2,4,7,9-tetramethyldec-5-yne-4,7-diol

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jan - Sept 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,4,7,9-tetramethyldec-5-yne-4,7-diol

EC Number:

204-809-1

EC Name:

2,4,7,9-tetramethyldec-5-yne-4,7-diol

Cas Number:

126-86-3

Molecular formula:

C14 H26 O2

IUPAC Name:

2,4,7,9-tetramethyldec-5-yne-4,7-diol

Constituent 2

Reference substance name:

2,4,7,9-Tetramethyl-5-dcyne-4,7-diol

IUPAC Name:

2,4,7,9-Tetramethyl-5-dcyne-4,7-diol

Test material form:

other: waxy solid

Details on test material:

Lot Number: 2910-109
Purity: 100 %

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
- Females (if applicable) nulliparous and non-pregnant: Naïve, purpose-bred, time-mated (Day mating is confirmed [copulatory plug] = Gestation Day [GD] 0)
- Age at study initiation: 10 to 13 weeks
- Weight at study initiation: 175 to 300 g
- Housing: Single/Individual; Individual housing of presumed pregnant females is required to adequately monitor the health of these females by allowing collection of individual food consumption and appropriate identification of cage observations in the event of early delivery .
Solid-bottom cages containing appropriate bedding material (Bed-OCobs ® or other suitable material). Nesting material will not be provided, as euthanasia is scheduled prior to anticipated parturition. For enrichment, animals will be provided items such as treats and/or a gnawing device, except when interrupted by study procedures/activities.
- Diet (e.g. ad libitum): PMI Nutrition International, LLC Certified Rodent LabDiet® 5002, ad libitum
- Water (e.g. ad libitum): Municipal tap water, treated by reverse osmosis and ultraviolet irradiation, ad libitum
- Acclimation period: Time-mated rats were received by Charles River on Gestation Day 1, 2, 3 and 4. The day on which confirmation of mating is made was designated Gestation Day 0. Each rat was inspected by a qualified technician upon receipt. All rats were permanently identified with a microchip.
Each rat was observed for mortality/moribundity twice daily. Cage-side observations were performed daily from receipt until Gestation Day 6 (except on days that detailed clinical bservations are performed). Prior to the start of dose administration, those animals judged to be suitable test subjects were identified. Detailed clinical observations and body weights on gestation Days 5 and 6, and food consumption from Gestation Day 5-6 were recorded for all rats (including alternates purchased for the study). The Gestation Day 0 body weight of each dam was recorded by the supplier.

DETAILS OF FOOD AND WATER QUALITY:
Diet: Results of analysis for nutritional components and environmental contaminants are provided by the supplier and are on file at the Testing Facility. It is considered that there are no known contaminants in the feed that would interfere with the objectives of the study.
Water: Periodic analysis of the water is performed, and results of these analyses are on file at the Testing Facility. It is considered that there are no known contaminants in the water that would interfere with the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 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: 21. Jan. To: 11. Feb. 2022

Administration / exposure

Route of administration:

oral: feed

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): PMI Nutrition International, LLC LabDiet Certified Rodent Diet 5002
- Storage temperature of food: 18-24°C

Analytical verification of doses or concentrations:

yes

Remarks:

by gas chromatography (GC) with flame ionization detection (FID)

Details on analytical verification of doses or concentrations:

Diet formulation samples were collected for analysis weekly for analysis of homogeneity and concentration.
Dose analysis results were verified prior to diet administration at each sampling interval, if
possible. If results are deemed unacceptable, the diet formulations will be prepared again and
analyzed.

Details on mating procedure:

n.a.; females were purpose-bred, time-mated

Duration of treatment / exposure:

Gestation Days 6 through 2

Frequency of treatment:

continuously

Duration of test:

Gestation Days 0 through 21

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Doses / concentrations 2

Dose / conc.:

1 500 ppm

Remarks:

in diet, corresponding to 109 mg/kg bw/d

Doses / concentrations 3

Dose / conc.:

5 000 ppm

Remarks:

in diet, corresponding to 354 mg/kg bw/d

Doses / concentrations 4

Dose / conc.:

15 000 ppm

Remarks:

in diet corresponding to 1058 mg/kg bw/d

No. of animals per sex per dose:

22

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: based on results dose range finding study
- Rationale for animal assignment (if not random): n.a.
- Fasting period before blood sampling for (rat) dam thyroid hormones: none
- Time of day for (rat) dam blood sampling: prior to noon

Examinations

Maternal examinations:

- Mortality: all animals; at least twice daily
- Detailed clinical observations: all animals; Gestation Days 5, 6, 9, 12, 15, 18, and 21; Mortality and all signs of overt toxicity were recorded on the day observed. The observations shall include, but are not limited to, evaluations for changes in appearance of skin and fur, eyes, mucous membranes, respiratory and circulatory system, autonomic and central nervous systems, somatomotor activity, and behavior.
- Cage-side observations: Once daily
- Individual body weights: all animals; Gestation Days 0 (by supplier), 5, 6, 9, 12, 15, 18 and 21
- Food consumption: all animals; Gestation Days 5, 6, 9, 12, 15, 18, and 21
- Thyroid hormone assessment: all animals on Gestation Day 21(T3, T4 and TSH)
- Macroscopic examination: all animals on Gestationd Day 21; The cranial, thoracic, abdominal, and pelvic cavities will be opened and the contents examined.
- Organ weights: uterus, thyroid gland and liver
- Microscopy: all animals; thyroid gland

Ovaries and uterine content:

The uterus of each female were excised and its adnexa trimmed. Corpora lutea were also counted and recorded.
The uterus of each female was opened and the number of viable and nonviable fetuses, early and late resorptions and total number of implantation sites was 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).

Fetal examinations:

External, internal, and skeletal fetal 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), 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.).

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

- Visceral (internal):
Approximately one-half of the fetuses in each litter was 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 (Stuckhardt and Poppe, 1984). This examination includes 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 sex of all fetuses wasconfirmed by internal examination. 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 and Alcian Blue by a method similar to that described by Dawson (Dawson, 1926) and Inouye (Inouye, 1976). The skeletal examination was made following this procedure.

Statistics:

Descriptive Statistical Analyses: Means, standard deviations, percentages, numbers, and/or incidences were reported, as appropriate by dataset.
Inferential Statistical Methods: All statistical tests were conducted at the 5% significance level. All pairwise comparisons were be conducted using two sided tests and will be reported at the 1% and 5% levels.
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 is not
significant or the Kruskal-Wallis test if it is significant. If the overall F-test or Kruskal-Wallis
test is 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 is found to be significant, then the above pairwise comparisons were conducted using Dunn’s test.
Group Pair-wise Comparison (General ANOVA)

Indices:

Body Weight Gains: Calculated between each scheduled interval as well as between the following intervals: GD 6-21
Food Consumption: Calculated for each corresponding body weight interval
Pre-Implantation Loss = (No. of corpora lutea – no. of implants x 100) / (No. of corpora lutea)
Post-Implantation Loss = (No. of implants – no. of live fetuses x 100) / (No. of implants)
Sex Ratio (% males) = (No. male fetuses x 100) / (Total no. of fetuses)
Litter % of Fetuses with Abnormalities = (No. of fetuses in litter with a given finding x 100) / (No. of fetuses in litter examined)

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

No test substance-related clinical observations were noted at the detailed clinical observations or cage-side observations at any concentration.

Mortality:

no mortality observed

Description (incidence):

All females in the control, 1500, 5000, and 15,000 ppm groups survived to the scheduled necropsy on Gestation Day 21.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the 15,000 ppm group, statistically significantly lower mean body weight gain was noted immediately following the initiation of exposure (Gestation Days 6–9), and was followed by statistically significant higher mean body weight gain during Gestation Days 9–12. Lower mean body weight gains were noted in this group during Gestation Days 15–21; the difference from the control group was statistically significant during Gestation Days 15–18. As a result of the fluctuations, mean body weight gains in the 15,000 ppm group was comparable to the control group when the overall exposure period (Gestation Days 6–21) was evaluated. The effects on mean body weight gains in the 15,000 ppm group were considered test substance-related but nonadverse because it had no impact on the overall body weight gain or mean absolute body weights.
Mean maternal body weights, body weight gains, adjusted body weights, and adjusted body weight gains in the 1500 and 5000 ppm groups and mean gravid uterine weight in the 1500 ppm were unaffected by test substance exposure.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean maternal food consumption and food utilization in the 15,000 ppm group were statistically significantly lower than the control group following the initiation of exposure (Gestation Days 6–9), and higher than or comparable to the control group during Gestation Days 9–15; differences were generally statistically significant. Mean food consumption and utilization in this group were generally comparable to or higher than the control group for the remainder of the exposure period (Gestation Days 15–21), with the following exception. A statistically significantly lower mean food utilization value was noted at 15,000 ppm compared to the control group during Gestation Days 15–18. The initial decrement in food consumption and food utilization at 15,000 ppm corresponded to the lower mean body weight gain during this interval; this change was considered test substance-related but nonadverse in the absence of an effect on absolute body weights. When the entire treatment period (Gestation Days 6–21) was evaluated, mean maternal food consumption and food utilization in this group were comparable to the control group.
Mean maternal food consumption and food utilization in the 1500 and 5000 ppm groups were unaffected by test substance exposure.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

effects observed, treatment-related

Description (incidence and severity):

In the 5000 and 15,000 ppm groups, nonadverse test substance-related effects on mean TSH, T3, and T4 concentrations were noted on Gestation Day 21. Lower mean T3 concentrations were noted in these groups compared to the control group (10.3 and 25.7% lower, respectively), and
were below the minimum range of the testing facility`s historical control data (version 2020.02; 221.4 pg/mL). Higher mean T4 concentrations in these groups were also noted compared to the control group (14.8 and 21.3% higher, respectively), but were within testing facility`s historical control data range (10,179.2 to 23,580.0 pg/mL).
Higher mean TSH concentrations were noted compared to the control group (14.0 and 54.5% higher, respectively), and were within testing facility`s historical control data range (1281.0 to 2153.1 pg/mL) at 5000 ppm, but exceeded the maximum value at 15,000 ppm.
This correlated with the nonadverse microscopic findings of hepatocellular hypertrophy in the liver at 5000 and 15,000 ppm and follicular cell hypertrophy and decreased colloid in the thyroid gland at 15,000 ppm. The
mechanism of thyroid gland follicular cell hypertrophy was considered to be enhanced thyroid hormone degradation secondary to test substance-related hepatic microsomal enzyme induction, specifically, induction of uridine diphosphoglucuronosyltransferase (UDP-GT) causing enhanced thyroid hormone metabolism and subsequent increased levels of TSH (Zabka et al., 2011).
In the 1500 ppm group, test substance-related lower mean T3 concentration was noted on Gestation Day 21 compared to the control group (10.2% lower) and was below the minimum range of the testing facility`s historical control data (221.4 pg/mL). This was considered nonadverse because there were no effects on thyroid gland weight or histopathology in the thyroid at this dose level.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related higher mean liver weights in the 5000 and 15,000 ppm group correlated microscopically with hepatocellular hypertrophy and macroscopically with liver enlargement in a single 15,000 ppm group female.
This was considered nonadverse in the absence of adverse microscopic findings.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related enlargement of the liver was observed in a single 15,000 ppm group female, which correlated with higher mean liver weights and hepatocellular hypertrophy noted microscopically. This was considered nonadverse because it was limited to a single animal in the absence of adverse microscopic findings.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Thyroid follicular cell hypertrophy was characterized by cuboidal follicular cells with vacuolated cytoplasm and occasional nuclear crowding. The finding was often accompanied by small diameter follicles containing decreased colloid. Thyroid gland findings had no effect on mean or individual thyroid gland weights.
Hepatocellular hypertrophy was characterized by large hepatocytes with increased eosinophilic hepatocellular cytoplasm in a centrilobular to panlobular distribution. The finding correlated with higher mean liver weights (absolute and relative to terminal body weight) and enlarged liver observed in a single 15,000 ppm group female.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

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:

not examined

Changes in number of pregnant:

not examined

Other effects:

not examined

Details on maternal toxic effects:

Intrauterine survival was unaffected by test substance at exposure levels of 1500, 5000, and 15,000 ppm. The mean litter proportion of postimplantation loss (primarily early resorptions) in the 5000 ppm and 15,000 ppm groups (6.40% and 8.95% per litter, respectively) were higher (not statistically significant) when compared to the concurrent control group (3.56% per litter), but was attributed to single animals (42.9% for a 5000 ppm animal and 66.7% for a 15,000 ppm animal) and within the testing facility`s historical control data range (2.61% to 12.60% per litter). Furthermore, the mean numbers of live fetuses in the same respective groups (12.1 and 12.0 per dam,) were comparable to the control group (13.2 per dam) and within the testing facility`s historical control data range (11.44 to 13.72 live fetuses per dam).
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)

Dose descriptor:

NOAEL

Effect level:

15 000 ppm

Based on:

test mat.

Remarks:

corresponding to 1058 mg/kg bw/d

Basis for effect level:

other: no adverse maternal effects observed

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related effects on intrauterine growth were noted in the 15,000 ppm group. Mean fetal weights (combined, males, and females) were lower (4.18%, 4.15%, and 3.75%, respectively) than the concurrent control group; differences were statistically significant for males and combined sexes and corresponded to the lower mean gravid uterine weight noted in this group. The mean fetal weights in this group were within the testing facility`s historical control data range, and therefore this effect was considered nonadverse. Intrauterine growth was unaffected by test substance at exposure levels of 1500 and 5000 ppm.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

not examined

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

No test substance-related external developmental variations were observed in fetuses in this study. Findings were limited to single fetuses in the control and 15,000 ppm groups.

Skeletal malformations:

no effects observed

Visceral malformations:

not examined

Description (incidence and severity):

No test substance-related visceral developmental malformations were observed in fetuses in this study.

Other effects:

not examined

Details on embryotoxic / teratogenic effects:

The numbers of fetuses (litters) available for morphological evaluation were 237(18), 248(19), 230(19), and 228(19) in the control, 1500, 5000, and 15,000 ppm groups, respectively.
Malformations were observed in 1(1), 1(1), 0(0), and 1(1) fetuses (litters) in these same respective dose groups and were considered spontaneous in origin. When the total malformations were evaluated on a proportional basis, no statistically significant differences from the control group were noted. Fetal malformations, when observed in the test substance-treated groups, occurred infrequently, did not occur in a dose-related manner, and/or were within the testing facility's historical control data ranges.
No test substance-related visceral developmental variations were observed in fetuses in this study. Visceral variations were limited to increased incidences of minimal to moderate dilatation of the ureter(s) in the 15,000 ppm group; these variations would not impact postnatal survival, the mean litter proportions were not statistically significantly different from the concurrent control group, and the values were within the testing facility's historical control data range, and therefore were considered the result of biological variability. Other fetal developmental variations, when observed in the test substance-treated groups, occurred infrequently, did not occur in a dose-related manner, and/or were within the testing facility's historical control data ranges.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

15 000 ppm

Based on:

test mat.

Remarks:

corresponding to 1058 mg/kg bw/d

Sex:

male/female

Basis for effect level:

other: no developmental toxic effect observed

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Treatment related:

not specified

Applicant's summary and conclusion

Conclusions:

In conclusion, there were no adverse maternal effects or adverse effects on intrauterine growth and survival or fetal morphology by test substance exposure up to 15,000 ppm. Based on these results, an exposure level of 15,000 ppm equivalent to 1058 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity and for prenatal developmental toxicity when 2,4,7,9-Tetramethyl-5-decyne-4,7-diol was administered orally in the diet to time-mated Crl:CD(SD) rats.

Executive summary:

The test substance was administered continuously in the diet during Gestation Days 6–21.

The following parameters and end points were evaluated in this study: mortality, clinical signs, body weights, body weight gains, gravid uterine weights, food consumption, food utilization, thyroid hormones, anogenital distance, organ weights, macroscopic and microscopic examinations, intrauterine growth and survival, and fetal morphology.

Mean calculated test substance consumption during GD 6 -21 (based on analytical determination of diet):

- 0 ppm: 0 mg/kg bw/d

- 1500 ppm: 109 mg/kg bw/d

- 5000 ppm: 354 mg/kg bw/d

- 15000 ppm: 1058 mg/kg bw/d

Test substance-related lower mean body weight gain with correspondingly lower mean food consumption and food utilization were noted in the 15,000 ppm group during Gestation Days 6-9, followed by higher mean body weight gains during Gestation Days 9–12 compared to the control group. Mean body weight gains, food consumption, and food utilization in this group were generally comparable to the control group for the remainder of the treatment period; any transient fluctuations noted did not impact mean absolute body weights. As a result, the initial decrements were considered nonadverse. Lower mean gravid uterine weight was noted in the 15,000 ppm group compared to the control group. The difference was considered test substancerelated because it correlated with lower mean fetal weight at this exposure level but was considered nonadverse because it was within the range of the testing facility's historical control data.

Mean maternal body weights, body weight gains, food consumption, food utilization, and gravid uterine weights in the 1500 and 5000 ppm groups and adjusted body weights and adjusted body weight gains in the 1500, 5000, and 15,000 ppm groups were unaffected by test substance exposure.

Test substance-related nonadverse effects on mean T3 concentrations were noted at all dose levels on Gestation Day 21; lower mean T3 concentrations were noted in these groups compared to the control group. Higher mean T4 and TSH concentrations in the 5000 and 15,000 ppm groups were also noted compared to the control group. This correlated with the nonadverse microscopic findings of hepatocellular hypertrophy in the liver at 5000 and 15,000 ppm and follicular cell hypertrophy and decreased colloid in the thyroid gland at 15,000 ppm. The mechanism of thyroid gland follicular cell hypertrophy was considered to be enhanced thyroid hormone degradation secondary to test substance-related hepatic microsomal enzyme induction, specifically, induction of uridine diphosphoglucuronosyltransferase (UDP GT) causing enhanced thyroid hormone metabolism and subsequent increased levels of TSH (Zabka et al., 2011).

Test substance-related maternal macroscopic findings included enlargement of liver noted in a single 15,000 ppm group female; this correlated with nonadverse hepatocellular hypertrophy noted microscopically in this group and thus was considered nonadverse.

Histopathological examinations revealed adaptive, nonadverse liver and thyroid gland changes. Hepatocellular hypertrophy was noted microscopically in the 5000 and 15,000 ppm groups, which correlated with higher mean liver weights and liver enlargement for a single 15,000 ppm group female. Thyroid gland findings consisted of follicular cell hypertrophy and decreased colloid, with no macroscopic or organ weight correlates.

Test substance-related slightly lower (up to 4.18%) mean fetal body weights (combined, males, and females) were noted in the 15,000 ppm group compared to the control group which corresponded to the lower mean gravid uterine weight noted in this group and were considered nonadverse because they were within the range of the testing facility's historical control data.

Intrauterine growth at 1500 and 5000 ppm and intrauterine survival at 1500, 5000, and 15,000 ppm were unaffected by test substance exposure.

No test substance-related external, visceral, or skeletal developmental malformations or variations were noted at any exposure concentration.