

Complex UVCB substance

IUPAC Name:

Thiophene, tetrahydro-, 1,1-dioxide, 3-(C9-11-isoalkyloxy) derivs., C10-rich

Test material form:

liquid

Details on test material:

- Physical description: clear yellow liquid
- Storage conditions: room temperature protected from light

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)

Details on test animals or test system and environmental conditions:

TEST SYSTEM, ANIMAL RECEIPT AND ACCLIMATION
- Sexually mature, virgin female Sprague Dawley [Crl:CD(SD)] rats were used as the test system on this study. This species and strain of animal is recognized as appropriate for developmental toxicity studies. Charles River Ashland has historical control data on the background incidence of fetal malformations and developmental variations in the Crl:CD(SD) rat. This animal model has been proven to be susceptible to the effects of developmental toxicants.
- The number of animals selected for this study (25 females/group) was based on the United States EPA Health Effects Test Guidelines OPPTS 870.3700, Prenatal Development Toxicity Study, August 1998 and the OECD Guidelines for the Testing of Chemicals: Guideline 414, Prenatal Developmental Toxicity Study, January 2001, which recommend evaluation of approximately 20 females with implantation sites at necropsy. Given the possibility of nongravid animals, unexpected deaths, or treatment-related moribundity and/or mortality, this was an appropriate number of animals to obtain a sample size of 20 at termination.
- Sexually mature, virgin female Crl:CD(SD) rats (150 females) were received in good health from Charles River Laboratories, Inc., Raleigh, NC, on 01 Nov 2016.
- The animals were approximately 69 days old upon receipt. Each female was examined by a qualified biologist on the day of receipt.
- On the day following receipt, all animals were weighed and clinical observations were recorded. Each animal was uniquely identified using a programmable microchip (BMDS system) which was implanted subcutaneously in the dorsoscapular region during the acclimation period.
- The animals were housed for a minimum of 14 days for acclimation purposes. During the acclimation period, the rats were observed twice daily for mortality and changes in general appearance and behaviour.

ANIMAL HOUSING
- Upon arrival, all rats were housed 2 animals/cage in clean, solid-bottom cages with bedding material (Bed-O'Cobs; The Andersons, Cob Products Division, Maumee, OH). The bedding material is periodically analysed by the manufacturer for contaminants. Analyses of the bedding material were provided by the manufacturer. No contaminants were present in the bedding at concentrations sufficient to interfere with the outcome of the study. The results of these analyses are maintained at Charles River.
- The rats were paired for mating in the home cage of the male.
- Following positive evidence of mating, the females were individually housed in clean, solid-bottom cages with bedding material. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals. The animal facilities at Charles River Ashland are fully accredited by AAALAC International.
- Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment and to aid in maintaining the animals’ oral health, and were sanitized weekly.

DIET, DRINKING WATER AND MAINTENANCE
- The basal diet used in this study, PMI Nutrition International, LLC Certified Rodent LabDiet 5002, was a certified feed with appropriate analyses performed by the manufacturer and provided to Charles River. Feed lots used during the study were documented in the study records.
- Feeders were changed and sanitized once per week.
- Municipal water supplying the facility was sampled for contaminants according to Charles River SOPs. The results of the diet and water analyses are maintained at Charles River.
- No contaminants were present in animal feed or water at concentrations sufficient to interfere with the objectives of this study.
- Reverse osmosis-purified (on-site) drinking water, delivered by an automatic watering system, and the
basal diet were provided ad libitum throughout the acclimation period and during the study.

ENVIRONMENTAL CONDITIONS
- All rats were housed throughout the acclimation period and during the study in an environmentally controlled room. The room temperature and relative humidity controls were set to maintain environmental conditions of 68 °F to 78 °F (20 °C to 26 °C) and 30% to 70%, respectively.
- Room temperature and relative humidity data were monitored continuously and were scheduled for automatic collection on an hourly basis.
- Actual mean daily temperature ranged from 71.7 °F to 73.3 °F (22.1 °C to 22.9 °C) and mean daily relative humidity ranged from 40.2% to 52.0% during the study.
- Fluorescent lighting provided illumination for a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod. The light status (on or off) was recorded once every 15 minutes.
- Air handling units were set to provide a minimum of 10 fresh air changes per hour.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Remarks:

Lot number 1FE0684; Expiry date 31 May 2017

Details on exposure:

PREPARATION
- The vehicle was dispensed approximately weekly for administration to the control group (Group 1) and for preparation of the test substance formulations.
- Aliquots were prepared for daily dispensation to the control group and stored at room temperature, protected from light.
- The vehicle was mixed throughout the preparation, sampling, and dose administration procedures.
- Dosing formulations were prepared at the test substance concentrations indicated in the table below.
- The test substance formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature protected from light.
- The test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures.
- The first test substance dosing formulations were visually inspected by the Study Director and were found to be visibly homogeneous and acceptable for administration.

ASSIGNMENT OF ANIMALS TO TREATMENT GROUPS AND BREEDING PROCEDURES
- At the conclusion of the acclimation period, all available females were weighed and examined in detail for physical abnormalities. At the discretion of the Study Director, each animal judged to be in good health and meeting acceptable body weight requirements was placed in a solid-bottom cage with bedding material with a resident male from the same strain and source for breeding.
- Resident males were untreated, sexually mature rats utilized exclusively for breeding. These rats were maintained under similar laboratory conditions as the females.
- A breeding record containing the male and female identification numbers and the dates of cohabitation was maintained.
- The selected females were approximately 12 weeks old when paired for breeding.
- Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm in a vaginal lavage and verified by a second biologist.
- Each mating pair was examined daily. The day on which evidence of mating was identified was termed Gestation Day 0 and the animals were separated.
- The experimental design consisted of 4 test substance-treated groups and 1 control group, composed of 25 rats/group.
- The bred females were assigned to groups using a WTDMS computer program, which randomized the animals based on stratification of the Gestation Day 0 body weights in a block design.
- Animals not assigned to study were transferred to the Charles River rat colony or euthanized by carbon dioxide inhalation and discarded.
- Body weight values of the selected females ranged from 223 g to 289 g on Gestation Day 0.

ORGANISATION OF TEST GROUPS, DOSAGE LEVELS AND TREATMENT REGIMEN
- The vehicle and test substance formulations were administered orally by gavage, via an appropriately sized flexible, disposable, plastic feeding tubes (Instech Solomon, Plymouth Meeting, PA), once daily during Gestation Days 6 to 19.
- The dose volume for all groups was 5 mL/kg. Individual dosages were based on the most recently recorded body weights to provide the correct mg/kg/day dose. All animals were dosed at approximately the same time each day.
- Study group assignments are shown in the table below.
- The dosage levels were selected based on the results of a previous dose range-finding study in rats.3 In that study, female rats were dosed from Gestation Day 6 through 19 at dosage levels of 0, 50, 175, 600, and 1000 mg/kg/day. There were no significant clinical findings noted in the previous study and mean body weights, body weight gains, and food consumption were unaffected by treatment. As a result, dosage levels of 175, 350, 600, and 1000 mg/kg/day were chosen for the current study.
- The selected route of administration for this study was oral (gavage) because this is a potential route of exposure for humans. Historically, this route has been used extensively for studies of this nature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

SAMPLING AND ANALYSIS
- Test substance homogeneity and stability following at least 8 days of room temperature storage was established to cover the range of concentrations used on this study. Therefore, stability assessments were not conducted at part of this study.
- Samples for homogeneity and/or concentration determination were collected from the top, middle, and bottom strata of the first and last test substance dosing formulations and from the middle stratum of the first and last control dosing formulations.
- One set of samples from each collection was subjected to the appropriate analyses.
- All remaining samples were stored frozen at room temperature as back-up.
- All analyses were conducted by the Charles River Analytical Chemistry Department using a validated high performance liquid chromatography method with charged aerosol detection.

Duration of treatment / exposure:

Gestation Days 6 to 19

Frequency of treatment:

Daily

Duration of test:

20 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

175 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

350 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

600 mg/kg bw/day (actual dose received)

Doses / concentrations 5

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

25 females per group

Control animals:

yes, concurrent vehicle

Details on study design:

- A flow chart summarising the study design is attached.

Examinations

Maternal examinations:

CLINICAL OBSERVATIONS AND SURVIVAL
- All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality. Individual clinical observations were recorded daily during Gestation Days 0 to 20 (prior to dose administration during the treatment period).
- Animals were also observed for signs of toxicity at approximately 1 hour following dose administration. The absence or presence of findings was recorded for all animals.

BODY WEIGHTS AND GRAVID UTERINE WEIGHTS
- Individual maternal body weights were recorded on Gestation Days 0 and 6–20 (daily). Group mean body weights were calculated for each of these days. Mean body weight changes were calculated for each corresponding interval and also for Gestation Days 6–9, 9–12, 12 15, 15–20, and 6–20.
- Gravid uterine weight was collected and net body weight (the Gestation Day 20 body weight exclusive of the weight of the uterus and contents) and net body weight change (the Gestation Day 0–20 body weight change exclusive of the weight of the uterus and contents) were calculated and presented for each gravid female at the scheduled laparohysterectomy

FOOD CONSUMPTION
- Individual food consumption was recorded on Gestation Days 0 and 6–20 (daily). Food intake was reported as g/animal/day and g/kg/day for the corresponding body weight change intervals.
- When food consumption could not be determined for an animal during a given interval (due to weighing error, food spillage, etc.), group mean values were calculated for that interval using the available data.
- The time periods when food consumption values were unavailable for a given animal were designated as “NA” on the individual report tables.

Ovaries and uterine content:

GESTATION DAY 20 LAPAROHYSTERECTOMY
- The laparohysterectomies and macroscopic examinations were performed blind to treatment group. All rats were euthanised on Gestation Day 20 by carbon dioxide inhalation. The thoracic, abdominal, and pelvic cavities were opened by a ventral mid-line incision, and the contents were examined. In all instances, the postmortem findings were correlated with the antemortem observations, and any abnormalities were recorded. The uterus and ovaries were then exposed and excised. The number of corpora lutea on each ovary was recorded (using magnification). The trimmed uterus was weighed and opened, and the number and location of all fetuses, early and late resorptions, and the total number of implantation sites were recorded. The placentae were also examined. The individual uterine distribution of implantation sites was documented using the following procedure. All implantation sites, including resorptions, were numbered in consecutive order beginning with the left distal to the left proximal uterine horn, noting the position of the cervix, and continuing from the right proximal to the right distal uterine horn.
- Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss.
- Maternal tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as indicated by the gross findings. Representative sections of corresponding organs from a sufficient number of control animals were retained for comparison. The carcass of each female was then discarded. Intrauterine data were summarized using 2 methods of calculation.
- An example of each method of calculation is attached.

Fetal examinations:

MORPHOLOGICAL EXAMINATION
- Fetal examinations were performed blind to treatment group. Each viable fetus was examined externally, individually sexed, weighed, euthanized by a subcutaneous injection of sodium pentobarbital in the scapular region, and tagged for identification. Fetal tags contained the Charles River study number, the female number, and the fetus number. The detailed external examination of each fetus included, but was not limited to, an examination of the eyes, palate, and external orifices, and each finding was recorded. Crown-rump measurements and degrees of autolysis were recorded for late resorptions, a gross external examination was performed (if possible), and the tissues were discarded.
- Each viable fetus was subjected to a visceral examination using a modification of the Stuckhardt and Poppe fresh dissection technique to include the heart and major blood vessels. The sex of each fetus was confirmed by internal examination. Fetal kidneys were examined and graded for renal papillae development. Heads from approximately one-half of the fetuses in each litter were placed in Harrison’s fixative for subsequent soft-tissue examination by the Wilson sectioning technique. The heads from the remaining one-half of the fetuses were examined by a midcoronal slice. All carcasses were eviscerated and fixed in 100% ethyl alcohol.
- Following fixation in alcohol, each fetus was stained with Alizarin Red S and Alcian Blue. Fetuses were then examined for skeletal malformations and developmental variations.
- External, visceral, and skeletal findings were recorded as 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) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life). 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.
- The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison and calculating the number of affected fetuses in a litter on a proportional basis (see details of calculations, attached).

Statistics:

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 findings of red and clear material around the mouth were noted in a dose-related manner in the 350, 600, and 1000 mg/kg/day groups approximately 1 hour following dose administration. These findings were generally noted throughout the treatment period, and were noted sparingly in the 350 mg/kg/day group. The observations generally did not persist to the daily clinical examinations and therefore, were not considered adverse.
- Other observations noted in the treated groups, including hair loss on various body surfaces, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.

Mortality:

no mortality observed

Description (incidence):

- All females in the control, 175, 350, 600, and 1000 mg/kg/day groups survived to the scheduled necropsy on Gestation Day 20.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

MATERNAL BODY WEIGHTS AND GRAVID UTERINE WEIGHTS
- Test substance-related, significantly (p < 0.05 or p < 0.01) lower mean body weight gains were noted for females in the 600 and 1000 mg/kg/day group during Gestation Days 6–9. During Gestation Days 9–12, mean body weight gains in these groups were significantly (p < 0.01) higher than the control group; however, these results did not occur in a dose-related manner and the differences were attributed to a lower control group mean during this period. Mean body weight gains in these groups were similar to the control group during Gestation Days 12–15. During Gestation Days 15–20, a test substance-related lower (significant at p < 0.01) mean body weight gain was noted in the 1000 mg/kg/day group, while mean body weight gain in the 600 mg/kg/day group was similar to the control group. As a result, mean body weight gain in the 1000 mg/kg/day group was significantly (p < 0.01) lower than the control group when the entire treatment period (Gestation Days 6–20) was evaluated and mean body weights in the 1000 mg/kg/day group were significantly (p < 0.05) lower than the control group on Gestation Days 9 (4.5%) and 20 (5.0%). Mean body weight gain in the 600 mg/kg/day group was similar to the control group when the entire treatment period (Gestation Days 6–20) was evaluated and mean body weights in this groups were similar to the control group throughout the study. Therefore, the lower mean body weight gain observed in the 600 mg/kg/day group during Gestation Days 6-9 was not considered adverse. Mean gravid uterine weight in the 1000 mg/kg/day group was significantly (p < 0.01) lower than the control group. However, the value was within the range of values in the Charles River Ashland historical control data (Version 2016.03) and was attributed to the non-test article-related lower mean number of viable fetuses (see Section 6.6.). Mean gravid uterine weight in the 600 mg/kg/day groups and net body weights and net body weight gains in the 600 and 1000 mg/kg/day group were similar to the control group.
- Mean maternal body weights, body weight gains, net body weights, net body weight gains, and gravid uterine weights in the 175 and 350 mg/kg/day groups were unaffected by test substance administration. During Gestation Days 9–12, mean body weight gains in these groups were significantly (p < 0.05) higher than the control group; however, these results did not occur in a dose-related manner and the differences were attributed to a lower control group mean during this period. 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 and g/kg/day, in the 1000 mg/kg/day group was significantly (p < 0.01) lower than the control group during Gestation Days 6–9. This was considered test substance-related and corresponded to the lower mean body weight gains noted for these females during this period. Mean food consumption in this group was similar to the control group throughout the remainder of the treatment period (Gestation Days 9–20).
- Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 175, 350, and 600 mg/kg/day groups was unaffected by test substance administration. Differences from the control group were slight and not statistically significant.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- All females survived to the scheduled necropsy on Gestation Day 20.
- No test substance-related internal findings were observed at any dosage level.
- Macroscopic findings observed in the test substance-treated groups occurred infrequently and/or in a manner that was not dose-related.

Maternal developmental toxicity

Other effects:

no effects observed

Description (incidence and severity):

GESTATION DAY 20 LAPAROHYSTERECTOMY
- Intrauterine growth and survival were unaffected by test substance administration at dosage levels of 175, 350, 600, and 1000 mg/kg/day.
- A significantly (p < 0.05) lower mean number of viable fetuses was noted in the 1000 mg/kg/day group. However, this value was within the range of values in the Charles River Ashland historical control data.
- Parameters evaluated included postimplantation loss, live litter size, mean fetal body weights, and fetal sex ratios. Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups. Differences from the control group were slight and not statistically significant.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Remarks:

maternal toxicity

Effect level:

600 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

- Fetus No. 2402-01 in the 1000 mg/kg/day group had exencephaly with open eyelid (right); this finding was confirmed skeletally, consisting of absent frontal (bilateral), parietal (bilateral), interparietal, and supraoccipital bones.
- Fetus No. 2388-05 in the 175 mg/kg/day group had microphthalmia (right).
- These findings were noted in single fetuses. In addition, the mean litter proportions of these findings were within the ranges of values in the Charles River Ashland historical control data (Version 2016.03) and were not statistically significantly different than the concurrent control group. Therefore, these findings were not considered test substance-related.
- No external developmental variations were observed in fetuses in this study.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

- No test substance-related skeletal malformations were noted. Fetus No. 2396-08 in the 1000 mg/kg/day group had sternoschisis (sternal bands not joined [entire length]), vertebral anomaly without associated rib anomaly (exxoccipital and cervical Arch No. 1 fused [bilateral]), and 6 cervical vertebrae (25 presacral vertebrae). In the 600 mg/kg/day group, Fetus No. 2403-10 had costal cartilage anomaly, consisting of right Costal Cartilage No. 1 and costal cartilage arising from 7th cervical rib fused and associate with sternum in normal No. 1 position. These findings occurred in single fetuses and the finding in the costal cartilage anomaly in the 600 mg/kg/day group did not occur in a dose-related manner. In addition, the mean litter proportions of these findings were within the ranges of values in the Charles River Ashland historical control data and were not statistically significantly different than the concurrent control group. Therefore, these findings were not considered test substance-related.
- No test substance-related skeletal developmental variations were noted. Findings observed in the test substance-treated groups were noted infrequently, similarly in the concurrent 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 Charles River Ashland historical control data.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

- No test substance-related visceral malformations were noted. In the 1000 mg/kg/day group, Fetus No. 2310-06 had hydrocephaly (increased cavitation of the lateral [bilateral] and third ventricles). In the 600 mg/kg/day group, Fetus No. 2325-03 had situs inversus (trachea, esophagus, heart, great and major vessels, lungs, liver, stomach, pancreas, spleen, kidneys, adrenals, and intestine laterally transposed). In the 350 mg/kg/day group, Fetus No. 2370-06 had retroesophageal aortic arch (aortic arch coursed retroesophageal immediately following the left carotid and returned in normal position adjacent to the ductus arteriosus). Fetus No. 2345-06 in the 350 mg/kg/day group had transposition of the great vessels, interventricular septal defect (2 mm opening in the anterior portion of the septum), and lobular dysgenesis of the lungs (1 lobe present [bilateral]). Interventricular septal defect (< 1 mm opening in the anterior portion of the septum) was also noted for Fetus No. 2400-05 in the 350 mg/kg/day group. These findings were noted in single fetuses and/or were not noted in a dose-related manner. In addition, the mean litter proportions of these findings were within the ranges of values in the Charles River Ashland historical control data (Version 2016.03) and were not statistically significantly different than the concurrent control group. In addition, interventricular septal defect was also noted for 2 fetuses (No. 2426-03 and 2426-16) in the concurrent control group (< 1 mm opening in the anterior portion of the septum). Therefore, these findings were not considered test substance-related.
- No test substance-related visceral developmental variations were noted. Findings observed in the test substance-treated groups were noted infrequently, similarly in the concurrent 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 Charles River Ashland historical control data. Renal papilla(e) not fully developed was noted for Fetus No. 2296-03 in the 175 mg/kg/day group, Fetus No. 2337-16 in the 350 mg/kg/day group, and Fetus Nos. 2325-04, 2362-03, 2411-13 in the 600 mg/kg/day group. This finding was not classified as either a malformation or developmental variation, was not included on the summary tables, and was not considered to be test substance-related because it occurred infrequently and in a manner that was not dose-related.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

FETAL MORPHOLOGICAL DATA
- The numbers of fetuses (litters) available for morphological evaluation were 361(24), 347(25), 354(24), 366(24), and 340(25) in the control, 175, 350, 600, and 1000 mg/kg/day groups, respectively.
- Malformations were observed in 3(2), 1(1), 3(3), 2(2), and 3(3) fetuses (litters) in these same respective dose groups and were considered spontaneous in origin.

Details on embryotoxic / teratogenic effects:

SUMMARY OF EXTERNAL, VISCERAL AND SKELETAL EXAMINATIONS
- The numbers of fetuses (litters) available for morphological evaluation were 361(24), 347(25), 354(24), 366(24), and 340(25) in the control, 175, 350, 600, and 1000 mg/kg/day groups, respectively.
- Malformations were observed in 3(2), 1(1), 3(3), 2(2), and 3(3) fetuses (litters) in these same respective dose groups and were considered spontaneous in origin.
- When the total malformations and developmental variations were evaluated on a proportional basis, no statistically significant differences from the concurrent control group were noted. Fetal malformations and developmental variations, when observed in the test substance-treated groups, occurred infrequently or at a frequency similar to that in the concurrent control group, did not occur in a dose-related manner, and/or were within the Charles River Ashland historical control data ranges.
- Based on these data, no fetal malformations or developmental variations were attributed to the test substance.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

maternal dose

Sex:

male/female

Basis for effect level:

other: based on lack of effects at any dosage level

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

ANALYSES OF DOSING FORMULATIONS

- The analysed dosing formulations were within Charles River SOP range for suspensions (85% to 115%) and were homogeneous.

- The test substance was not detected in the analysed vehicle formulation that was administered to the control group (Group 1).

- Results of the analyses of dosing formulations are summarised in the table attached.

Applicant's summary and conclusion

Conclusions:

Based on the adverse effects on body weight, body weight gain, and food consumption at 1000 mg/kg/day, a dosage level of 600 mg/kg/day was considered to be the no-observedadverse-effect level (NOAEL) for maternal toxicity. Due to the lack of effects at any dosage level, a dosage level of 1000 mg/kg/day, the highest dosage level evaluated, was considered to be the NOAEL for embryo/fetal development when test item was administered orally by gavage to bred Crl:CD(SD) rats.

Executive summary:

GUIDELINE

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 toxicity and developmental toxicity. The protocol was designed in general accordance with the United States EPA Health Effects Test Guidelines: OPPTS 870.3700, Prenatal Developmental Toxicity Study, Aug 1998, and the OECD Guideline for the Testing of Chemicals Guideline 414, Prenatal Developmental Toxicity Study, 22 Jan 2001.

 

METHODS

Test substance in the vehicle (corn oil) was administered orally by gavage to 4 groups of 25 bred female Crl:CD(SD) rats once daily from Gestation Days 6–19. Dosage levels were 175, 350, 600, and 1000 mg/kg/day administered at a dose volume of 5 mL/kg. A concurrent control group composed of 25 bred females received the vehicle on a comparable regimen. The females were approximately 13 weeks of age at the initiation of dose administration. All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. On

Gestation Day 20, a laparohysterectomy was performed on each female. The uteri, placentae, and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations.

 

RESULTS

All females in the control, 175, 350, 600, and 1000 mg/kg/day groups survived to the scheduled necropsy on Gestation Day 20. Test substance-related clinical findings of red and clear material around the mouth were noted in a dose-related manner in the 350, 600, and 1000 mg/kg/day groups approximately 1 hour following dose administration; these findings were noted sparingly in the 350 mg/kg/day group. The observations generally did not persist to the daily clinical examinations and therefore, were not considered adverse. No other test substance-related clinical findings were noted.

 

Test substance-related lower mean body weight gains were noted in the 600 and 1000 mg/kg/day groups during Gestation Days 6–9 and in the 1000 mg/kg/day group during Gestation Days 15–20, resulting in lower mean body weights in the 1000 mg/kg/day group on Gestation Day 9 (4.5%) and 20 (5.0%). Corresponding effects on food consumption were noted for females in the 1000 mg/kg/day group during Gestation Days 6–9. Mean body weights in the 600 mg/kg/day group were similar to the control group throughout the study. Therefore, the lower mean body weight gain observed in the 600 mg/kg/day group during Gestation Days 6–9 was not considered adverse. No other test substance-related effects were noted on mean body weights, body weight gains, gravid uterine weights, net body weights, and net body weight gains. No test substance-related maternal macroscopic findings were noted at the scheduled necropsy on Gestation Day 20. Intrauterine growth and survival and fetal morphology were unaffected by maternal test substance administration.

 

CONCLUSION

Based on the adverse effects on body weight, body weight gain, and food consumption at 1000 mg/kg/day, a dosage level of 600 mg/kg/day was considered to be the no-observedadverse-effect level (NOAEL) for maternal toxicity. Due to the lack of effects at any dosage level, a dosage level of 1000 mg/kg/day, the highest dosage level evaluated, was considered to be the NOAEL for embryo/fetal development when test item was administered orally by gavage to bred Crl:CD(SD) rats.