Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 Jun 2019 to 1 Jul 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
under GLP conditions
Justification for type of information:
Following an ECHA decision CCH-D-2114394631-45-01/F on EC:263-214-5 (3,7-dimethylnona-2,6-dienenitrile), it was requested to conduct additional toxicological studies:
- In vitro gene mutation study in mammalian cells, OECD 476;
- Screening for reproductive/developmental toxicity in rats, oral route, OECD 421,
- Sub-chronic toxicity study (90-day), oral route, in rats, OECD 408,
- Pre-natal developmental toxicity study, oral route, rats or rabbits, OECD 414,
- Identification of degradation products.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
guideline OECD from June 2018
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
United States Environmental Protection Agency (EPA) Health Effects Test Guidelines, August 1998
GLP compliance:
yes (incl. QA statement)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
impurity
Type:
impurity
Test material form:
liquid
Specific details on test material used for the study:
Lemonile (CAS No. 61792-11-8)
Purity >=98%
colorless to pale yellow liquid
Expiry date: 7 Feb. 2021

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD(SD)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc. (Raleigh, NC)
- Age at study initiation: A minimum of 70 days at the initiation of breeding
- Weight at study initiation: min 200g (201 and 250 g on Gestation Day 0)
- Housing: individually housed in clean solid-bottom cages with bedding material (Bed O’Cobs® or other suitable material)
- Diet (e.g. ad libitum): PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 meal offered ad libitum prior to dose initiation and is used to prepare the test and basal (control) diets throughout the study. Test and basal (control) diets will be offered ad libitum. Fresh test diet from the current batch in frozen storage provided daily.
- Water (e.g. ad libitum): treated by reverse osmosis and ultraviolet irradiation, is available ad libitum during the study
- Acclimation period: animals received on Gestation Day 1, 2, 3, or 4. Each rat is observed twice daily for changes in general appearance and behavior. Detailed clinical observations from receipt until Gestation Day 6, body weights on Gestation Day 5, and 6, and food consumption from Gestation Day 5-6 will be recorded for all rats (including extra rats obtained for the study). After receipt at the Testing Facility, the Crl:CD(SD) rats were acclimated prior to the initiation of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26°C
- Humidity (%): 30- 70%
- Air changes (per hr): The ventilation rate will be set at a minimum of 10 room air changes per hour, 100% fresh air.
- Photoperiod (hrs dark / hrs light): 12-h light/dark photoperiod

IN-LIFE DATES: From: 21 Jun 2019 to 18 Jul 2019
Experimental termination date: 01 Jul 2020

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF TEST SUBSTANCE:
Test substance dietary formulations were prepared at appropriate concentrations to meet dose level requirements. An appropriate amount of the test substance for each group was added to an appropriate amount of rodent feed on a weight/weight basis (no adjustment for purity), transferred into a Hobart mixer, and mixed to form a premix. The resulting premix was then mixed thoroughly with the remaining amount of feed in a Hobart mixer to achieve a total batch of homogeneous diet at the appropriate concentration/group. The dietary formulations were prepared every 3–6 days, divided into aliquots for daily dispensation, and stored frozen (target of -20°C) until use. Details of the preparation and dispensing of the test substance have been retained in the Study Records.

PREPARATION OF CONTROL DIET:
Basal Diet/Carrier: PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 (meal, on a weight/weight basis and prepared per SOP T2-011).
For administration to Group 1 control animals, an appropriate amount of PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 was weighed out every 3–6 days, placed in a labeled bag, divided into aliquots for daily dispensation, and stored frozen (target of -20°C) until use. Details of the dispensing of the control diet have been retained in the Study Records.

RATE OF PREPARATION:
Diet prepared: weekly. Formulated diets will be stored in a sealed container in a freezer set to maintain - 20°C prior to use (for a period not to exceed established stability).

EXPOSURE:
The control and test diets were offered continuously during Gestation Days 6 through 21
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed by gas chromatography with flame ionization detection using a validated analytical procedure.

Concentration Analysis
Duplicate sets of samples (50 g) for each sampling time point were transferred to the analytical laboratory; the remaining samples were retained at the Testing Facility as backup samples. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% of theoretical concentration. After acceptance of the analytical results, backup samples were discarded.

Homogeneity Analysis
Duplicate sets of samples (50 g) for the first sampling time point were transferred to the analytical laboratory; the remaining samples were retained at the Testing Facility as backup samples. Homogeneity results were considered acceptable if the relative standard deviation of the mean concentration was ≤ 10% and if mean sample concentration results were within or equal to ± 15% of theoretical concentration. After acceptance of the analytical results, backup samples were discarded.

Stability Analysis
Test substance formulations have been previously shown to be stable over the range of concentrations used on this study when stored in a sealed container for at least 10 days in a freezer at -20°C.1 Therefore, stability of test substance formulations were not assessed on this study.

Details on mating procedure:
On 28 Jun 2019, 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 approximately 11–12 weeks old at receipt and weighed between 201 and 250 g on gestation Day 0.
Duration of treatment / exposure:
The test item and control item were administered to the appropriate animals by inclusion in the diet ad libitum from Day 6 to Day 21 post-coitum, inclusive.
Frequency of treatment:
Test item was included in the diet provided daily ad libitum.
Duration of test:
16 days
Doses / concentrationsopen allclose all
Dose / conc.:
200 ppm
Remarks:
corresponds to 14 mg/kg bw
Dose / conc.:
400 ppm
Remarks:
corresponds to 27 mg/kg bw
Dose / conc.:
1 200 ppm
Remarks:
corresponds to 75 mg/kg bw
No. of animals per sex per dose:
25 females per group.
The Crl:CD(SD) rat is recognized as appropriate for developmental toxicity studies. Charles River Ashland has historical data on the background incidence of fetal malformations and developmental variations in the Crl:CD(SD) rat. This animal model has been proven to be susceptible to the effects of developmental toxicants.

The number of animals selected for this study was based on the US EPA Health Effects Test Guidelines OPPTS 870.3700, Prenatal Developmental Toxicity Study, August 1998 and the OECD Guidelines for the Testing of Chemicals: Guideline 414, Prenatal Developmental Toxicity Study, Jan 2001, which recommends evaluation of approximately 20 females with implantation sites at necropsy. Given the possibility of nongravid animals, unexpected deaths, or test substance-related moribundity and/or mortality, this was an appropriate number of animals to obtain a samples size of 20 at termination.
Control animals:
yes, plain diet
Details on study design:
Justification for Test System and Number of Animals
The Crl:CD(SD) rat is recognized as appropriate for developmental toxicity studies. Charles River Ashland has historical data on the background incidence of feta malformations and developmental variations in the Crl:CD(SD) rat. This animal model has been proven to be susceptible to the effects of developmental toxicants.

The number of animals selected for this study was based on the US EPA Health Effects Test Guidelines OPPTS 870.3700, Prenatal Developmental Toxicity Study,August 1998 and the OECD Guidelines for the Testing of Chemicals: Guideline 414, Prenatal Developmental Toxicity Study, Jan 2001, which recommends evaluation of approximately 20 females with implantation sites at necropsy. Given the possibility of nongravid animals, unexpected deaths, or test substance-related moribundity and/or mortality, this was an appropriate number of animals to obtain a samples size of 20 at termination.

- Dose selection rationale:
The route of administration was oral because oral ingestion is a potential route of exposure for humans. Historically, this route has been used extensively for studies of this nature.

Dietary concentration selection for this study was based on a previous range-finding rat embryo/fetal developmental toxicity study in which Lemonile was administered at dietary concentrations of 0, 100, 300, 500, and 1000 ppm in the diet. All animals survived until the scheduled necropsy on Gestation Day 21. The female rats that were fed 1000 ppm test diet showed decreased body weight gain and food consumption. Based on these results, the high-concentration level was expected to produce some maternal and fetal toxicity, but not excessive lethality that would prevent meaningful evaluation. The mid- and low-concentration levels were expected to produce graded toxic effects.

Dose Range-Finding Prenatal Developmental Toxicity Study of Lemonile in Rats (Study No. 01179011). Charles River, Ashland, OH, 2020:
Animals were administered the test substance continuously in the diet from Gestation Days 6 through 21at dose levels of 0, 100, 300, 500 and 1000 ppm of Lemonile (8 females/group).
The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, gravid uterine weights, food consumption, gross necropsy, intrauterine growth and survival, and external fetal morphology.

Average compound consumption during the treatment period (Gestation Days 6–21) was 7, 21, 34, and 68 mg/kg/day in the 100, 300, 500, and 1000 ppm groups, respectively. All females in the control, 100, 300, 500, and 1000 ppm groups survived to the scheduled necropsy on Gestation Day 21. There were no remarkable clinical observations noted at the daily examinations at any exposure level. Lower mean body weight gains during Gestation Days 6–15 (and consequently for the overall treatment period [Gestation Days 6-21]) and lower mean food consumption during Gestation Days 6–12 were noted in the 1000 ppm group compared to the control group. Thereafter, mean body weight gains and food consumption in this group were comparable to the control group, and mean absolute body weights were comparable to the control group throughout gestation. In the 500 ppm group, a lower mean body weight gain was noted during Gestation Days 6–9, and lower mean food consumption was noted during Gestation Days 6–12 compared to the control group; mean body weight gains and foodconsumption in this group were comparable to the control group thereafter. Mean maternal body weight gains and food consumption in the 100 and 300 ppm groups, and mean absolute body weights, corrected body weights, corrected body weight gains, and gravid uterine weights in the 100, 300, 500, and 1000 ppm groups were comparable to the control group.
No maternal macroscopic findings were noted for females in the test substance-treated groups at the scheduled necropsy.There were no test substance-reaated effects on intrauterine growth and survival or external fetal morphology in the 100, 300, 500, and 1000 ppm groups.

In conclusion, lower mean body weight gains and food consumption at 500 and 1000 ppm had no remarkable effect on mean absolute body weights. There were no effects on intrauterine growth and survival and fetal morphology at any exposure level. Therefore, exposure levels of 200, 400, and 1200 mg/kg/day were selected for a definitive prenatal developmental toxicity study of Lemonile administered orally via the diet to time-mated Crl:CD(SD) rats.

Examinations

Maternal examinations:
Viability
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, once in the morning and once in the afternoon (see Appendix 1 – Study Protocol and Deviations). Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.

Observations
The animals were removed from the cage, and a detailed clinical observation was performed once daily, beginning on the day of receipt and lasting through euthanasia (see Appendix 1 – Study Protocol and Deviations.During the dosing period, these observations were performed prior to dosing.

Body Weights
Animals were weighed individually on Gestation Days 0 (by supplier) and 5–21 (daily). Gravid uterine weight was collected and corrected body weight (the Gestation Day 21 body weight exclusive of the weight of the uterus and contents) and corrected body weight change (the Gestation Day 0–21 body weight change exclusive of the weight of the uterus and contents) were calculated and presented for each gravid female at the scheduled laparohysterectomy.

Food Consumption
Food consumption was quantitatively measured on Gestation Days 5–21 (daily).

Food Evaluation
The mean amounts of test substance consumed (mg/kg/day) per dose group were calculated from the mean food consumed (g/kg/day) and the appropriate target concentration of test substance in the food (mg/kg).

Thyroid Hormones
Sample Collection: Blood samples for thyroid hormone analysis were collected (prior to noon in order to avoid diurnal fluctuations in thyroid hormone levels) from the jugular vein into tubes without anticoagulant. Group 1 to 4 on Gestational day 21.
Blood samples were maintained at room temperature and allowed to clot. Serum was isolated in a refrigerated centrifuge and stored in a freezer set to maintain a target of -70°C. Blood samples were analyzed for Triiodothyronine (Total T3), Thyroxine (Total T4), Thyroid-stimulating hormone (TSH).

Animals surviving until scheduled euthanasia were weighed and euthanized by carbon dioxide inhalation.

Necropsy
Animals were subjected to a complete necropsy examination, which included evaluation of the thoracic, abdominal, and pelvic cavities with their associated organs and tissues.

Organ Weights
The thyroid gland (following fixation) and liver were weighed at the scheduled necropsy for all females.

Tissue Collection and Preservation
The thyroid gland from all animals was collected and preserved in 10% neutral buffered formalin for subsequent histopathologic examinations. In addition, 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.

Histology
Tissue trimming was performed at the Testing Facility. The thyroid gland from all females in the control and high-concentration groups was embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin.

Histopathology
Pathological evaluation was performed by a board-certified veterinary pathologist. The thyroid gland was examined microscopically from all females in the control and high-concentration group.
Ovaries and uterine content:
The uterus was weighed, and the ovaries and uterus were examined for number and distribution of corpora lutea, implantation sites, live and dead fetuses, and early and late resorptions. The placentae were also examined. Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss.

Intrauterine data were summarized using 2 methods of calculation as indicated below.

Group Mean Litter Basis:
Postimplantation Loss/Litter = (No. Dead Fetuses, Resorptions (Early/Late)/Group)/ No. Gravid Females/Group

Proportional Litter Basis:
Summation Per Group (%) = (Sum of Postimplantation Loss/Litter (%))/ No. Litters/Group

Where:
Postimplantation Loss/Litter (%) = (No. Dead Fetuses, Resorptions (Early/Late)/Litter)/ No. Implantation Sites/Litter x 100
Fetal examinations:
Fetal Examinations:
Fetal examinations were conducted without knowledge of treatment group. External, internal, and skeletal fetal findings were recorded as either developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or occur at high incidence, representing slight deviations from normal), malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life), or incidental (minor changes in coloration, mechanical damage to specimen, etc.). Representative photographs of all malformations, as appropriate, were included in the Study Records. Corresponding low magnification photographs, depicting both the malformed fetus and a comparison control fetus, or normal littermate, were also included in the Study Records as needed and as appropriate for comparison, when possible.

The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison and calculating the number of affected fetuses in a litter on a proportional basis as follows:

Summation per Group (%) = Sum of Viable Fetuses Affected/Litter (%) No. Litters/Group

Where:
Viable Fetuses Affected/Litter (%) = No. Viable Fetuses Affected/Litter No. Viable Fetuses/Litter x 100

External:
Each viable fetus was examined in detail, sexed, weighed, tagged (see Appendix 1 – Study Protocol and Deviations), and euthanized by a subcutaneous injection of sodium pentobarbital in the scapular region. The anogenital distance of all viable fetuses was measured. Anogenital distance was defined as the distance from the caudal margin of the anus to the caudal margin of the genital tubercle.

Visceral (Internal):
The sex of all fetuses was confirmed by internal examination. Approximately one-half of the fetuses in each litter were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe.6 This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development.7 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:
The remaining fetuses (approximately one-half from each litter, excluding any carcasses without heads) were eviscerated and fixed in 100% ethyl alcohol. Following fixation in alcohol, fetuses were stained with Alizarin Red S and Alcian Blue.1 The skeletal examination was made following this procedure.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and are reported at the 1% and 5% levels. Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated by sex and occasion or by litter. Descriptive statistics number, mean, standard deviation, percentage, and/or incidence were reported whenever possible. Calculated values on the Provantis tables may not be reproducible from the individual values presented because all calculations are conducted using non-rounded values. Inferential statistics were performed according to the matrix below when possible (see Appendix 1 – Study Protocol and Deviations), but excluded semi quantitative data, and any group with less than observations. Data obtained from nongravid animals were excluded from statistical analysis.

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 daily examinations at any exposure level. Observations noted in the test substance-exposed groups, including red fur staining and a thin fur cover on various body surfaces, occurred infrequently and/or in a manner that was not exposure-related. With the exception of Female No. 4525 in the 1200 ppm group, all females were gravid.
Description (incidence and severity):
Not a dermal study.
Mortality:
no mortality observed
Description (incidence):
All females in the control, 200, 400, and 1200 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 1200 ppm group, lower mean body weight gains or mean body weight losses were generally noted throughout the study; differences were statistically significant on Gestation Day 6–7 and when the Gestation Days 6–9, 12–15, and 6–21 cumulative intervals were evaluated compared to the control group. As a result, mean absolute body weights and the mean corrected body weight change in the 1200 ppm group were statistically significantly lower (5.1% to 9.0% for mean absolute body weights and 50.6% for corrected body weight change) than the control group during Gestation Days 7–21. In addition, a statistically significantly lower mean corrected body weight gain was noted at 1200 ppm group resulting in a statistically significantly lower mean corrected body weight (9.4%) in this group compared to the control group. Furthermore, the mean gravid uterine weight in the 1200 ppm group was 8.10% lower than the control group; the difference was not statistically significant. The effects on body weight, body weight gain, corrected body weight, corrected body weight gain, and gravid uterine weight in the 1200 ppm group were considered test substance-related and adverse.

A statistically significantly lower mean body weight gain was noted in the 400 ppm group when the Gestation Day 6–9 cumulative interval was evaluated, and statistically significantly lower mean body weight gains were noted in the 200 and 400 ppm groups when the entire treatment period (Gestation Days 6–21) was evaluated compared to the control group. However, these differences had no effect on absolute mean body weights and were therefore considered test substance-related but not adverse. Mean corrected body weights, corrected body weight gains, and gravid uterine weights in the 200 and 400 ppm groups were unaffected by test substance administration. Differences from the control group were slight and not statistically significant.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In the 1200 ppm group, mean food consumption, evaluated as g/animal/day, was statistically significantly lower generally throughout the exposure period and when the Gestation Days 6–9, 9–12, 12–15, 15–21, and 6–21 cumulative intervals were evaluated compared to the control group. The lower mean food consumption in this group corresponded to the lower mean absolute body weights and were considered test substance-related and adverse.

Mean maternal food consumption in the 200 and 400 ppm groups were lower than the control values generally during the exposure period; values were generally statistically significant. However, these changes were not of sufficient magnitude to affect mean absolute body weights or body weight gains in these groups, and were therefore considered test substance-related but not considered adverse.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
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 test substance-related alterations in thyroid or liver weight.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Review of the gross necropsy observations revealed no observations that were considered to be associated with administration of the test substance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test substance-related histologic changes. Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test substance. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Thyroid Hormones
There were no test substance-related effects on thyroid hormone values at any exposure level. Differences from the control group were not statistically significant and were considered to be the result of normal biological variation.

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:
not examined
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Effects on intrauterine growth at 1200 ppm were considered test substance-related and advers. Intrauterine survival in the 1200 ppm groups was unaffected by test substance administration.
Details on maternal toxic effects:
Ovarian and Uterine Examinations
In the 1200 ppm group, mean male (5.53 g), female (5.19 g), and combined (5.35 g) fetal weights were statistically significantly lower than the concurrent control group (5.89, 5.59, and 5.74 g, respectively) and the minimum mean value in the Charles River Ashland historical control data (5.955, 5.653, and 5.817 g, respectively; version 2019.01). The effects on intrauterine growth at 1200 ppm were considered test substance-related and adverse. Intrauterine survival in the 1200 ppm groups was unaffected by test substance administration.

Intrauterine growth and survival were unaffected by test substance administration at exposure levels of 200 and 400 ppm. Parameters evaluated included mean litter proportions of postimplantation loss, mean number of live fetuses, mean fetal body weights, and fetal sex rations. Differences from the control group were slight and not statistically significant, with the following exception. In the 400 ppm group, statistically significantly lower mean male fetal weight was noted compared to the concurrent control group; however, based on the absence of other effects on female fetal weight and intrauterine survival at 400 ppm, this difference was not considered test substance-related and was attributed to biological variation.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Remarks:
Maternal Toxicity, Embryo/fetal development
Effect level:
400 ppm
Based on:
test mat.
Remarks:
equivalent to 27 mg/kg/day
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the 1200 ppm group, mean male (5.53 g), female (5.19 g), and combined (5.35 g) fetal weights were statistically significantly lower than the concurrent control group (5.89, 5.59, and 5.74 g, respectively) and the minimum mean value in the Charles River Ashland historical control data (5.955, 5.653, and 5.817 g, respectively; version 2019.01).
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Intrauterine survival was unaffected by test substance administration.
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
In the 1200 ppm group, mean male (5.53 g), female (5.19 g), and combined (5.35 g) fetal weights were statistically significantly lower than the concurrent control group (5.89, 5.59, and 5.74 g, respectively) and the minimum mean value in the Charles River Ashland historical control data (5.955, 5.653, and 5.817 g, respectively; version 2019.01).
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
Fetal Morphological Data
The numbers of fetuses (litters) available for morphological evaluation were 332(25), 314(25), 315(25), and 309(24) in the control, 200, 400, and 1200 ppm, respectively. Malformations were observed in 1(1), 0(0), 2(2), and 4(3) fetuses (litters) in these same respective exposure levels and were considered spontaneous in origin.

No test substance-related external malformations were noted for fetuses in this study. In the 1200 ppm group, Fetus No. 4513-10 was noted with a thread-like tail (5 mm in length; skeletally consisting of absent caudal vertebrae) and Fetus No. 4509-03 was noted with a small lower jaw (skeletally consisting of small and fused mandibles). These malformations were not considered test substance-related because they were noted in single fetuses, the mean litter proportions were not statistically significant compared to the concurrent control group, and/or were within the range of values for equivalent malformations in the Charles River Ashland historical control data (version 2019.01). No other external malformations were noted for fetuses in this study. No external developmental variations were observed in fetuses in this study.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related skeletal malformations were noted for fetuses in this study. In addition to the skeletal malformations discussed in Section 8.12.1., skeletal malformations of absent lumbar vertebrae for 3 fetuses (Nos. 4513-10, 4514-03, and 4514-07) and an absent sacral
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related visceral malformations were noted for fetuses in this study. Visceral malformations were limited to fused right cranial lobes of the lung for Fetus No. 3522-03 in the 400 ppm group and was not considered test substance-related because it was noted in a single fetus and was not noted in an exposure-related manner. No test substance-related visceral developmental variations were noted. Findings observed in the test substance-exposed groups were noted infrequently, similarly in the control group, were not observed in an exposure-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 range of values for equivalent developmental variations in the Charles River Ashland historical control data.vertebra for Fetus No. 4513-10 in the 1200 ppm group and an absent thoracic vertebra and supernumerary cervical vertebrae for Fetus No. 3503-12 in the 400 ppm group were noted. These malformations were not considered test substance-related because they were noted infrequently or in single litters, were not observed in an exposure-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 range of values for equivalent malformations in the Charles River Ashland historical control data. In the control group, Fetus No. 1518-13 was noted with fused cervical centra. No other skeletal malformations were noted for fetuses in this study.

No test substance-related skeletal developmental variations were noted. Findings observed in the test substance-exposed groups were noted infrequently, similarly in the control group, were not observed in an exposure-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.
Other effects:
no effects observed
Description (incidence and severity):
Anogenital Distance
The anogenital distance (absolute and relative to the cube root of fetal body weight) in the 200, 400, and 1200 ppm groups were similar to the control group values. Differences from the control group were slight and not statistically significant.
Details on embryotoxic / teratogenic effects:
The numbers of fetuses (litters) available for morphological evaluation were 332(25), 314(25), 315(25), and 309(24) in the control, 200, 400, and 1200 ppm, respectively. Malformations were observed in 1(1), 0(0), 2(2), and 4(3) fetuses (litters) in these same respective exposure levels and were considered spontaneous in origin. When the total malformations and developmental variations were evaluated on a proportional basis, no statistically significant differences from the control group were noted. Fetal malformations and developmental variations, when observed in the test substance-exposed groups, occurred infrequently or at a frequency similar to that in the control group, did not occur in an exposure-related manner, and/or were within the Charles River Ashland historical control data ranges. Based on these data, no fetal malformations or developmental variations were attributed to the test substance.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Remarks:
Embryo/Fetal
Effect level:
400 ppm
Based on:
test mat.
Remarks:
equivalent to 27mg/kg/d
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes

Overall developmental toxicity

Developmental effects observed:
yes
Lowest effective dose / conc.:
200 ppm
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
no
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
Based on adverse effects on maternal body weights and food consumption, and intrauterine growth at 1200 ppm, a dosage level of 400 ppm, equivalent to 27 mg/kg/day, was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity and embryo/fetal development when Lemonile was administered orally (via the diet) to time mated Crl:CD(SD) rats.
Executive summary:

The objective of this study was to determine the potential of the test substance to induce developmental toxicity after maternal exposure from implantation to 1 day prior to expected parturition, to characterize maternal toxicity at the exposure levels tested, and to determine a no-observed-adverse-effect level (NOAEL) for maternal and developmental toxicity.

Animals were administered the test substance continuously in the diet from Gestation Day 6 through 21. The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, gravid uterine weights, food consumption, thyroid hormones, gross necropsy findings, thyroid weights and histopathology, intrauterine growth and survival, anogenital distance, and fetal morphology.

Average test substance consumption during the treatment period (Gestation Days 6–21) was 14, 27, and 75 mg/kg/day in the 200, 400, and 1200 ppm groups, respectively.

All females in the control, 200, 400, and 1200 ppm groups survived to the scheduled necropsy. There were no test substance-related clinical observations at any exposure level during the study.

In the 1200 ppm group, lower mean body weight gains or mean body weight losses were generally noted throughout the study; differences were statistically significant when the Gestation Days 6–9 and 12–15 cumulative intervals and when the entire study (Gestation Days 6–21) were evaluated compared to the control group. Corresponding statistically significantly lower mean food consumption was noted in the 1200 ppm group generally throughout the study. As a result, mean absolute body weights at 1200 ppm were statistically significantly lower (5.1% to 9.0%) than the control group during Gestation Days 7–21. In addition, statistically significantly lower mean corrected body weight and corrected body weight gain (50.6% lower compared to control value), and lower (not statistically significant) gravid uterine weight were noted in the 1200 ppm group compared to the control group. The effects on maternal body weight and food consumption in the 1200 ppm group were considered test substance-related and adverse. In the 200 and 400 ppm groups, lower mean food consumption was generally noted compared to the control group generally throughout the treatment period; differences were generally statistically significant. As a result, a statistically significantly lower mean body weight gain during the Gestation Day 6–9 cumulative interval was noted in the 400 ppm group and statistically significantly lower mean body weight gains were noted in the 200 and 400 ppm groups when the entire treatment period (Gestation Days 6–21) were evaluatedcompared to the control group. The effects on body weight gain and food consumption were considered test substance-related but nonadverse because mean absolute body weights in these groups were unaffected. In the 1200 ppm group, mean male, female, and combined fetal weights were statistically significantly lower compared to the concurrent control group and lower than the minimum mean value in the Charles River Ashland developmental historical control data. The effects on fetal weight at 1200 ppm were considered test substance-related and adverse. Intrauterine growth at 200 and 400 ppm were unaffected by test substance administration.

There were no test substance macroscopic findings, effects on thyroid gland or liver weight, microscopic findings in the thyroid gland, or effects on thyroid hormone concentration (T3, T4, or TSH) at any exposure level.

Based on adverse effects on maternal body weights and food consumption, and intrauterine growth at 1200 ppm, a dosage level of 400 ppm, equivalent to 27 mg/kg/day, was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity and embryo/fetal development when Lemonile was administered orally (via the diet) to time mated Crl:CD(SD) rats.