Alkenes, C6-11 (branched), hydroformylation products, distn. residues, heavy cracked fraction

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

Deviations from test guideline were not specified. Protocol and SOP deviations were reported and none of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Alkenes, C6-11 (branched), hydroformylation products, distn. residues, heavy cracked fraction

EC Number:

701-314-7

Molecular formula:

CnH2n+2O2. n=24-33

IUPAC Name:

Alkenes, C6-11 (branched), hydroformylation products, distn. residues, heavy cracked fraction

Details on test material:

Commercial product

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Sprague Dawley CD (Crl:CD[SD])

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: time-mated females were received from Charles River Laboratories, Raleigh, NC
- Age at study initiation: approximately 59-79 days old at receipt
- Weight at study initiation: 187-262 g at study randomization
- Housing: animals were co-housed (where possible) in solid-bottomed cages by dose group (no more than 2 per cage)
- Diet: ad libitum (Certified Rodent Diet® #5002 pelleted food, PMI® Nutrition International)
- Water: ad libitum
- Acclimation period: the rats arrived on GDs 1, 2, 3, or 4 and were acclimated prior to initiation of dosing on GD 6

ENVIRONMENTAL CONDITIONS
- Temperature: 68°F to 79°F (20°C to 26°C), During the study, the temperature was out of range (as low as 61°F). This deviation did not impact the outcome of the study because the variations from the target temperature were minimal and did not persist.
- Humidity: 30-70%
- Air changes: Ten or greater air changes per hour with fresh air (no air recirculation)
- Photoperiod: 12 hour light/12 hour dark cycle

IN-LIFE DATES: From 04 JAN 2019 to 24 JAN 2019

Administration / exposure

Route of administration:

oral: gavage

Details on exposure:

PREPARATION OF DOSE FORMULATIONS:
The study substance dosing formulations were prepared at least once weekly and dispensed up to 7 days from the date of preparation for dose administration. The study substance dose aliquots were stored in a refrigerator set to maintain 4°C and dispensed daily. The prepared test substance dose aliquots were each removed from the refrigerator to acclimate to ambient conditions and stirred for at least 30 minutes before dose administration and continuously during dosing.
VEHICLE
- Concentration in vehicle: 50, 325 and 500 mg/mL in corn oil
- Amount of vehicle (if gavage): 2 mL/kg
- Lot/batch no.: Q9367

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The dosing formulations were analysed for concentration and homogeneity by GC-FID. Trial formulations were prepared in corn oil at 51.4 mg/g (50 mg/mL) and 553 mg/g (500 mg/mL). Formulations were suspensions. The concentrations analyzed in the trial formulations were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). The formulations were homogeneous (i.e. coefficient of variation ≤ 10%). Formulations were stable when stored at room temperature under normal laboratory light conditions for at least 24 hours, in a refrigerator (2-8°C) for at least 8 days, and in a freezer (≤ -15°C) for at least 21 days (3 weeks).
Resuspension homogeneity was demonstrated (i.e. coefficient of variation ≤ 10%).

The dosing suspensions of VAMMAR D10 prepared in corn oil were analyzed following the first and last preparations and were found to be homogeneous under the conditions of this study. The first preparation of the dosing suspensions of 50, 325 and 1000 mg/mL were analyzed and
found to be 94%, 98% and 100% of the target concentrations, respectively. The homogeneity values obtained for the first preparation were 3.1% and 2.4% relative standard deviation (RSD) for the 50 and 500 mg/mL formulations, respectively. The last preparation of the dosing suspensions of 50, 325 and 500 mg/mL were analyzed and found to be 99%, 100% and 100% of the target concentrations, respectively.

Details on mating procedure:

Female rats were naturally bred at the supplier, by breeder male rats of the same source and strain, before shipment to the Testing Facility. The day of confirmed mating was designated gestation day 0.

Duration of treatment / exposure:

Suspensions of the study substance or the corn oil were administered via oral gavage once daily from gestation day 6 through 20.

Frequency of treatment:

once daily

Duration of test:

16 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

650 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

24/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Dose levels were selected by the Sponsor based on the results of the dose range-finding study (Charles River Study No. 20157649) which found no effects up to the limit dose of 1000 mg/kg/day in rats.

Examinations

Maternal examinations:

The animals were checked for viability at least twice daily during the study. The animals were observed for general appearance at least once during the acclimation period, daily before each dose was administered, and on the day of scheduled euthanasia. Postdose observations were recorded between 1 and 2 hours postdose. Body weights were recorded on gestation days 0 (supplier), 6, 9, 12, 15, 18, and 21. Food consumption was recorded on gestation days 6, 9, 12, 15, 18, and 21. All surviving female rats were sacrificed on gestation day 21 and blood samples were collected for hormone analysis of thyroxine T3, T4, and TSH. The thoracic, abdominal, and pelvic cavities were examined at gross necropsy. Individual thyroid weights were also recorded. Ovarian and uterine contents were examined and recorded. The following tissues were collected: esophagus, heart, kidney, liver, lung, spleen, stomach, thyroid, and trachea; however they were not examined. Thyroids were examined microscopically.

Ovaries and uterine content:

On gestation day 21, all surviving females were euthanized, gravid uterine weights were recorded, and uterine contents of each female were examined (i.e., number and distribution of corpora lutea, implantation sites, placentae [size, color, or shape], live and dead fetuses, and early and late resorptions).

Fetal examinations:

Fetal body weights and sex ratios, and fetal external, visceral, coronal and skeletal morphology were evaluated. All fetuses were examined for sex and external abnormalities. Late resorptions and dead fetuses were examined for external abnormalities and sex to the extent possible. The body weight of each fetus was recorded. Approximately one-half of the fetuses in each litter were examined for visceral abnormalities by using a modification of the microdissection technique of Staples. Each fetus was fixed in Bouin's solution and the heads were subsequently examined by free-hand sectioning; head sections were stored in alcohol. The decapitated carcasses were not retained. The remaining fetuses (approximately one-half of the fetuses in each litter) were examined for skeletal abnormalities after staining with alizarin red S.

Statistics:

Any data collected during the predose period was not tabulated, summarized or statistically analyzed. All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels, unless otherwise noted.

Maternal body weight, body weight gains, food consumption, hormone variables, and litter observations were analysed with parametric/non-parametric analysis. Levene’s test was used to assess the homogeneity of group variances. The groups were compared using an overall one-way ANOVA F test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal-Wallis test was found significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively.

Ovarian/uterine parameters and fetal malformations/variations were subjected to non-parametric analysis. The groups were compared using an overall Kruskal-Wallis test. If the overall Kruskal-Wallis test was found significant, then the above pairwise comparisons were conducted using Dunn’s test.

For parental indices and mortality, a Fisher’s exact test was used to conduct pairwise group comparisons of interest.

Indices:

Pre Implantation Loss (%) = (No. of corpora lutea – no. of implants)/(No. of corpora lutea) x 100

Post Implantation Loss (%) = (No. of implants – no. of live fetuses)/(No. of implants) x 100

Historical control data:

Historical control data were attached (see attached in the information panel) including results from 82 full studies and 50 dose range studies for maternal reproductive data, 21 studies for fetal abnormalities, 31 studies for fetal soft tissue abnormalities, 76 studies for fetal skeletal abnormalities, and 69 studies for fetal skeletal ossification sites. See attachment in information panel.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There was an increased incidence in the clinical sign: salivation in rats in the 650 mg/kg/day and 1000 mg/kg/day groups (10 and 17 animals, compared to 0 in controls), respectively. These increases were not considered to be adverse because they may have been related to palatability of the test substance and there were no remaining test substance-related clinical signs observed in these groups. There were no test substance-related clinical signs in the 100 mg/kg/day group.

The remaining clinical signs (i.e., abnormal breathing sounds, ungroomed fur, fur loss, thin fur cover, and a scab on the skin) were considered unrelated to administration of the test substance because the observations recorded were singular occurrences within a group, were present at incidences that lacked a dose-response relationship and/or were similar to those seen in vehicle-treated rats.

Mortality:

no mortality observed

Description (incidence):

There were no substance-related deaths in any group. Two females (1 female in the 100 mg/kg/day group and 1 in the 1000 mg/kg/day group) were euthanized and sacrificed early on gestation day 21 due to early delivery of their litters. There were no stillbirths. Therefore, the early deliveries were not considered test substance-related. All remaining animals survived until scheduled termination.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the 1000 mg/kg/day group, there was increased mean maternal body weight gain (+15 g) compared to controls from gestation day (GD) 6 to 21 (corrected for gravid uterine weight) that was statistically significant (p≤ 0.01). However, this was most likely due to a slight transient increase during the GD 6 to 9 interval (16.8 ± 5.5 g compared to control 11.5 ± 6.9 g ) that was statistically significant (p≤ 0.01). Therefore, mean maternal body weight gain was not considered to be adverse.

There were no changes in mean maternal body weight in the 100-mg/kg/day and 650-mg/kg/day groups. The remaining variations in mean maternal body weight and body weight gain were similar to those seen in vehicle-treated rats and were unrelated to treatment.

Summary of Gravid Uterine Weights and Corrected Body Weights - Gestation
Group 1 2 3 4
Dose (mg/kg/day) 0 100 650 1000
No. Animals per Group 24 23 24 23
Bodyweight on Day 6 (g) [G] Mean(SD) 244.5 (15.7) 242.9 (16.4) 2 40.3 (14.4) 244.0 (17.2)
Terminal Body Weight (g) [G] Mean(SD) 388.3( 36.1) 396.3 (30) 395.9 (24.9) 412.0 (32.2)
Gravid Uterus Weight (g) [G] Mean(SD) 96.97(19.2) 98.49 (13.15) 97.38 (13.22) 106.10 (8.96)
Corrected Bodyweight (g) [G] Mean(SD) 291.4 (22.4) 297.8 (21) 298.5 (18.9) 305.9 (26.6)
Corrected BWG (GD 6-21) (g) [G1] Mean (SD) 46.9 (17.9) 54.9 (10.4) 58.2 * (14) 61.9 **(15.8)



Bodyweight Gain (Interval)
Day(s) Relative to Mating (Litter: A)
6 → 9 [G] 9 → 12 [G1] 12 → 15 [G1] 15 → 18 [G1] 6 → 18 [G1] 18 → 21 [G1] 6 → 21 [G1]
0 Mean 11.5 19.3 19.4 40.2 90.4 53.4 143.8
mg/kg/ SD 6.9 7.0 7.5 9.7 18.6 13.6 30.0
day N 24 24 24 24 24 24 24
Group 1 - - - - - - -
100 Mean 15.1 22.5 19.5 41.8 99.0 55.3 154.3
mg/kg/ SD 3.8 6.4 5.9 7.3 15.6 6.8 19.9
day N 24 24 24 24 24 24 24
Group 2 - - - - - - -
650 Mean 13.5 23.3 21.5 42.5 100.8 54.8 155.6
mg/kg/ SD 3.3 4.8 5.7 6.7 13.0 10.9 21.3
day N 24 24 24 24 24 24 24
Group 3 - - - - - - -
1000 Mean 16.8** 21.4 21.9 45.7 105.8** 61.4 167.2**
mg/kg/ SD 5.5 6.5 11.1 10.5 13.4 11.0 20.7
day N 24 24 24 24 24 24 24

* Significantly different from the vehicle control group value (p≤0.05).
** Significantly different from the vehicle control group value (p≤0.01).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In the 1000 mg/kg/day group, there was a statistically significant increase (p≤0.05) in food consumption during the interval from gestation day (GD) 6 to 21 (24.8 g compared to 22.8 g in control). However, this increase in mean maternal food consumption was due to a transient but statistically significant increase during the intervals GD 12 to 15 (25.1 g compared to 22.9 g in control) and GD 18 to 21 (27.8 g compared to 24.2 g in control). Therefore, these transient increases were not considered to be adverse as they were minimal (<15%).

There were no test substance-related effects on mean maternal food consumption in the 100 mg/kg/day or 650 mg/kg/day groups. The remaining variations in food consumption were similar to those seen in vehicle-treated pregnant rats and were unrelated to treatment.

Summary of Food Consumption by Interval

Interval 6 → 9 [G] 9 → 12 [G1] 12 → 15 [G1] 15 → 18 [G1] 6 → 18 [G1] 18 → 21 [G1] 6 → 21 [G1]
Dose (mg/kg/day)

0 mg/kg/day Mean (SD) 19.1 (2.9) 21.6 (2) 22.9 (2.1) 26.3 (2.6) 22.5 (2.2 24.2 (2.4) 22.8 (2.2)
100 mg/kg/day Mean (SD) 20.7 (1.6) 22.3 (2.1) 23.6 (1.7) 26.9 (1.9) 23.4 (1.6) 25.0 (1.9) 23.7 (1.5)
650 mg/kg/day Mean (SD) 19.8 (1.5) 22.5 (1.4) 23.9 (2.0) 26.8 (1.5) 23.3 (1.4) 25.7 (2.0) 23.7 (1.4)
1000 mg/kg/day Mean (SD) 20.0 (1.4) 23.4 (2.3) 25.1* (1.9) 27.9 (2.1) 24.1 (1.6) 27.8** (1.8) 24.8* (1.6)

* Significantly different from the vehicle control group value (p≤0.05).
** Significantly different from the vehicle control group value (p≤0.01).

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:

effects observed, non-treatment-related

Description (incidence and severity):

In the 1000 mg/kg/day group, there was a slight decrease in mean T3 (210.6 ±74.2 pg/mL compared to control 428.5 ± 212.0 pg/mL) and T4 concentrations (17760.9 ± 5127.8 compared to 21869.5 ± 6787.5 in controls). There was a slight increase in Thyroid Stimulating Hormone (TSH) concentrations (8.8 ± 2.7 compared to 7.4 ± 3.6 in controls). These values were not statistically significant and when plotted individually, the GD 21 thyroid hormone levels (i.e., T4 and TSH) were withn or slightly outside (i.e., T3) the range of concurrent controls. Hence the mean values were skewed. Since there were no other adverse findings in the dams, the variation in thyroid hormone levels in the 100-, 650- and 1000-mg/kg/day groups were not considered adverse.

Table 1: Thyroid Endpoint Mean (SD Range)
Control 100 mg/kg/day 650 mg/kg/day 1000 mg/kg/day
TSH (ng/mL) 7.4 (3.1 - 21) 7.7 ( 5.5 - 13.6) 8.0 (4.7 - 18.7) 8.8 (4.7 – 13.1)
T3 (pg/mL) 428.5 (159 - 837) 327 (154-523) 252 (117-472 ) 210.6 (111 – 342a)
T4 (pg/mL) 21869 (10800 - 38100) 21548( 12400- 29300) 19902 (9450-33500) 17761 (11100 - 32600)
Thyroid Weight (g) 0.04 (0.01 – 0.06) 0.04 (0.03-0.05) 0.04 (0.02-0.06) 0.04 (0.28 – 0.06b)

a.Four animals were below concurrent control range.
b.Excludes 2 female weights (i.e., 0.08 g and 0.074 g) that skewed the mean. Mean thyroid weight with females included was 0.041 g and excluded was 0.038 g compared to control weight of 0.038 g.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no substance-related changes in thyroid weight in the 100-, 650-, or 1000-mg/kg/day dose groups.

Summary of Organ Weight Data - FemaleTerminal Euthanasia (GD 21)
Group 1 2 3 4
Dose (mg/kg/day) 0 100 650 1000
No. Animals per Group 24 23 24 23
Thyroid (No. Weighed) 23 22 23 23
Absolute value (g) 0.0377 0.0389 0.0404 0.0418
% of control weighta -- 103 107 111
% of body weighta -- 1.29 5.23 4.58
a All values expressed as percent difference from control group means.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross pathological findings in any group.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the 100-, 650-, and 1000-mg/kg/day groups, there were test substance-related histopathologic changes: hypertrophied thyroid follicular cells associated with thyroid follicular cells often exhibiting a dark basophilic cytoplasm and basophilic colloid secretion in the thyroid glands in 4, 22, 21 animals, respectively compared to 0 in control. However, the overall architecture of the thyroid glands was relatively well preserved; there was no evidence of hyperplasia. Since there were no other statistically significant and/or adverse findings associated with the thyroid (i.e., hormone, thyroid weight changes) in the dams, the variations seen in the thyroid glands in the 100-, 650- and 1000-mg/kg/day groups were considered to be a physiological adaptation and were not considered adverse. In addition, no historical control data is available to determine whether a change in thyroid hormone levels are considered a consequence to the animal.

Histopathological findings: neoplastic:

not examined

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

There were no abortions.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no substance-related effects on embryonic/fetal survival. The variations in embryonic/fetal survival assessed by the numbers of corpora lutea, implantations, and live fetuses per female and the derived peri- and postimplantation loss calculations were similar to those seen in vehicle-treated dams and were unrelated to treatment.

In the 0-, 100-, 650-, and 1000-mg/kg/day groups, Mean % Preimplantation loss was 8.23%, 7.82%, 7.97% and 5.9%, respectively and % Postimplantation loss was 4.17%, 3.83%, 2.61% and 4.19%, respectively.
In the 0-, 100-, 650-, and 1000-mg/kg/day groups, Mean number Preimplantation loss was 0.08,0.08, 0.08 and 0.06, respectively and mean number of Postimplantation loss was 0.04, 0.04, 0.03 and 0.04, respectively.
In the 100-, 650-, and 1000-mg/kg/day groups, % difference from control for Preimplantation loss was -5.01%, -3.14%, -29.5%, respectively and % difference for Postimplantation loss was -8.15%, -37.51% and 0.4% respectively.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There were no litters with total litter losses in any group.

Early or late resorptions:

no effects observed

Description (incidence and severity):

There were no substance-related effects on embryonic/fetal survival. The variations in embryonic/fetal survival assessed by the numbers of early or late resorptions were similar to those seen in vehicle-treated dams and were unrelated to treatment.

In the 0-, 100-, 650-, and 1000-mg/kg/day groups, the mean number of early resorptions 0.5, 0.4, 0.3, 0.5, respectively and late resorptions was 0, 0, 0, 0.1, respectively.

Dead fetuses:

no effects observed

Description (incidence and severity):

There were no dead fetuses in any group.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

There were no substance-related effects in pregnancy duration in any group. There were 2 females (i.e., (1 female in the 100 mg/kg/day group and 1 in the 1000 mg/kg/day group) that delivered early (prior to cesarean section) on gestation day 21 (GD 21). All remaining females survived until termination on GD 21 (i.e., date of scheduled cesarean section).

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

There was a 100% pregnancy in each dose group. In the 0-, 100-, 650-, and 1000-mg/kg/day groups, there were 24 pregnant females in each group. There were no nonpregnant females.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Basis for effect level:

other: there were no study substance-related adverse maternal findings at the highest dose tested

Results (fetuses)

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

At 650 mg/kg/day, there were statistically significant increases (p≤ 0.01) in the in the mean combined fetal body weights and the male and female fetal body weights. These increases were not considered to be test substance related because they were not dose dependent.

Summary of Fetal Body Weight Data
Group 1 2 3 4
Dose (mg/kg/day) 0 100 650 1000
Mean Fetal Weight (both) (g) [G] Mean (SD) 5.880 (0.4) 6.001 (0.3) 6.219** (0.4) 6.111 (0.3)
Mean Fetal Weight (M) (g) [G] Mean (SD) 6.005 (0.6) 6.195 (0.3) 6.421** (0.4) 6.276 (0.3)
Mean Fetal Weight (F) (g) [G] Mean (SD) 5.700 (0.4) 5.830 (0.3) 6.034** (0.4) 5.910 (0.3)

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There were no substance-related effects on mean number of live female/male offspring. The variations in live offspring were similar to those seen in vehicle-treated dams and were unrelated to treatment.

In the 0-, 100-, 650-, and 1000-mg/kg/day groups, the mean number of live female offspring was 6, 6.5, 6.1, 6.0, respectively and live male offspring was 6.4, 5.8, 5.6, 6.8, respectively.
In the 100-, 650-, and 1000-mg/kg/day groups, the % difference from control for live female offspring was 8%, 1%, and 0%, respectively and for live male offspring was -9%,-12%, and -6%, respectively.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There were no substance-related effects on fetal sex ratios. The variations in fetal sex ratios were similar to those seen in vehicle-treated dams and were unrelated to treatment.

In the 0-, 100-, 650-, and 1000-mg/kg/day groups, the mean number of live male fetus/litter % was 52%, 48%, 48%, 52%, respectively; and therefore, the mean number of live female fetus/litter % was 48%, 52%, 52%, 48%, respectively.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There were no substance-related effects on litter size or weights. The variations in litter size and weights were similar to those seen in vehicle-treated dams and were unrelated to treatment.

In the 0-, 100-, 650-, and 1000-mg/kg/day groups, the mean number of implantations was 12.9 12.7, 12.0, and 13.3, respectively and mean total fetal weight was 5.9 g, 6.0 g, 6.2 g, and 6.1 g, respectively.

Summary of Fetal Body Weight Data
Group 1 2 3 4
Dose (mg/kg/day) 0 100 650 1000
Mean Fetal Weight (both) (g) [G] Mean (SD) 5.880 (0.4) 6.001 (0.3) 6.219** (0.4) 6.111 (0.3)
Mean Fetal Weight (M) (g) [G] Mean (SD) 6.005 (0.6) 6.195 (0.3) 6.421** (0.4) 6.276 (0.3)
Mean Fetal Weight (F) (g) [G] Mean (SD) 5.700 (0.4) 5.830 (0.3) 6.034** (0.4) 5.910 (0.3)

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no substance-related fetal external malformations or variations observed. The external abnormalities observed (i.e., 1 fetus in the 100 mg/kg/day group had a small right eye, 1 fetus in the 650 mg/kg/day had discoloration in the left lens) were singular occurrences in a fetus and were unrelated to treatment.

Exam Type: Fixed Head Control 100 mg/kg/day 650 mg/kg/day 1000 mg/kg/day
Number of Fetuses Examined: 142 136 133 140
Number of Fetuses Evaluated: 297 282 280 294
Number of Litters Examined: 24 23 24 23
Number of Litters Evaluated: 24 23 24 23
Incidental
Number of Fetuses 0 0 1 0
Litter % of Fetuses [k] 0.00 0.00 0.69 0.00
Number of Litters 0 0 1 0
Variation
Number of Fetuses 0 0 1 0
Litter % of Fetuses [k] 0.00 0.00 1.04 0.00
Number of Litters 0 0 1 0
Malformation
Number of Fetuses 0 1 0 0
Litter % of Fetuses [k] 0.00 0.62 0.00 0.00
Number of Litters 0 1 0 0
All classifications
Number of Fetuses 0 1 2 0
Litter % of Fetuses [k] 0.00 0.62 1.74 0.00
Number of Litters 0 1 2 0

In the male fetuses at 650 mg/kg/day and 1000 mg/kg/day, there were statistically significant increases (p≤0.01) in anogenital distances as compared to the control group value. These increases were not considered to be adverse because they were not dose dependent and the increase at 650 mg/kg/day correlated with the increased fetal body weights at that dose. There was also a statistically significant increase (p≤0.01) in anogenital distance in the female fetuses at 1000 mg/kg/day as compared to the control group value. This increase was not considered to be adverse because it was minimal (8%).

Summary of Fetal Anogenital Distance Control 100 mg/kg/day 650 mg/kg/day 1000 mg/kg/day

ANOGENITAL DISTANCE (MILLIMETERS)
N 24 22 25 21
MALE PUPS MEAN (SD) 2.90 (0.25) 3.01 (0.23) 3.11 (0.21)** 3.09 (0.14)**
% Diff from control - 3.73 7.24 6.64
N 24 22 25 21
FEMALE PUPS MEAN (SD) 1.32 (0.12) 1.32 (0.16) 1.39 (0.12) 1.43 (0.09)**
% Diff from control - -0.53 5.31 7.70

* Significantly different from the vehicle control group value (p≤0.05).
** Significantly different from the vehicle control group value (p≤0.01).

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no substance-related fetal skeletal malformations or variations, or changes in mean number of sites of incomplete ossification. The skeletal abnormalities observed and the incidences of fetuses with sites of incomplete ossification were singular occurrences within a group, were present at incidences that lacked a dose-response relationship and were unrelated to test substance treatment.

Exam Type: Skeletal Control 100 mg/kg/day 650 mg/kg/day 1000 mg/kg/day
Number of Fetuses Examined: 155 146 147 154
Number of Fetuses Evaluated: 297 282 280 294
Number of Litters Examined: 24 23 24 23
Number of Litters Evaluated: 24 23 24 23
Variation
Number of Fetuses 16 24 17 21
Litter % of Fetuses [k] 9.66 15.84 11.26 13.28
Number of Litters 10 14 13 13
All classifications
Number of Fetuses 16 24 17 21
Litter % of Fetuses [k] 9.66 15.84 11.26 13.28
Number of Litters 10 14 13 13

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no substance-related fetal visceral or coronal malformations or variations observed. The visceral abnormality observed was in 1 fetus in the 100 mg/kg/day and 1000 mg/kg/day groups that had absent innominate arteries and the coronal abnormalities observed was in 1 fetus in the 650 mg/kg/day group that had both dilated lateral and third ventricles. These variations were not considered to be study substance-related because they occurred in a single fetus, were within historical control range (i.e., dilated ventricles and absent innominate arteries range: 0-1 fetus), and were not observed in a dose response manner.

Exam Type: FreshVisBody Control 100 mg/kg/day 650 mg/kg/day 1000 mg/kg/day
Number of Fetuses Examined: 142 136 133 140
Number of Fetuses Evaluated: 297 282 280 294
Number of Litters Examined: 24 23 24 23
Number of Litters Evaluated: 24 23 24 23
Variation
Number of Fetuses 0 1 0 1
Litter % of Fetuses [k] 0.00 0.62 0.00 0.72
Number of Litters 0 1 0 1
All classifications
Number of Fetuses 0 1 0 1
Litter % of Fetuses [k] 0.00 0.62 0.00 0.72
Number of Litters 0 1 0 1

Details on embryotoxic / teratogenic effects:

There were no developmental toxicity findings at any dose.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: there were no study substance-related adverse fetal findings at the highest dose tested

Overall developmental toxicity

Key result

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

The maternal and developmental NOAEL in rats was 1000 mg/kg/day, the highest dose tested, as no study substance-related adverse maternal or developmental effects were observed.

Executive summary:

The objectives of this study were to detect adverse effects of the VAMMAR on pregnant Sprague-Dawley rats and development of the embryo and fetus consequent to exposure of the female from implantation to closure of the hard palate. Pregnant females were administered doses of 0 (vehicle control), 100, 650, or 1000 mg/kg/day by oral gavage once daily from Gestation Day (GD) 6 through 20 (24 rats/group). The females were sacrificed on GD 21 and the following parameters and endpoints were evaluated: viability, maternal clinical signs, maternal body weight and body weight gain, maternal food consumption, maternal thyroid hormone evaluation, gross necropsy findings, maternal thyroid weight and histopathology, ovarian and uterine examination, fetal sex ratio, fetal body weight, fetal anogenital distance, and fetal abnormalities (external, visceral, coronal and skeletal).

There were no test substance-related mortalities, or gross necropsy findings in any dose group. Two females delivered their litters early and were euthanized on GD 21; there were no study substance-related adverse observations in these females.

There was an increased incidence in the clinical sign: salivation in rats in the 650 mg/kg/day and 1000 mg/kg/day groups (10 and 17, compared to 0 in controls) respectively. These increases were not considered to be adverse because they may have been related to palatability of VAMMAR and there were no remaining VAMMAR-related clinical signs observed. There were no VAMMAR-related clinical signs in the 100 mg/kg/day group.

In the 1000 mg/kg/day group, there was increased mean maternal body weight gain (+15 g) compared to controls from gestation day (GD) 6 to 21, which corresponded to a minimal increases in mean maternal food consumption from GD 12 to 15 and 18 to 21 (+2.2 g and +3.6 g, respectively compared to controls). These parameters were not considered to be adverse because they did not have a significant impact on the overall mean gestation body weight. There were no changes in mean maternal body weight in the 100-mg/kg/day and 650-mg/kg/day groups. The remaining variations in mean maternal body weight and body weight gain were similar to those seen in vehicle-treated rats and were unrelated to treatment.

In the 100-, 650-, and 1000-mg/kg/day groups, there were VAMMAR-related histopathologic changes in the maternal thyroid glands in 4, 22, 21 animals, respectively compared to 0 in control in hypertrophied thyroid follicular cells, but the overall architecture of the thyroid glands was relatively well preserved there was no evidence of hyperplasia. Although there was a slight increase in mean thyroid weight, it was not statistically significant in the 1000-mg/kg/day group, the mean was skewed by 2 females that fell outside of concurrent controls. Corresponding hormone levels for these 2 females were within the concurrent control range. Also, in the 1000-mg/kg/day group, thyroid hormone levels (i.e., TSH and T4) were withn or (T3) slightly outside the range of concurrent controls. Since there were no other adverse findings in the dams, the effects seen on the thyroid gland in the 100-, 650- and 1000-mg/kg/day groups were not considered adverse.

There were no VAMMAR-related changes in developmental toxicity endpoints of embryo/fetal viability, sex, body weight, or examination of fetal anogenital distance, external, visceral, coronal, skeletal or sites of incomplete ossification in the 100-, 650-, and 1000-mg/kg/day groups.  The fetal examination findings observed were similar to concurrent controls, were not observed in a dose-response manner and/or were within historical control of the testing laboratory.

In conclusion, female (Crl:CD[SD]) rats were administered VAMMAR at doses of 0 (corn oil), 100, 650, or 1000 mg/kg/day by oral gavage once daily on GD 6 through 20. There were no adverse VAMMAR-related changes. Therefore, the maternal and developmental no-observed-adverse-effect level forVAMMAR was 1000 mg/kg/day.