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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22.May2020-23.Nov2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2020
Reference Type:
publication
Title:
Characterization of xenobiotic-induced hepatocellular enzyme induction in rats: Anticipated thyroid effects and unique pituitary gland findings.
Author:
Zabka TS, Fielden MR, Garrido R, et al.
Year:
2011
Bibliographic source:
Toxicol Pathol. 2011; 39:664-677.

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Isoundecan-1-ol
EC Number:
257-376-6
EC Name:
Isoundecan-1-ol
Cas Number:
51750-47-1
Molecular formula:
C11H24O
IUPAC Name:
3,5 dimethyl nonanol-1

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
On 29 May 2020 and 02 Jun 2020, time-mated female Crl:CD(SD) rats were received from Charles River Laboratories, Inc., Raleigh, NC on Gestation Day 1, 2, 3, or 4. The animals were 11–13 weeks old and weighed between 231 and 296 g at the initiation of dosing on Gestation Day 6. After receipt at the Testing Facility, the Crl:CD(SD) rats were acclimated prior to the initiation of dosing. Animals were assigned to groups by a stratified randomization scheme designed to achieve
similar group mean body weights. Animals in poor health, at extremes of body weight range, and/or reduced food consumption were not assigned to groups. Animals were individually housed in solid-bottom cages containing appropriate bedding material. Targeted conditions for the animal room environment were set to the following: temperature to 68-78F (20-26C), humiditiy at 30-70%m and a 12h light/dark cycle. Food (PMI Nutrition International, LLC Certified Rodent LabDiet® 5002) was provided ad libitum, as was municipal tap water (treated by reverse osmosis and ultraviolet irradiation). Periodic analyses of the certified feed and of the water supply to ensure that there was not a presence of contaminants that could interfere with the outcome of the study.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Female rats were administered the control substance or test substance formulations via gavage once daily on gestation days (GD) 6 through 20.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analyzed dosing formulations contained 101% to 107% of the test substance which was within the protocol-specified range of target concentrations for suspensions (85% to 115%) and were homogeneous. The test substance was not detected in the analyzed vehicle formulation that was administered to the control group (Group 1).
Details on mating procedure:
Dams were supplied as naïve, purpose-bred, time-mated (Day mating is confirmed [copulatory plug] = Gestation Day [GD] 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 by oral gavage
Duration of test:
16 days
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
Doses were informed by a dose range-finding study conducted prior to this study (Charles River Laboratories, Study ID 438045, 2020).

Examinations

Maternal examinations:
The animals were examined at least twice daily for mortality, beginning upon arrival through termination. Detailed clinical observations occurred once daily, beginning with the day of animal arrival and continuing through the day of euthanasia. Clinical observations occurred approximately 1-2h post dose, wherein animals were observed for findings that were potentially related to treatment or that might change before the next scheduled observation.

Individual body weights were collected on all animals on gestation days 5-21 (daily). Food consumption measurements were made for all animals on gestation days 5-21 (daily) and were reported as g/animal/day for each corresponding body weight interval during gestation.

Thyroid hormone assessments: Blood samples were taken from the jugular vein on GD 21. Serum was separated and stored in a freezer set to maintain a target of -70°C. Collected prior to noon on each day of collection, around the same time each day, and within a 2 hour window on each collection day, to reduce variability due to normal diurnal variations in physiological levels of thyroid hormones. Blood collections were performed in an animal ante room, the necropsy laboratory, or as far away from live animals as possible to minimize stress-induced hormone fluctuations. Three hormones were measured: Triiodothyronine (T3), Thyroxine (T4), and Thyroid Stimulating Hormone (TSH). T3 and T4 concentrations were conducted using a validated UHPLC/MS/MS assay, and TSH was measured using a validated Luminex Bead-based assay.

Ovaries and uterine content: On GD 21, laparohysterectomies and macroscopic examinations were performed blind to treatment group. All surviving females were euthanized and subjected to a gross necropsy. The thoracic, abdominal, and pelvic cavities were opened and the contents examined. The uterus of each dam was excised and its adnexa trimmed. Corpora lutea were also counted and recorded. Gravid uterine weights were obtained and recorded. The uterus of each dam was opened and the number of viable and nonviable fetuses, early and late resorptions, and total number of implantation sites were recorded, and the placentae were examined. The individual uterine distribution were 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.

Macroscopic and histopathologic examination: For all animals, the liver and thyroid gland were excised, weighed (thyroid postfixation), and preserved in 10% neutral buffered formalin. Gross lesions were collected and preserved in 10% neutral-buffered formalin for possible future histopathologic examination. Representative sections of corresponding organs from a sufficient number of controls were retained for comparison, if possible. Thyroids for all animals in the controls and all dose groups were subjected to microscopic examination.


Ovaries and uterine content:
On GD 21, laparohysterectomies and macroscopic examinations were performed blind to treatment group. All surviving females were euthanized and subjected to a gross necropsy. The thoracic, abdominal, and pelvic cavities were opened and the contents examined. The uterus of each dam was excised and its adnexa trimmed. Corpora lutea were also counted and recorded. Gravid uterine weights were obtained and recorded. The uterus of each dam was opened and the number of viable and nonviable fetuses, early and late resorptions, and total number of implantation sites were recorded, and the placentae were examined. The individual uterine distribution were 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.
Fetal examinations:
Fetal examinations were conducted without knowledge of treatment group. External, internal, and skeletal fetal findings were recorded as either developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or occur at high incidence, representing slight deviations from normal), malformations (those structural anomalies that alter general body conformity, disrupt or interfere
with normal body function, or may be incompatible with life), or incidental (minor changes in coloration, mechanical damage to specimen, etc.).

External examinations: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 relative values (to the cube root of fetal body weight) were reported. The crown-rump length of late resorptions (advanced degree of autolysis) was measured, the degree of autolysis recorded, a gross external examination performed (if possible), and the tissue was discarded.

Visceral (internal) examinations:The sex of all fetuses was confirmed by internal examination. Approximately one-half of the fetuses in each litter were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development. 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. Following examination, the carcasses and cephalic slices were discarded.

Skeletal examinations: Each eviscerated fetus, following fixation in alcohol, was macerated in potassium hydroxide and stained with Alizarin Red S and Alcian Blue. The skeletal examination was made following this procedure.
Statistics:
Means, standard deviations, percentages, numbers, and/or incidences were reported, as appropriate by dataset. Calculated values on the tables may not be reproducible from the individual values presented because all calculations are conducted using non-rounded values. Data obtained from nongravid animals were excluded from statistical analysis. 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. Pairwise comparisons included analysis of each dosed group against the control. Analyses were performed according to the matrix below when possible but excluded any group with less than 3 observations.

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

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Two females in the 1000 mg/kg/day group were euthanized early due to maternal toxicity exhibited as body weight loss, low/no food consumption, and clinical observations (i.e. reduced activity).

One female was euthanized on Gestation Day 8 following a body weight loss (12 g) and low food consumption (3 g/day) during Gestation Days 6–8. Decreased activity was noted for this female prior to sacrifice on Gestation Day 8 at 2 hours following dose administration; no gross findings were noted at necropsy.

The other female was euthanized on Gestation Day 9 following a body weight loss (40 g) and no food consumption (0 g/day) during Gestation Days 6–9. Decreased activity was also noted for this female prior to death during Gestation Days 8–9 at the daily examinations and 2 hours following dose administration. Gross findings including multifocal dark red and pale white foci in the glandular and nonglandular regions of the stomach were noted. Due to maternal toxicity, this moribundity was considered test substance-related and adverse.

In addition, another female in the 1000 mg/kg/day group was found dead on Gestation Day 18. Gross findings included a perforated esophagus and
accumulation of material in the thoracic cavity, consistent with a dosing (i.e. gavage) accident, which was considered to be the cause of death and not test substance-related.

All other animals survived to the terminal euthanasia.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two females in the 1000 mg/kg/day group were euthanized early due to maternal toxicity exhibited as body weight loss, low/no food consumption, and clinical observations (i.e. reduced activity).

One female was euthanized on Gestation Day 8 following a body weight loss (12 g) and low food consumption (3 g/day) during Gestation Days 6–8. Decreased activity was noted for this female prior to sacrifice on Gestation Day 8 at 2 hours following dose administration; no gross findings were noted at necropsy.

The other female was euthanized on Gestation Day 9 following a body weight loss (40 g) and no food consumption (0 g/day) during Gestation Days 6–9. Decreased activity was also noted for this female prior to death during Gestation Days 8–9 at the daily examinations and 2 hours following dose administration. Gross findings including multifocal dark red and pale white foci in the glandular and nonglandular regions of the stomach were noted. Due to maternal toxicity, this moribundity was considered test substance-related and adverse.

In addition, another female in the 1000 mg/kg/day group was found dead on Gestation Day 18. Gross findings included a perforated esophagus and accumulation of material in the thoracic cavity, consistent with a dosing (i.e. gavage) accident, which was considered to be the cause of death and not test substance-related.

All other animals survived to the terminal euthanasia.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related, statistically significantly lower mean body weight gains and mean body weight losses were noted in the 500 and 1000 mg/kg/day groups relative to the control group following the initiation of dosing during Gestation Days 6–9. Mean body weight gains in these groups were generally similar to the control group during Gestation Days 9–12. Slightly lower mean body weight gains in the 500 mg/kg/day group and lower mean body weight gains in the
1000 mg/kg/day group were generally noted throughout the remainder of the dosing period relative to the control group (occasionally statistically significant at 1000 mg/kg/day). When the entire treatment period (Gestation Days 6-21) was evaluated, mean body weight gains in the 500 and 1000 mg/kg/day groups were statistically significantly lower than the control group, resulting in mean body weights that were 4.4% and 10.1% lower, respectively, than the control group on Gestation Day 21. Due to the magnitude of difference from the control group, the test substance-related effects on body weight were considered adverse at 1000 mg/kg/day but not at 500 mg/kg/day. Mean corrected body weight gain and mean corrected body weight in the 1000 mg/kg/day group were statistically significantly lower than the control group. Test substance-related lower mean gravid uterine weights were noted in the 500 and 1000 mg/kg/day groups relative to the control group; the differences were statistically significant. The lower gravid uterine weights in these groups corresponded to the lower mean fetal weights noted in these groups (see Section 8.9.) as well as lower mean maternal body weight gain at the end of gestation. Mean corrected body weight gain and mean corrected body weight in the 500 mg/kg/day group were comparable to the control group.

Mean maternal body weights, body weight gains, corrected body weights, corrected body weight gains, and gravid uterine weights in the 100 mg/kg/day groups were unaffected by test substance administration. Differences from the control group were slight and not statistically significant.

Ctrl 100mg/kg/d 500mg/kg/d 1000mg/kg/d
N 24 25 25 22
Terminal Body Weight (g)
Mean 385.2 390.1 368.2 346.4 **
SD 23.1 31.1 27.1 36.1
%Diff - 1.3 -4.4 -10.1

Gravid Uterus Weight (g)
Mean 97.70 95.36 82.08** 78.69**
SD 12.71 15.22 20.30 18.94
%Diff - -2.40 -15.99 -19.46

Corrected Bodyweight (g)
Mean 287.5 294.7 286.2 267.7 **
SD 17.0 22.6 18.9 29.3
%Diff - 2.5 -0.5 -6.9

Corrected BWG (0-TBW) (g)
Mean 63.2 70.7 62.4 42.7 **
SD 11.7 15.9 15.3 23.3
%Diff - 11.8 -1.3 -32.5

** Statistically significantly different from control group (ANOVA and Dunnett) (p ≤ 0.01)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Mean maternal food consumption, evaluated as g/animal/day, in the 500 and 1000 mg/kg/day groups was statistically significantly lower relative to the control group during Gestation Days 6-9. Mean food consumption in these groups was generally slightly lower (500 mg/kg/day) or lower (1000 mg/kg/day) throughout the remainder of the dosing period relative to the control group (occasionally statistically significant at 1000 mg/kg/day). When the entire treatment periodduring Gestation Days 6–21 was evaluated, mean food consumption was statistically significantly lower than the control group in the 1000 mg/kg/day group and slightly (not statistically significant) lower than the control group in the 500 mg/kg/day group. The lower mean food consumption noted at 500 and 1000 mg/kg/day corresponded to the lower mean body weight gains and body weight losses.

Mean maternal food consumption in the 100 mg/kg/day groups was unaffected by test substance administration. Differences from the control group were slight and not statistically significant.

See attached tables and below section on "Any other information on results incl. tables".
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, treatment-related
Description (incidence and severity):
Test substance related effects on thyroid hormone levels were noted in the 1000 mg/kg/day group. Mean maternal TSH level in the 1000 mg/kg/day group was 71.3% higher than the control group; the difference was statistically significant. Mean T3 and T4 levels in the 1000 mg/kg/day group were 34.3% and 29.2% lower, respectively, compared to the control group; the differences in T3 were statistically significant.

Mean TSH level in the 500 mg/kg/day group was comparable to the control group. Mean T3 level in this group was 29.7% lower than the control group; the difference was statistically significant. Mean T4 level in this group was comparable to the control group. Due to the absence of effects on TSH and T4 levels, the significantly lower T3 levels in this group were not considered test substance related.

These changes in hormone thyroid levels were noted in association with non-adverse microscopic findings of minimal to mild thyroid follicular cell hypertrophy/hyperplasia and considered likely to be adaptive changes consistent with the induction of hepatic microsomal enzymes and where associated increases in thyroxin metabolism and TSH and decreases in T3 and/or T4 can be seen. As such, these changes in thyroid hormone levels were considered nonadverse.

No test substance-related effects on mean TSH, T3, or T4 levels were noted at 100 mg/kg/day. Differences from the control group were not statistically significant.

Serum analysis on GD 21 (mean, %ctrl):
T3 (pg/mL): ctrl 259.9; 100mg/kg/d 219.6 (-15.5%); 500mg/kg/d 182.6** (-29.7%); 1000mg/kg/d 170.7** (-34.3%)
Total T4 (pg/mL): ctrl 8941.0; 100mg/kg/d 8620 (-3.6%); 500mg/kg/d 9917.0 (10.9%); 1000mg/kg/d 6330.0 (-29.2%)
TSH (pg/mL): ctrl 1802.0; 100mg/kg/d 1603.0 (-11.0%); 500mg/kg/d 2056.0 (14.1%); 1000mg/kg/d 3086.0** (71.3%)
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):
There was a dose-dependent, test substance-related effect of higher mean absolute liver weights noted in the 100, 500, and 1000 mg/kg/day group animals. These findings were considered adaptive and nonadverse, as they are consistent with the induction of hepatic microsomal enzymes (see attachment in Section 13.2 with supporting microsomal data from the subchronic study on this test substance) and where associated increases in thyroxin metabolism and TSH and decreases in T3 and/or T4 can be seen. Absolute liver weight changes were as follows (mean in g, (% ctrl)): ctrl 13.87; 100mg/kg/d 15.5* (9.28%); 500mg/kg/d 16.37** (17.81); 1000mg/kg/d 17.58** (26.75). Asterisks denote statistical significance from control.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related gross findings were noted. The gross findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to administration of the test substance.

In one female sacrificed early due to maternal toxicity in the 1000mg/kg/d group, gross findings included multifocal dark and pale white foci in the glandular and non-glandular regions of the stomach. There were no gross findings in the other female in the 1000mg/kg/d group that was sacrificed early due to maternal toxicity.

In the female found dead on Day 18 (in the 1000mg/kg/d dose group), gross findings included a perforation in the esophagus and material accumulation in the thoracic cavity, which is consistent with a dosing accident.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
There was a dose-dependent test substance-related effect of microscopic findings of minimal to mild hypertrophy/hyperplasia (follicular cell) in the thyroid glands of the 100, 500, and 1000 mg/kg/day group animals. These findings were considered adaptive and nonadverse as a response to liver enzyme induction upon test substance administration, resulting in decreased T3 and T4 and increased TSH (See Section 13.2 attachment: human relevance document).

The observed increases in liver weight and resultant changes in thyroid hormones in this study correspond with data in a subchronic study on the test substance (see Repeat Dose: Oral section of dossier for robust study summary and data tables), in which these same changes were observed, as well as induction of liver enzymes (quantified in microsomal analyses as a separate study, see Toxicokinetic section of dossier for robust study summary and data tables).

See attached data tables and in "Any other information on results including tables" section of dossier.
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
No abortions were observed in this study.
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. Preimplantation losses in the control group were outside of the historical control range; Postimplantation losses across all groups fell within the historical control range for Charles River Ashland (see below).

In the 0-, 100-, 500-, and 1000-mg/kg/day groups, Mean % Preimplantation loss was 15.75%, 15.76%, 19.28% and 18.04%, respectively and % Postimplantation loss was 3.16%, 8.49%, 7.07% and 7.95%, respectively.

In the 100-, 500-, and 1000-mg/kg/day groups, % difference from control for Preimplantation loss was 0.08%, 22.43%, 14.55%, respectively and % difference for Postimplantation loss was 169.0%, 124.03% and 151.78% respectively.

Historical control range for mean % Preimplantation loss: 2.12-9.22%
Historical control range for mean % Postimplantation loss: 2.61-12.60%
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There were no total litter losses by resorption observed in this study.
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-, 500-, and 1000-mg/kg/day groups, the mean number of early resorptions 0.4, 1.1, 0.7, 1.0, respectively and late resorptions was 0, 0, 0, 0, respectively.
Dead fetuses:
no effects observed
Description (incidence and severity):
No dead fetuses were observed in this study.
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 (1000 mg/kg/day group) that were sacrificed early (prior to cesarean section) on gestation day 21 (GD 21), and one dam that was found dead on GD18 (esophageal perforation due to dosing accident).

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-, 500-, and 1000-mg/kg/day groups, there were 24, 25, 25, and 25 pregnant females in each group, respectively. There were no nonpregnant females.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
> 500 mg/kg bw/day (actual dose received)
Basis for effect level:
body weight and weight gain
food efficiency

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related lower mean fetal weights were noted at 500 and 1000 mg/kg/day compared to the control group (statistically significant at 1000mg/kg/d). The lower mean fetal weights at 500 mg/kg/day were not considered adverse due to the slight magnitude of difference from the concurrent control group and the lack of concurrent effects on fetal morphology.

See attached tables and section on "Any other findings on results incl. tables" below.
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.

Number of live fetuses per litter mean (%ctrl):

Control: 12.5
100mg/kg/d: 12.3 (-2.1%)
500mg/kg/d: 11.1 (-11.7%)
1000mg/kg/d: 11.9 (-5.4%)

Means were not statistically different from control group. There were no dead fetuses observed in dams that were sacrificed on GD 21.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on the ratios of males/females in this study; there were no statistically significant differences in live males or females in any dose group relative to the control group.

Number of live male fetuses (mean, %ctrl)
0mg/kg/d (6.0)
100mg/kg/d (6.4, 8.1%)
500mg/kg/d (5.3, -11.4%)
1000mg/kg/d (6.4, 6.8%)

Number of live female fetuses (mean, %ctrl)
0mg/kg/d (6.6)
100mg/kg/d (5.8, -11.3%)
500mg/kg/d (5.8, -11.9%)
1000mg/kg/d (5.5, -16.5%)
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
There were no substance-related effects on litter size. The variations in litter size were similar to those seen in vehicle-treated dams and were unrelated to treatment. In the 0-, 100-, 500-, and 1000-mg/kg/day groups, the mean number of implantations was 13.0, 13.4, 11.8, and 12.8, respectively.

Mean total fetal weights were lower in the 500mg/kg/d and 1000mg/kg/d dose groups, but were only statistically significant at 500mg/kg/d for females (statistically significant for both sexes combined, males, and females at 1000mg/kg/d) compared to controls. The lower mean fetal weights at 500 mg/kg/day were not considered adverse due to the slight magnitude of difference from the concurrent control group and the lack of concurrent effects on fetal morphology.

See attached tables and section on "Any other findings on results incl. tables" below.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related external developmental malformations or variations were observed in fetuses in this study.

One fetus in the 100 mg/kg/day group had malformations of short tail, absent anus, and short trunk (malformation). This finding was not considered test substance-related because it occurred only in a single fetus in the low-dose group. One fetus in the control group had localized subcutis edema (variation).

Exam Type: Fixed Head Control 100 mg/kg/day 500 mg/kg/day 1000 mg/kg/day
Number of Fetuses Examined: 301 307 277 261
Number of Fetuses Evaluated: 301 307 277 275
Number of Litters Examined: 24 25 25 22
Number of Litters Evaluated: 24 25 25 23

Variation
Number of Fetuses 1 0 0 0
Litter % of Fetuses [k] 0.28 0.00 0.00 0.00
Number of Litters 1 0 0 0
Malformation
Number of Fetuses 0 1 0 0
Litter % of Fetuses [k] 0.00 0.29 0.00 0.00
Number of Litters 0 1 0 0
All classifications
Number of Fetuses 1 1 0 0
Litter % of Fetuses [k] 0.28 0.29 0.00 0.00
Number of Litters 0 1 0 0


There were no test substance-related changes to mean anogenital distance in either sex at any dose level.

Summary of Fetal Anogenital Distance Control 100 mg/kg/day 500 mg/kg/day 1000 mg/kg/day

ANOGENITAL DISTANCE (MILLIMETERS)
N 24 25 24 22
MALE PUPS MEAN (SD) 3.05 (0.13) 3.08 (0.11) 3.11 (0.09) 3.03 (0.20)
% Diff from control - 1.00 2.07 -0.69
N 24 25 24 22
FEMALE PUPS MEAN (SD) 1.34 (0.05) 1.35 (0.06) 1.36 (0.05) 1.34 (0.07)
% Diff from control - 1.03 1.43 0.20


Differences noted in AGD relative to cube root of body weight were considered secondary to test substance-related significantly lower body weights. AGD relative to cube root of body weight was statistically significantly longer in male and female fetuses at 500mg/kg/d (p≤0.05) and 1000mg/kg/d (p≤0.01). These changes are not considered to be adverse due to the small magnitude of change from the control group.

Summary of Fetal AGD Relative to Cube Root of Body Weight
Control 100 mg/kg/day 500 mg/kg/day 1000 mg/kg/day

Adjusted AGD/Wt Ratio
N 24 25 25 21
MALE PUPS MEAN (SD) 1.69 (0.07) 1.70 (0.08) 1.75* (0.07) 1.77** (0.10)
% Diff from control - 0.74 3.59 4.69
N 24 25 25 21
FEMALE PUPS MEAN (SD) 0.74 (0.05) 0.77 (0.05) 0.78* (0.05) 0.80** (0.05)
% Diff from control - 4.14 5.22 7.83

* 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):
Malformations:
No test substance-related skeletal developmental malformations were noted. One fetus in the 100 mg/kg/day group had absent ribs, thoracic, lumbar, sacral, and caudal vertebrae, and fused thoracic arch and centra (the same fetus that exhibited an external malformation). These findings were not considered test substance-related because they occurred only in a single fetus in the low-dose group. One fetus in the control group had an absent cervical arch and fused cervical centra noted. No other skeletal malformations were noted in the study.

Variations:
A test substance-related higher mean litter proportion of short thoracolumbar supernumerary rib was noted in the 1000 mg/kg/day group (see incidence below); the difference from the control group was statistically significant. This finding was not considered adverse because it was not expected to impact survival. Other findings observed in the test substance-treated groups were not observed in a dose-related manner, occurred similarly in the control group, 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.

Exam Type: Skeletal Control 100 mg/kg/day 500 mg/kg/day 1000 mg/kg/day
Number of Fetuses Examined: 151 156 139 132
Number of Fetuses Evaluated: 301 307 277 275
Number of Litters Examined: 24 25 25 22
Number of Litters Evaluated: 24 25 25 23
Incidental
Number of Fetuses 2 0 3 4
Litter % of Fetuses [k] 1.39 0.00 3.24 3.07
Number of Litters 2 0 3 4
Variation
Number of Fetuses 19 20 17 43
Litter % of Fetuses [k] 12.46 12.71 12.47 34.15**
Number of Litters 11 13 11 19
Malformations
Number of Fetuses 1 1 0 0
Litter % of Fetuses [k] 0.69 0.50 0.00 0.00
Number of Litters 1 1 0 0
All classifications
Number of Fetuses 21 20 20 46
Litter % of Fetuses [k] 13.84 12.71 15.70 36.57*
Number of Litters 11 13 13 20

* Significantly different from the vehicle control group value (p≤0.05).
** Significantly different from the vehicle control group value (p≤0.01).
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Malformations:
No test substance-related visceral developmental malformations were noted. One fetus in the 500 mg/kg/day group had retroesophageal subclavian artery noted. One fetus in the control and 100 mg/kg/day groups, respectively, had situs inversus noted; the fetus from the control group also had fused lung lobe. The findings observed in the test substance-treated groups were noted infrequently, similarly in the control group, were not observed in a doserelated
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 and were therefore not attributed to the test substance. No other visceral malformations were noted.

Variations:
A higher mean litter proportion of dilatation of the ureters was noted in the 1000 mg/kg/day group compared to the control group; the difference was not statistically significant. This finding was considered test substance-related but not adverse because it was not expected to impact survival. Other findings observed in the test substance-treated groups occurred infrequently, similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the Charles River Ashland historical control data.

Exam Type: FreshVisBody Control 100 mg/kg/day 500 mg/kg/day 1000 mg/kg/day
Number of Fetuses Examined: 150 151 138 129
Number of Fetuses Evaluated: 301 307 277 275
Number of Litters Examined: 24 25 25 22
Number of Litters Evaluated: 24 25 25 23
Variation
Number of Fetuses 6 2 5 21
Litter % of Fetuses [k] 3.97 1.67 3.33 15.09
Number of Litters 4 2 5 8
Malformation
Number of Fetuses 1 1 1 0
Litter % of Fetuses [k] 0.60 0.67 0.67 0.00
Number of Litters 1 1 1 0
All classifications
Number of Fetuses 7 3 5 21
Litter % of Fetuses [k] 4.56 2.33 3.33 15.09
Number of Litters 5 3 5 8
Details on embryotoxic / teratogenic effects:
The numbers of fetuses (litters) available for morphological evaluation were 301(24), 307(25), 277(25), and 275(23) in the control, 100, 500, and 1000 mg/kg/day groups, respectively.

There were no test substance-related external, visceral, or skeletal malformations or external developmental variations noted at any dose level. Higher mean litter proportions of visceral and skeletal developmental variations (dilatation of the ureters and short thoracolumbar supernumerary rib, respectively) in the 1000 mg/kg/day group were statistically significant compared to the concurrent control group. These findings were considered test substance-related but not adverse because they were not considered adverse as they are not expected to impact survival and/or resolve postnatally.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
> 500 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Remarks on result:
other:
Remarks:
Reduced fetal weight occurred in the presence of a weight of evidence of maternal toxicity

Overall developmental toxicity

Developmental effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes

Any other information on results incl. tables

Maternal Outcomes:

Mean daily food consumption (g/animal/day) GD 6 -21

 Dose group (mg/kg/d)  0  100  500  1000
 Mean  15.10  15.75  14.14  12.58*
 SD  1.08  1.78  1.78  3.32
 %Diff from ctrl  -  4.25  -6.36  -16.70

* Statistically significantly different from control group (ANOVA & Dunnett) (p ≤ 0.05)

 Dose (mg/kg/d)  0  100  500  1000

Terminal Body Weight (g)

       
 Mean

385.2

 390.1  368.2  346.4**
 SD

23.1

 31.1  27.1  36.1
 %Diff from ctrl

 -

 1.3  -4.4  -10.1

Gravid Uterus Weight (g)

       
 Mean

97.70

 95.36  82.08**  78.69**
 SD

12.71

 15.22  20.30  18.94
 %Diff from ctrl

 -

 -2.40  -15.99  -19.46

Corrected Bodyweight (g)

       

Mean 

287.5

 294.7  286.2  267.7**
 SD

17.0

 22.6  18.9  29.3
 %Diff from ctrl

-

 2.5  -0.5  -6.9

Corrected BWG (0-TBW) (g)

       
 Mean

63.2

 70.7  62.4  42.7**
 SD

11.7

 15.9  15.3  23.3
 %Diff from ctrl

 -

 11.8  -1.3  -32.5

** Statistically significantly different from control group (ANOVA and Dunnett) (p ≤ 0.01)

Histopathological Findings (dams):

Dose (mg/kg/day)

 0  100  500  1000

No. Animals per Group

 25  25  25  22

Thyroid gland (No. Examined)

 (25)  (25)  (25)  (22)

Hypertrophy/hyperplasia; follicular cell

 0  4  6*  12**
 Minimal  -  2  4  9**
 Mild  -  2  2  3

- = No noteworthy findings.

* = Statistically significantly different from the control group at ≤ 0.05 using Fisher’s Exact test.

** = Statistically significantly different from the control group at ≤ 0.01 using Fisher’s Exact test.

Summary of Fetal Body Weight Data:

 Dose (mg/kg/d)  0  100  500  1000

Mean Fetal Weight (both) (g) [G] Mean (%ctrl)  

5.856

5.798 (-1.0)

5.560 (-5.1)

4.91** (-16.15)

Mean Fetal Weight (male) (g) [G] Mean (%ctrl)  

6.003

5.906 (-1.6)

5.672 (-5.5)

5.01** (-16.59)

Mean Fetal Weight (female) (g) [G] Mean (%ctrl)  

5.716

5.670 (-.81)  

5.346* (-6.4)

4.78** (-16.36)

Historical Control Mean in g (Range)

Sexes Combined: 5.986 (5.761-6.203)

Male: 6.140 (5.881-6.378)

Female: 5.827 (5.610-6.034)

Applicant's summary and conclusion

Conclusions:
Based on test substance-related maternal toxicity in the form of morbidity, mean body weight losses, lower mean body weight gains, and lower mean food consumption at 1000 mg/kg/day, a dose level of 500 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity when the test substance (Isoundecanol) was administered orally by gavage to time-mated Crl:CD(SD) rats.

Based on lower mean fetal body weights at 1000 mg/kg/day, a dose level of 500 mg/kg/day was considered to be the NOAEL for developmental toxicity. However, the the lower mean fetal body weights at 1000mg/kg/d were observed in the presence of maternal toxicity in the form of decreased food consumption, lower mean body weight gains, and mean body weight losses.
Executive summary:

The objectives of this study were to detect adverse effects of the test substance 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, 500, or 1000 mg/kg/day by oral gavage once daily from Gestation Day (GD) 6 through 20 (25 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, and skeletal).

No test substance-related gross findings were noted at necropsy.

Two females in the 1000 mg/kg/day group were euthanized early due to maternal toxicity on Gestation Days 8 and 9 following body weight losses, low food consumption, and clinical observations of decreased activity at the daily and/or 2-hour postdose examinations. Additionally, 1 of these 2 females was noted with dark red and pale white foci in the stomach at necropsy. Due to the clinical observations, body weight loss, and low food consumption, this moribundity was considered test substance-related and adverse. An additional female in this group was found dead on Gestation Day 18 and was noted at necropsy with a perforation of the esophagus; therefore, the cause of death for this female was considered a dosing error, unrelated to test substance administration. All other animals survived to scheduled necropsy.

Test substance-related clinical observations of wet fur around the mouth were noted in the 500 and 1000 mg/kg/day groups at the 2 hour post-dose examinations in a dose responsive manner; this transient finding did not persist into the daily examinations and was therefore not considered adverse. There were no test substance-related clinical observations noted in the 100 mg/kg/day group.

Test substance-related lower mean body weight gains and mean body weight losses with corresponding lower food consumption were noted in the 500 and 1000 mg/kg/day groups relative to the control group during Gestation Days 6–8 and 6–9, respectively; differences were statistically significant. Slightly lower mean body weight gains in the 500 mg/kg/day group and lower mean body weight gains in the 1000 mg/kg/day group with corresponding lower food consumption were generally noted from Gestation Day 12 -21 relative to the control group. When the entire treatment period (Gestation Days 6-21) was evaluated, mean body weight gains in both the 500 and 1000 mg/kg/day groups were significantly lower than the control group, resulting in mean body weights that were 4.4% and 10.1% lower, respectively than the control group on Gestation Day 21 (significant at 1000 mg/kg/day). Due to the magnitude of difference from the control group, the test substance-related body weight and food consumption effects were considered adverse only at 1000 mg/kg/day. Test substance-related (statistically significant) lower mean gravid uterine weights were noted in the 500 and 1000 mg/kg/day groups relative to the control group, corresponding to the lower mean fetal body weights noted in these groups. Mean corrected body weight gain and mean corrected body weight in the 1000 mg/kg/day group were also lower than the control group; differences were statistically significant. Mean corrected body weight and corrected body weight gain in the 500 mg/kg/day group were similar to the control group; therefore, the lower maternal body weight gains noted during the treatment period were primarily attributed to the lower gravid uterine weights noted in this group. Mean maternal body weights, body weight gains, corrected body weights, corrected body weight gains, gravid uterine weights, and maternal mean food consumption in the 100 mg/kg/day group were unaffected by test substance administration.

Test substance related significantly higher TSH levels (71.3%) and corresponding significantly lower T3 (34.3%) and T4 (29.2%) levels were noted in the 1000 mg/kg/day group compared to the control group. These changes were noted in association with microscopic findings in the thyroid of minimal to mild follicular cell hypertrophy/hyperplasia in the thyroid gland that were considered likely to be secondary, adaptive changes consistent with the induction of hepatic microsomal enzymes and where associated increases in thyroxin metabolism and TSH and decreases in T3 and/or T4 can be seen (Zabka et al., 2011) and as such, non-adverse. Further supporting this is the dose-dependent test substance-related effect of higher mean absolute liver weights noted in the 100, 500, and 1000 mg/kg/day group animals (9.28%, 17.81%, and 26.75% higher, respectively, than the control group. Higher liver weights in response to the test substance correlates with significantly increased microsomal enzyme concentrations in liver samples from the subchronic study on the test substance (Charles River Laboratories, 2020, Study ID 438038). See also the documentation on human relevance (Section 13.2) in an attachment showing supporting evidence that rats exhibit sensitivity to the induction of liver enzymes in response to xenobiotic exposure that leads to a secondary, overactive thyroid response; this sensitivity is not seen in humans.

Mean T3 level in the 500 mg/kg/day group was 29.7% lower than the control group and was not considered test substance related due to a lack of corresponding effects on TSH and T4 levels. Mean TSH and T4 levels in the 500 mg/kg/day group were comparable to the control group. No test substance-related effects on thyroid hormone levels were noted at 100 mg/kg/day.

In the 500 and 1000 mg/kg/day groups, mean fetal body weights were up to 6.48% and 16.59% lower, respectively, than the control group. The test substance-related effects on mean fetal weights at 1000 mg/kg/day were considered adverse because of the magnitude of the difference from the concurrent control group. However, the significantly lower mean fetal body weights at 1000mg/kg/d were observed in the presence of maternal toxicity in the form of decreased food consumption, lower mean body weight gains, and mean body weight losses.

Due to the small magnitude of the difference from the concurrent control group and the lack of test substance-related effects on fetal morphology and survival, the test substance-related effects on mean fetal weights at 500 mg/kg/day were considered nonadverse. Intrauterine growth and survival were unaffected by test substance administration at the dose level of 100 mg/kg/day; differences from the control group were slight and not statistically significant.

There were no test substance-related effects on mean anogenital distance at any dose level. Differences noted in anogenital distance relative to cubed root of body weight were considered secondary to test substance-related lower body weights.

No test substance-related external, visceral, or skeletal developmental malformations were observed in fetuses in this study. Higher mean litter proportions of visceral and skeletal developmental variations (dilatation of the ureters and short thoracolumbar supernumerary rib, respectively) were noted in the 1000 mg/kg/day group compared to the concurrent control group. These findings were considered test substance-related but not adverse as these variations are not expected to impact survival. Rib variations have often been associated with maternal toxicity or stress. Therefore, the developmental significance of rib variations in the high dose fetuses probably relates to the transient maternal toxicity observed in the high dose females during the treatment phase of this study. Thus, this variation was not considered to be an adverse effect on the fetuses. 

Based on test substance-related maternal toxicity in the form of morbidity, mean body weight losses, lower mean body weight gains, and lower mean food consumption at 1000 mg/kg/day, a dose level of 500 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity when the test substance (Isoundecanol) was administered orally by gavage to time-mated Crl:CD(SD) rats.

Based on lower mean fetal body weights at 1000 mg/kg/day, a dose level of 500 mg/kg/day was considered to be the NOAEL for developmental toxicity. However, the significantly lower mean fetal body weights at 1000mg/kg/d were observed in the presence of maternal toxicity in the form of decreased food consumption, lower mean body weight gains, and mean body weight losses. The plausibly-linked undernutrition of the dams as a result of decreased food consumption were correlated with the observed fetal body weight decreases. Maternal body weight changes are likely also due to liver hypertrophy in the dams, as a dose-dependent, statistically significant increase in liver weight was observed in this study, and correlates with observations at this dose in the 90 -day study on the test substance (in males and females, liver weights were significantly increased with a concurrent observation of hepatocellular hypertrophy; Charles River Laboratories, Study ID 00438038, 2020), and resultant significant liver enzyme induction (see Toxicokinetics section of this dossier for quantification; microsomal enzyme induction study on liver samples from the 90 -day study). These are adaptive changes in liver in response to the high oral doses observed in non-pregnant females and adult males (in the 90 -day study) as well as increased liver weights in dams in this study. It is plausible that this, in turn, is affecting food consumption and liver metabolism such that it compromises systemic nutrition of the rat fetuses. Thus, the decreased fetal weights at high doses are due to a non-specific secondary mechanism (maternal stress and the disruption of homeostasis), which describes the maternal toxicity observed (Regulation (EC) No 1272/2008 of the European Parliament and of the Council on classification, labelling and packaging of substances and mixtures, updated 6.07.2019). Supporting this conclusion is the lack of malformations observed in fetuses at any dose, including 1000mg/kg/d. Additionally, it should be noted that maternal effects observed were not due to a mean effect, nor were the decreased fetal weights. Individual dams with lower terminal body weight at 1000mg/kg/d correlated with lower litter weights (R2 = 0.5835; see attachment in "attached background material" section of the dossier for analysis against control dams and litters (R2 = 0.0028)).

In addition, no test substance-related findings in reproductive organs or function in adult males and females in the 90 -day study on the test substance were observed, including weight changes and/or histopathology to testes, epididymides, seminal vesicles, uterus, ovaries, vagina, or spermatogenesis parameters (Charles River Laboratories, Study ID 00438038, 2020).

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