Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

In a OECD 421 Oral (Dietary) Reproduction/Developmental Toxicity Screening Study of Lemonile in Rats, animals were administered the test substance continuously in the diet. Males were exposed for 14 days prior to mating and continuing through the day of euthanasia. Females were exposed for 14 days prior to mating and continuing through Lactation Day 13.  

Lemonile had no effects on F0 reproductive performance (mating, fertility, copulation, and/or conception indices), as well as mean estrous cycle lengths and precoital intervals, when tested at 200, 400 and 1000 ppm. No test substance-related effects were noted on mean gestation length or parturition at any exposure level.

There were no test substance-related effects on the mean number of pups born, pup survival, live litter size, mean sex ratio, anogenital distance, areolae/nipple anlagen (males), T4 concentration on PND 13, or thyroid gland weight at any exposure level. There were no clinical observations or gross necropsy findings that could be attributed to F0 maternal exposure of the test substance at any concentration. The 1000 ppm concentration level corresponded to actual consumption of 56 mg/kg/day for F0 males during the premating period and 55, 69, and 152 mg/kg/day for F0 females during the premating, gestation, and lactation periods, respectively.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 Aug 2019 to 25 Jun 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.
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
guideline OECD from 29 Jul 2016
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Identification: Lemonile (CAS No. 61792-11-8)
Physical Description: Clear, colorless liquid
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD(SD)
Details on species / strain selection:
The Crl:CD(SD) rat was chosen as the animal model for this study as it is an accepted rodent species for preclinical toxicity testing by regulatory agencies.

At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
On 20 Aug 2019 and 03 Sep 2019, female and male Crl:CD(SD) rats, respectively, were received from Charles River Laboratories, Inc., Raleigh, NC. The animals were approximately 10 weeks old and weighed between 181 and 393 g at the initiation of exposure.

Justification for Test System and Number of Animals
The Crl:CD(SD) rat is recognized as appropriate for reproduction studies. Charles River Ashlandhas reproductive historical control data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants. The number of animals selected for his study was based on the OECD Guideline for the Testing of Chemicals: Guideline 421, Reproduction/Development Toxicity Screening Test, 29 Jul 2016, which recommends that evaluation of each group be initiated with at least 10 males and 12–13 females per group. Females were evaluated for estrous cyclicity during the pretest period and any females that failed to exhibit normal 4–5 day estrous cycling (e.g., EDDDE) during the pretest period were excluded from the study, therefore, the extra females were included to yield at least 10 females per group. Given the possibility of nongravid animals, unexpected deaths, total litter losses, or test substance-related moribundity and/or mortality, this was an appropriate number of animals to obtain a sample size of 8 at termination.

- Age at study initiation (P generation): males/females were 70 days old.
- Weight at study initiation: (P generation) Males: between 181 and 393 g at the initiation of exposure.
- Housing: On arrival, animals were group housed (up to 2 animals of the same sex) until cohabitation. During cohabitation, animals were paired for mating in he home cage of the male. Following the breeding period, animals were individually housed. Animals were housed in solid-bottom cages containing appropriate bedding equipped with an automatic watering valve throughout the study. Each cage was clearly labeled with a color-coded cage card indicating study, group, animal, cage number(s), exposure level, and sex. Cages were arranged on the racks in group order. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). The animal facilities at Charles River Ashland are accredited by AAALAC International.
- Food (e.g. ad libitum): PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 (meal) was provided ad libitum to all animals prior to the initiation of dosing and to control group animals throughout the exposure period. The feed was analyzed by the supplier for nutritional components and environmental
contaminants. Results of the analysis are provided by the supplier and are on file at the Testing Facility. It is considered that there are no known contaminants in the feed that would interfere with the objectives of the study.
- Water (e.g. ad libitum): Municipal tap water after treatment by reverse osmosis and ultraviolet irradiation was freely available to each animal via an automatic watering system. Water bottles were provided, if required. Periodic analysis of the water is performed, and results of these analyses are on file at the Testing Facility. It is considered that there are no known contaminants in the water that could interfere with the outcome of the study.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23(+/-3)°C
- Humidity (%): 50 (+/-20)%
- Air changes (per hr): min 10 times/h
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

IN-LIFE DATES: From19 Aug 2019 to 12 Nov 2019
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Remarks:
PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 meal.
Details on exposure:
ORAL DIETARY STUDY
The route of administration was oral (dietary), because oral ingestion is a potential route of exposure in humans. Historically, this route has been used extensively for studies of this nature.

The control and test diets were offered continuously throughout the study. The test substance was administered as a constant concentration (ppm) in the diet. Males were exposed for 14 days prior to mating, throughout mating, and continuing until euthanasia. Females were exposed for 14 days prior to mating and continuing through Lactation Day 13. Females with no evidence of mating were exposed through euthanasia.

The F1 animals were not directly exposed to the test substance at any time during the study; the offspring of the F0 parental generation were potentially exposed to the test substance in utero and while nursing.

Dose Formulation and Analysis
Preparation of Control Diet: For administration to Group 1 control animals, an appropriate amount of PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 was weighed out 2 or 3 times weekly, 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.

Preparation of Test Substance: Test substance dietary formulations were prepared based on Sponsor instructions at appropriate concentrations to meet exposure level requirements. An appropriate amount of the test substance for each group was added to an appropriate amount of rodent feed on a weight/weiht basis (Batch No. PE00231703 was used during Study Days 0–24 and a correction factor of 15% was used during Study Days 4 to 24; Batch No. PE00251069 was used starting on Study Day 25 until the end of the study with no correction factor applied), 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 2 to 3 times weekly, 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.
Details on mating procedure:
Males were exposed for 14 days prior to mating, throughout mating, and continuing until euthanasia. Females were exposed for 14 days prior to mating and continuing through Lactation Day 13. Females with no evidence of mating were exposed through euthanasia.
After a minimum of 14 days of exposure, the animals were paired on a 1:1 basis within each group. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm in a vaginal lavage. Vaginal lavages were performed daily during the mating period until evidence of mating was observed. If evidence of mating was not apparent after 14 days, the animals were separated, with no further opportunity for mating. Animals cohabited over a 12-hour dark cycle were considered to have been paired for 1 day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected for analysis as indicated in Table 1 (see below).

Analytical Method:
Analyses were performed by a gas chromatography method using flame ion detection using a validated analytical procedure (Akalkotkar, 2020, 01179013).

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 sample. 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 each sampling time point were transferred to the analytical laboratory; the remaining samples were retained at the Testing Facility as backup samples (for exceptions, see Appendix 1). 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 (Akalkotkar, 2020, 01179013). Therefore, stability of test substance formulations was not assessed on this study.
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 1 onwards for a minimum of 29 days. Males were exposed for 29 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period.
Females were exposed for 14 days prior to mating and continuing through Lactation Day 13*. Females with no evidence of mating were exposed through euthanasia.
*Premating period (Study Days 0–15), gestation period (Gestation Days 0–20), lactation period (Lactation Days 1–13).
Frequency of treatment:
Daily in ad libitum diet
Dose / conc.:
200 ppm
Remarks:
equivalent to:
F0 males: 13 mg/kg/day;
F0 females: 13 mg/kg/day (premating)/ 14 mg/kg/day (gestation) / 29 mg/kg/day (lactation)
Dose / conc.:
400 ppm
Remarks:
equivalent to:
F0 males: 25 mg/kg/day;
F0 females: 25 mg/kg/day (premating) / 28 mg/kg/day (gestation) / 58 mg/kg/day (lactation)
Dose / conc.:
1 000 ppm
Remarks:
equivalent to:
F0 males: 56 mg/kg/day;
F0 females: 55 mg/kg/day (premating) / 69 mg/kg/day (gestation) / 152 mg/kg/day (lactation)
No. of animals per sex per dose:
10 females and 10 males per dose level
Control animals:
yes, concurrent no treatment
Details on study design:
DOSE SELECTION
The exposure levels for this study were determined from results of previous studies. In the rat embryo/fetal developmental toxicity study in which Lemonile was administered at doses of 0, 200, 400, and 1200 ppm in the diet (Wang, 01179012). Mean absolute body weights in the 1200 ppm group were up to 9.0% lower than the control group during Gestation Days 7–21. Mean fetal body weights (male, female, and combines sexes) were lower in the 1200 ppm group compared to the concurrent control group.

Based on these results, 0, 200, 400 and 1000 ppm dose levels were selected. The high-dosage level is expected to produce some toxicity, but not excessive lethality that would prevent meaningful evaluation. The mid-dose level is expected to produce minimal toxic effects. The low-dosage level is expected to produce no observable indications of toxicity.- Dose selection rationale:
- Rationale for animal assignment (if not random):
- Fasting period before blood sampling for clinical biochemistry:
- Other:
Positive control:
no
Parental animals: Observations and examinations:
The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, food consumption, estrous cycles, reproductive performance, parturition, litter viability and survival, anogenital distance, areolae/nipple anlagen, thyroid hormones, gross necropsy findings, organ weights, and histopathologic 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. 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 throughout the study. During social housing, some observations (e.g., fecal observations) may not have been attributable to an individual animal.

Body Weights
Animals were weighed individually daily throughout the study and prior to the scheduled necropsy. Once evidence of mating was observed, female body weights were recorded on Gestation Days 0–20 and on Lactation Days 1–13.

Food Consumption
Food consumption was quantitatively measured daily until cohabitation. Once evidence of mating was observed, female food consumption was recorded on
Gestation Days 0–20 and Lactation Days 1–13.

Food Evaluation
The mean amounts of test substance consumed (mg/kg/day) by each sex 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). Food efficiency (body weight gained as a percentage of food consumed) was calculated and reported.

Breeding Procedures
After a minimum of 14 days of exposure, the animals were paired on a 1:1 basis within each group. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm in a vaginal lavage. Vaginal lavages were performed daily during the mating period until evidence of mating was observed. If evidence of mating was not apparent after 14 days, the animals were separated, with no further opportunity for mating. Animals cohabited over a 12-hour dark cycle were considered to have been paired for 1 day.

Parturition
The day parturition was initiated was designated Lactation Day 0 (Postnatal Day [PND] 0 for pups). During the period of expected parturition, females were observed twice daily for initiation and completion of parturition and for dystocia or other difficulties. All females were allowed to deliver naturally. Beginning on the day parturition was initiated, the numbers of stillborn and live pups were recorded. Individual gestation length was calculated using the date delivery was first observed.


Oestrous cyclicity (parental animals):
Estrous Cycles
For all females, vaginal lavages were performed daily for 14 days prior to randomization and continuing until evidence of mating was observed or until the end of the mating period. The slides were microscopically examined to determine the stage of the estrous cycle. The average cycle length was calculated for complete estrous cycles (i.e., the total number of returns to metestrus [M] or diestrus [D] from estrus [E] or proestrus [P] for 14 consecutive days before cohabitation and until the detection of evidence of mating). Estrous cycle length was determined by counting the number of days from the first M or D in a cycle to the first M or D in a subsequent cycle. The cycle during which evidence of mating was observed for a given animal was not included in the individual mean estrous cycle length calculation. Vaginal lavages were also performed on the day of necropsy to determine the stage of the estrous cycle.
Litter observations:
Viability
Litters were observed for general health/mortality and moribundity twice daily, once in the morning and once in the afternoon. A daily record of litter size was maintained. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.

Litter parameters were defined as follows:
- Mean Live Litter Size = Total No. of Viable Pups on PND 0/ No. of Litters with Viable Pups PND 0

- Postnatal Survival Between Birth and PND 0 or PND 4 (% Per Litter) =Sum of (Viable Pups Per Litter on PND 0 orPND 4/(No. of Pups Born Per Litter)/ No. of Litters Per Group) x 100

Postnatal Survival for All Other Intervals (% Per Litter) = Sum of (Viable Pups Per Litter at End of Interval N/Viable Pups Per Litter at Start of Interval N) / (No. of Litters Per Group) x 100

Where N = PND 0–1, 1–4 (pre-selection), 4 (post-selection)–7, 7–10, 10–13, and 4 (post-selection)–13

Observations:
The animals were removed from the cage, and a detailed clinical observation was performed on PND 1, 4, 7, 10, and 13.

Sex Determination:
Pups were individually sexed on PND 0, 4, and 13. 4.8.2.4.

Body Weights:
Pups were weighed individually on PND 1, 4, 7, 10, and 13.

Preweaning Developmental Landmarks

Anogenital Distance:
The anogenital distance of all pups was measured on PND 1. Anogenital distance was defined as the distance from the caudal margin of the anus to the caudal margin of the genital tubercle (Gallavan et al., 1999).

Assessment of Areolas/Nipple Anlagen
On PND 13, all male pups were evaluated for the presence of nipples/areolae (Gray et al., 1999). The number of nipples was recorded.

Thyroid Hormone Analysis
Sample Collection: Blood samples for thyroid hormone analyses were collected via cardiac puncture from animals anesthetized with isoflurane into tubes without anticoagulants PND4 (at least 2/sex/litter) and PND13 (1/sex/litter).
Postmortem examinations (parental animals):
Scheduled Euthanasia
All surviving animals, including females that failed to deliver, were euthanized by carbon dioxide inhalation.

Thyroid Hormone Analysis
sample Collection: Blood samples for thyroid hormone analyses were collected from the jugular vein into tubes without anticoagulants. Group 1 to 4. Males at week 4 (termination), females on Lactation day 13.

Necropsy
Animals were subjected to a complete necropsy examination, which included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera. The numbers of former implantation sites were recorded for females that delivered or had macroscopic evidence of implantation. The number of unaccounted-for sites was calculated for each female by subtracting the number of pups born from the number of former implantation sites observed. Uteri of females without macroscopic evidence of implantation were opened and placed in 10% ammonium sulfide solution for detection of early implantation loss (Salewski, 1964).

Organ Weights
The organs identified above were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together, unless otherwise indicated. Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios were calculated. Adrenal glands, Brain, Epididymidesa, Heart, Kidneys, Liver, Ovaries with oviducts, Pituitary gland, Prostate gland, Seminal vesicle (with coagulating gland and fluid), Spleen, Testesa, Thymus gland, Thyroids with parathyroids.

Tissue Collection and Preservation
Representative samples of the tissues identified were collected from all animals and preserved in 10% neutral buffered formalin, unless otherwise indicated. Tissues collected were:Brain, Coagulating gland, Kidneys (2), Liver, Mammary glands, Ovaries and oviducts, Pituitary gland, Prostate gland, Seminal vesicles, Testes with epididymidesa (2) and vas deferens, Thyroids (with parathyroids, if present, Uterusb with cervix and vagina, All gross lesions.

Histology
Tissue trimming was performed at the Testing Facility. Tissues identified below from all animals in the control and high-dose groups, as well as gross lesions from all groups, were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin. In addition, PAS staining was used for the testes and epididymides. Tissues for Histopathology: Brain, Coagulating gland, Mammary glands, Ovaries and oviducts, Pituitary gland, Prostate gland
Seminal vesicles, Testes with epididymides and vas deferens, Uterus with cervix and vagina, All gross lesions.

Histopathology
Pathological evaluation was performed by a board-certified veterinary pathologist. Tissues identified above for microscopic examination were evaluated from all animals in the control and high-dose groups. Gross lesions were examined from all groups.
Postmortem examinations (offspring):
Scheduled Euthanasia
On PND 4, surviving animals were euthanized via an intraperitoneal injection of sodium pentobarbital and discarded.
On PND 13, surviving animals were euthanized via an intraperitoneal injection of sodium pentobarbital.

Necropsy
On PND 13, 1 pup/sex/litter was subjected to a complete necropsy examination, with emphasis on developmental morphology and organs of the reproductive system. All other animals were discarded without examination.

Organ Weights
The Thyroid (with parathyroids, if present) were weighed at necropsy from 1 pup/sex/litter at the scheduled euthanasia.

Tissue Collection and Preservation
Representative samples of the Thyroid (with parathyroids, if present) were collected from 1 pup/sex/litter at the scheduled euthanasia and preserved in 10% neutral buffered formalin.
Statistics:
Each mean was presented with the standard deviation (S.D.) and the number of animals or cages (N) used to calculate the mean. Statistical analyses were not conducted on F0 daily female body weight data after 1 or more animals had entered the gestation phase. Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the means and standard deviations on the summary and individual tables may differ slightly. Therefore, the use of reported individual values to calculate subsequent parameters or means will, in some instances, yield minor variations from those listed in the report data tables. Data obtained from nongravid animals were excluded from statistical analyses. All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group by sex.
Parental mating, fertility, copulation, and conception indices were analyzed using the Chi-square test with Yates’ correction factor. Parental and offspring body weights and body weight changes, parental food consumption, food efficiency, estrous cycle lengths, precoital intervals, gestation lengths, former implantation sites, unaccounted-for sites, live litter size on PND 0, numbers of pups born, absolute and relative organ weights, thyroid hormone values, anogenital distance (absolute and relative to the cube root of body weight), and number of nipples/areolae values were subjected to a parametric one-way ANOVA to determine intergroup differences. If the ANOVA revealed significant (p < 0.05) intergroup variance, Dunnett's test was used to compare the test substance-treated groups to the control group. Mean litter proportions of postnatal survival and pup sexes at birth (percentage of males per litter) were subjected to the Kruskal-Wallis nonparametric ANOVA.
Reproductive indices:
Male Mating Index
Female Mating Index
Male Fertility Index
Female Fertility Index
Male Copulation Index
Female Conception Index
Estrous Cycle Length (days)
Pre-Coital Interval (days)
Gestation Length and Parturition
Offspring viability indices:
live litter size
Postnatal survival
Clinical signs:
effects observed, non-treatment-related
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 hair loss and scabbing on various body surfaces, occurred infrequently, similarly in the control group, and/or in a manner that was not exposure-related.
Description (incidence and severity):
Not dermal study.
Mortality:
no mortality observed
Description (incidence):
All F0 males and females in the control, 200, 400, and 1000 ppm groups survived to the scheduled necropsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males: Effects test substance-related but nonadverse
In the 1000 ppm group males, statistically significant mean body weight losses or lower mean body weight gains were noted compared to the control group sporadically during the study. As a result, lower mean body weight gains were noted in this group when the premating (Study Days 0–15) and entire treatment (Study Days 0–29) periods were evaluated and mean absolute body weights were 5.4% to 9.3% lower than the control group during Study Days 5–29; differences were generally statistically significant. The effects on mean absolute body weight and body weight gains in the 1000 ppm group were considered test substance-related but nonadverse based on the generally low magnitude of change.

Mean body weights and body weight gains in the 200 and 400 ppm group males were unaffected by test substance exposure throughout the study. Statistically significantly lower mean body weight gains were noted at 200 and 400 ppm compared to the control group during Study Days 11–12 and 6–7, respectively, and at 200 ppm when the premating period (Study Days 0-15) was evaluated. These differences were transient in nature, not dose-responsive manner, and/or
had no effect on mean absolute body weights, and therefore were not considered test substance-related.

Females: Effects test substance-related but nonadverse

Weekly:
In the 400 and 1000 ppm groups, mean body weight losses were noted during Study Days 0–1 and 0–3, respectively; differences from the control group were generally statistically significant. Slightly lower mean body weight gains or body weight losses were generally noted for the remainder of the premating period (Study Days 3–15). As a result, a lower mean body weight gain and a mean body weight loss were noted at 400 and 1000 ppm, respectively, when the entire premating period (Study Days 0–15); differences from the control group were statistically significant. Mean absolute body weights were 4.6% to 8.7% lower at 400 ppm during Study Days 4–15 and 5.8% to 11.9% lower at 1000 ppm during Study Days 2–15; differences from the control group were not statistically significant. The effects on body weight and body weight gain in the 400 and 1000 ppm groups during the premating period were considered test substance-related and nonadverse based on the low magnitude of change.

In the 200 ppm group females, lower (not statistically significant) mean body weight gains or mean body weight losses were generally noted throughout the premating period, resulting in a statistically significantly lower mean body weight gain when the entire premating period (Study Day 0–15) was evaluated. However, there was no effect on mean absolute body weights in this group, and therefore the differences were not considered test substance-related.


Gestation:
A statistically significantly lower mean body weight gain was noted at 1000 ppm during Gestation Day 2–3 compared to the control group, this difference corresponded to lower food consumption during this interval. As a result, the mean absolute body weights in the 1000 ppm groups were up to 7.9% lower than the control group, during Gestation Days 0–6. Mean absolute body weights in the 400 ppm group were up to 7.1% than the controls during Gestation Days 0-1. However, none of the differences from the control group were statistically significant and they were attributed to the effects on mean absolute body weights during the premating period therefore, these changes were considered test substance-related but nonadverse. Mean body weights and body weight gains in the 200 ppm group were unaffected by test substance exposure during gestation.

Lactation
Mean absolute body weights in the 400 and 1000 ppm groups were up to 6.9% and 7.3% lower (not statistically significant) than the control group throughou lactation. These differences were attributed to the effects on mean absolute body weights noted in these groups during the premating period therefore, these changes were considered test substance-related but nonadverse. A higher mean body weight gain was noted at 200 ppm during Lactation Day 7–8 and a mean body weight loss was noted at 400 ppm on Lactation Day 10–11; differences from the control group were statistically significant. These differences were transient and/or not dose responsive, and therefore were not considered test substance-related.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males:Effect substance-related and nonadverse

Mean food consumption, evaluated as g/animal/day, in the 1000 ppm group males was lower than the control group throughout the premating period; differences were generally statistically significant. The lower mean food consumption corresponded to the lower mean body weight gains noted in this group and was considered test substance-related and nonadverse because there were no adverse effects on mean absolute body weights and body weight gains in this group.

Mean food efficiency at 1000 ppm was unaffected by test substance exposure. A statistically significant lower mean food efficiency value was noted at 1000 ppm on Study Day 1–2; however, this difference was transient in nature and mean food efficiency values were similar to those in the control group thereafter, and therefore this difference was not attributed to test substance exposure.
Mean food consumption and food efficiency in the 200 and 400 ppm group males were unaffected by test substance exposure. In the 400 ppm group males, mean food consumption was slightly lower than the control group throughout the premating period; differences were occasionally statistically significant. As a result, mean food consumption in this group was statistically significantly lower than the control group when the entire premating period (Study Days 0–15) was evaluated. These differences occurred in the absence of an effect on mean absolute body weight, and therefore were not considered test substance-related.

Females

Weekly:
In the 1000 ppm group, lower mean food consumption, evaluated as g/animal/day, was noted throughout the premating period (Study Days 0–15); differences were generally statistically significant. In addition, statistically significantly lower mean food efficiency values were noted compared to the control group during Study Days 0–2. The effects on food consumption and food efficiency corresponded with the lower mean body weight gains or mean body weight losses noted at 1000 ppm during the premating period and were considered test substance-related and nonadverse based on the lack of adverse effects on mean absolute body weight. In the 400 ppm group, lower mean food consumption was noted sporadically throughout the premating period; differences were statistically significantly during Study Days 0–2 and on Study Day 5–6 compared to the control group. In addition, a statistically significantly lower mean food efficiency value was noted at 400 ppm on Study Day 0–1 compared to the control
group. The lower mean food consumption and food efficiency values generally corresponded with the lower mean body weight gains noted at 400 ppm and were considered test substance-related but nonadverse based on the transient nature and generally low magnitude of change.
Mean food consumption and food efficiency in the 200 ppm group females was unaffected by test substance exposure during the premating period. Statistically significantly lower mean food consumption was noted in this group on Study Day 5–6 compared to the control group. This difference was transient and had no effect on mean absolute body weights, and therefore was not attributed to test substance exposure.

Gestation
Lower mean food consumption (statistically significant) and lower food efficiency was noted in the 1000 ppm group during Gestation Days 2–3 compared to the control group, which corresponded to the lower body weight gains during this interval. Due to lack of adverse effect on the body weight, this change was considered test substance-related but nonadverse. Mean maternal food consumption, evaluated as g/animal/day, and food efficiency in the 200 and 400 ppm groups was unaffected by test substance exposure during gestation. Differences from the control group were slight and not statistically significant, with the following exception.

Lactation
Mean maternal food consumption, evaluated as g/animal/day, and food efficiency in the 200, 400, and 1000 ppm groups was unaffected by test substance exposure during lactation. Differences from the control group were slight and not statistically significant, with the following exceptions. A statistically significantly higher mean food efficiency value was noted in the 200 ppm group on Lactation Day 7–8 and a statistically significantly lower food efficiency value was noted in the 400 ppm group on Lactation Day 10–11 compared to the control group. These differences were transient in nature and not dose-responsive manner, and therefore were not considered test substance-related.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Thyroid Hormone Analysis
There were no test substance-related effects on thyroid hormone values in the F0 males at any exposure level. Differences from the control group were considered to be the result of normal biological variation and were not considered to be of toxicological significance.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related microscopic findings were noted. The microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence and severity in control and treated group animals and, therefore, were considered unrelated to exposure to the test substance.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Estrous Cycle Length (days):
Control: 4.5
Group 1 - 200 ppm: 4.8
Group 2 - 400 ppm: 4.4
Group 3 - 1000 ppm: 4.5
Charles River Historical Control range: 4.2 (3.9–5.2)
Reproductive performance:
no effects observed
Description (incidence and severity):
No test substance-related effects on reproductive performance were observed at any exposure concentration. No statistically significant differences were noted between the control and test substance-exposed groups. Three and 1 mating pairs in the control and 200 ppm groups, respectively, did not produce a litter.

The mean numbers of days between pairing and coitus in the test substance-exposed groups were similar to the control group value. The mean lengths of estrous cycles in these groups were also similar to the control group value. None of these differences were statistically significant.
There were no adverse effects indicative of systemic toxicity and no test substance-related effect on reproductive performance for F0 males and females at any exposure level; therefore, a concentration of 1000 ppm was considered to be the no-observed-adverse-effect level (NOAEL) for F0 systemic and reproductive toxicity of Lemonile when administered in the diet to male and female Crl:CD(SD) rats.
Key result
Dose descriptor:
NOAEL
Remarks:
Systemic, Repro, Developmental
Effect level:
>= 1 000 ppm
Based on:
test mat.
Remarks:
equivalent to 56 mg/kg/d for males and 55, 69 and 152 mg/kg/d for females during premating, gestation, and lactation periods respectively
Sex:
male/female
Basis for effect level:
other: Body weight gain were considered treatement related but not adverse
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
The general physical condition (defined as the occurrence and severity of clinical observations) of all F1 pups in this study was unaffected by test substance exposure.
Description (incidence and severity):
Not a dermal study
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The mean number of pups born, live litter size and the percentage of males at birth in the 200, 400, and 1000 ppm groups were similar to the control group values. Postnatal survival in the 200, 400, and 1000 ppm groups were unaffected by test substance exposure.

Three (3), 3(2), and 2(2), pups (litters) in the control, 200, and 400 ppm groups, respectively, were found dead or euthanized in extremis. One (1) and 4(2) pups (litters) in the 200 and 400 ppm groups, respectively, were missing and presumed to have been cannibalized. No internal findings that could be attributed to parental test substance exposure were noted at the necropsies of pups that were found dead or euthanized in extremis. Aside from the absence of milk in the stomach, no internal findings were noted.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related F1 pup body weight changes were noted. Due to high pup weight in 3 control litters during PND 1–10, the mean pup body weight in this group is higher than the Charles River Ashland historical control range. Therefore the noted statistically significantly lower mean pup body weight in the 400 and 1000 ppm groups were not considered to be substance-related. In addition, all the mean pup body weight in 200, 400 and 1000 ppm groups on PND 13 are comparable to the control values and during PND 1–13 values in the treatment groups were well within and close to the mean of the Charles River Ashland historical control range.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Thyroid Hormone Analysis (PND 13)
There were no test substance-related effects on thyroid hormone values in the F1 males and females at any exposure level on PND 13. Differences from the control group were considered to be the result of normal biological variation and were not of toxicological significance.
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
The anogenital distances (absolute and relative to the cube root of pup body weight) in the 200, 400, and 1000 ppm groups were similar to the control group values. Differences from the control group were slight and not statistically significant.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
No nipples were noted in the F1 male pups at any exposure level when evaluated on PND 13.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no test substance-related effects on thyroid weights in the F1 males and females at any exposure level on PND 13. Differences from the control group were considered to be the result of normal biological variation and were not considered to be of toxicological significance.
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
no effects observed
Behaviour (functional findings):
no effects observed
Developmental immunotoxicity:
not examined
There were no test substance-related effects on F1 pup body weight. Noted changes were due to high pup body weight in 3 control litters. In addition, in all treatment groups, the mean pup body weight gains were unaffected and the mean absolute pup body weights in all groups were within the Charles River Ashland historical control data ranges Therefore, the statistically significant changes noted on mean pup body weight at all dosages were not considered to be test substance-related.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 ppm
Based on:
test mat.
Remarks:
corresponded to actual consumption of 56 mg/kg/day for F0 males during the premating period and 55, 69, and 152 mg/kg/day for F0 females during the premating, gestation, and lactation periods, respectively.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed in F1 generation
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
200 ppm
Treatment related:
no

Results of Reproductive Performance

Parameter Dosage Level (ppm)  CRL HCa
Mean (Range)
0 200 400 1000
Male Mating Index (%)  90.0  100.0  100.0  100.0  98.0 (83.3–100.0)
Female Mating Index (%)  90.0  100.0  100.0  100.0  98.0 (83.3–100.0)
Male Fertility Index (%)  70.0  90.0  100.0  100.0  93.9 (80.0–100.0)
Female Fertility Index (%)  70.0  90.0  100.0  100.0  93.9 (80.0–100.0)
Male Copulation Index (%)  77.8  90.0  100.0  100.0 95.7 (80.0–100.0)
Female Conception Index (%)  77.8  90.0  100.0  100.0  95.7 (80.0–100.0)
Estrous Cycle Length (days)  4.5  4.8  4.4  4.5  4.2 (3.9–5.2)
Pre-Coital Interval (days)  1.9  3.0  2.0  3.9  2.7 (1.4–4.5)
aCharles River Ashland historical control data (version 2019.04)
Conclusions:
Under the conditions of this screening study, there were no adverse effects indicative of systemic toxicity and no test substance-related effect on reproductive performance for F0 males and females at any exposure level; therefore, a concentration of 1000 ppm was considered to be the no-observed-adverse-effect level (NOAEL) for F0 systemic and reproductive toxicity of Lemonile when administered in the diet to male and female Crl:CD(SD) rats. There were no adverse effects in the F1 generation that could be attributed to F0 parental exposure of the test substance, and therefore the NOAEL for F1 neonatal toxicity was 1000 ppm. The 1000 ppm concentration level corresponded to actual consumption of 56 mg/kg/day for F0 males during the premating period and 55, 69, and 152 mg/kg/day for F0 females during the premating, gestation, and lactation periods, respectively.
Executive summary:

The objective of this study was to provide preliminary information on the potential adverse effects of the test substance on male and female reproduction within the scope of a screening study. This encompassed gonadal function, mating behavior, conception, parturition, and lactation of the parental generation and the development of offspring from conception through day 13 of postnatal life.


The study design was as follows: 10 animals/sex/group treated at dose levels of 0, 200 ppm, 400 ppm, 1000 ppm, in an Oral Dietary Study.


Animals were administered the test substance continuously in the diet. Males were exposed for 14 days prior to mating and continuing through the day of euthanasia. Females were exposed for 14 days prior to mating and continuing through Lactation Day 13. The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, food consumption, estrous cycles, reproductive performance, parturition, litter viability and survival, anogenital distance, areolae/nipple anlagen, thyroid hormones, gross necropsy findings, organ weights, and histopathologic examinations.


Mean compound consumption was 13, 25, and 56 mg/kg/day in the 200, 400, and 1000 ppm group F0 males, respectively, during the premating exposure period (Study Days 0–15). Mean compound consumption was 13, 25, and 55 mg/kg/day during the premating period (Study Days 0–15), 14, 28, and 69 mg/kg/day during gestation (Gestation Days 0–20), and 29, 58, and 152 mg/kg/day during lactation (Lactation Days 1–13) in the 200, 400, and 1000 ppm group F0 females, respectively.


All F0 males and females in the control, 200, 400, and 1000 ppm groups survived to the scheduled necropsy. No test substance-related clinical observations were noted at the daily examinations at any exposure level. Test substance-related slightly lower mean body weight gains or mean body weight losses were noted for F0 males in the 1000 ppm group compared to the control group generally throughout the exposure period and when the premating period (Study Days 0–15) and entire exposure period (Study Days 0–29) were evaluated. Corresponding lower mean food consumption was noted for F0 males at 1000 ppm during the premating period. As a result, mean absolute body weights for F0 males at 1000 ppm were up to 9.3% lower than the control group during Study Days 5–29. The magnitude of these effects is due in part to a single male in the control group with a much higher body weight throughout the majority of dosing period compared to the other control group males. The effects on body weight and food consumption for males at 1000 ppm were considered nonadverse based on the low magnitude of change. Mean absolute body weights, body weight gains, and food consumption for F0 males in the 200 and 400 ppm groupwere unaffected by test substance exposure. For F0 females, lower mean body weight gains or mean body weight losses with corresponding lower mean food consumption were noted in the 400 and 1000 ppm groups compared to the control group throughout the premating period (Study Days 0–15) in a dose-responsive manner. As a result, mean absolute body weights that were up to 7.1% and 11.9% lower than the control group on Study Day 15 (end of premating period) in these same respective groups and remained slightly lower (up to 7.1% and 7.9%, respectively) throughout the gestation and lactation periods


with no corresponding effects on food consumption with exception during Gestation Days 2–3 at 1000 ppm. The effects on body weight gains and food consumption for F0 females at 400 and 1000 ppm group were considered test substance-related but nonadverse based on the low magnitude of change and because the food consumption effect did not persist during gestation or lactation. Mean absolute body weights, body weight gains, and food consumption were unaffected by test substance exposure in the 200 ppm group during the premating period and at all exposure levels during the gestation (Gestation Days 0–20) and lactation (Lactation Days 1-13) exposure periods.


F0 reproductive performance (mating, fertility, copulation, and/or conception indices), as well as mean estrous cycle lengths and precoital intervals, were unaffected by test substance exposure at all exposure levels. No test substance-related effects were noted on mean gestation length or parturition at any exposure level. There were no test substance-related effects on T4 concentration for F0 males at any exposure level.


No test substance-related findings were noted in gross observations, organ weights, or in histopathology. The mean number of unaccounted-for sites and former implantation sites was unaffected by test substance exposure at all concentrations. No test substance-related findings were noted in in F1 pup body weights and body weight gains at any dosage. Mean body weight and body weight gains for F1 pups in the control groups were higher than the Charles River Ashland historical control data range. Due to higher values in the controls group, mean absolute body weights for F1 male and female pups at 200, 400 and 1000 ppm were up to 10.7%, 13.4% and 15.6% lower, respectively, than the control group during PND 4–10. However, all values in the treatment group were close to the mean value of the Charles River Ashland historical control data and therefore these changes were not considered as related to test substance.


There were no test substance-related effects on the mean number of pups born, pup survival, live litter size, mean sex ratio, anogenital distance, areolae/nipple anlagen (males), T4 concentration on PND 13, or thyroid gland weight at any exposure level. There were no clinical observations or gross necropsy findings that could be attributed to F0 maternal exposure of the test substance at any concentration. Under the conditions of this screening study, there were no adverse effects indicative of systemic toxicity and no test substance-related effect on reproductive performance for F0 males and females at any exposure level; therefore, a concentration of 1000 ppm was considered to be the no-observed-adverse-effect level (NOAEL) for F0 systemic and reproductive toxicity of Lemonile when administered in the diet to male and female Crl:CD(SD) rats. There were no adverse effects in the F1 generation that could be attributed to F0 parental exposure of the test substance, and therefore the NOAEL for F1 neonatal toxicity was 1000 ppm. The 1000 ppm concentration level corresponded to actual consumption of 56 mg/kg/day for F0 males during the premating period and 55, 69, and 152 mg/kg/day for F0 females during the premating, gestation, and lactation periods, respectively.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
69 mg/kg bw/day
Study duration:
subchronic
Experimental exposure time per week (hours/week):
168
Species:
rat
Quality of whole database:
Klimish 1
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

OECD 421 An Oral (Dietary) Reproduction/Developmental Toxicity Screening Study of Lemonile in Rats:

Dams:

Thyroid Hormone Analysis: There were no test substance-related effects on thyroid hormone values in the F0 males at any exposure level. Differences from the control group were considered to be the result of normal biological variation and were not considered to be of toxicological significance.

 

F1:

Clinical Observations: The general physical condition (defined as the occurrence and severity of clinical observations) of all F1 pups in this study was unaffected by test substance exposure.

 

Offspring Body Weights: No test substance-related F1 pup body weight changes were noted.

 

Anogenital Distance: The anogenital distances (absolute and relative to the cube root of pup body weight) in the 200, 400, and 1000 ppm groups were similar to the control group values.

 

Areolae/Nipple Anlagen: No nipples were noted in the F1 male pups at any exposure level when evaluated on PND 13.

 

Thyroid Hormone Analysis (PND 13): There were no test substance-related effects on thyroid hormone values in the F1 males and females at any exposure level on PND 13.

 

OECD 414 - An Oral (Dietary) Prenatal Developmental Toxicity Study of Lemonile in Rats:

External Malformations and Variations: No malformations or external developmental variations observed in fetuses in this study were considered treatment-related.

 

Visceral Malformations and Variations: No test substance-related visceral malformations were noted for fetuses in this study. Visceral malformations were limited and not considered treatment related. No test substance-related visceral developmental variations were noted.

 

Skeletal Malformations and Variations: No test substance-related skeletal malformations or skeletal variations were noted for fetuses in this study.

 

Fetal body weight: 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 would normally be considered to be adverse. However, these effects were considered to be secondary to the reduced food consumption of the dams, particularly as there were no other toxicological correlates, and are therefore considered to be not adverse. Intrauterine growth at 200 and 400 ppm were unaffected by test substance administration.

Link to relevant study records
Reference
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.
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)
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
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
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
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.

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.
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 maternal body weight and food consumption in the 1200 ppm group are considered likely to be related to issues of palatability as this is often observed with fragrance substances and are therefore considered not 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 effects on maternal body weight and food consumption in the 1200 ppm group are considered likely to be related to issues of palatability as this is often observed with fragrance substances and are therefore considered not 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.
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.
Key result
Dose descriptor:
NOAEL
Remarks:
Maternal Toxicity, Embryo/fetal development
Effect level:
>= 1 200 ppm
Based on:
test mat.
Remarks:
equivalent to 75 mg/kg/day
Basis for effect level:
body weight and weight gain
food consumption and compound intake
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.
Key result
Dose descriptor:
NOAEL
Remarks:
Embryo/Fetal
Effect level:
>= 1 200 ppm
Based on:
test mat.
Remarks:
equivalent to 75 mg/kg/d
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
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
Conclusions:
In conclusion, based on the results of this study, the following NOAEL were established:
Maternal NOAEL: at least 1200 ppm (corresponding to an actual test intake of 75 mg/kg bw/day).
Developmental NOAEL: at least 1200 ppm (corresponding to an actual test intake of 75 mg/kg bw/day).
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 are considered likely to be related to issues of palatability as this is often observed with fragrance substances and are therefore considered not 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 evaluated compared to the control group. The effects on body weight gain and food consumption were considered test substance-related but non adverse 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 would normally be considered to be adverse. However, these effects were considered to be secondary to the reduced food consumption of the dams, particularly as there were no other toxicological correlates, and are therefore considered to be not 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.

In conclusion, based on the results of this study, the following NOAEL were established:

Maternal NOAEL: at least 1200 ppm (corresponding to an actual test intake of 75 mg/kg bw/day).

Developmental NOAEL: at least 1200 ppm (corresponding to an actual test intake of 75 mg/kg bw/day).

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
75 mg/kg bw/day
Study duration:
subacute
Experimental exposure time per week (hours/week):
168
Species:
rat
Quality of whole database:
Klimish 1
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

OECD 414 Prenatal Developmental study:

An Oral (Dietary) Prenatal Developmental Toxicity Study of Lemonile in Rats was performed in compliance with GLP. The test article was assessed in rats, following OECD 414 guideline. Animals were administered with Lemonile continuously in the diet at 200, 400 and 1200 ppm. 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.

 

Animals were administered the test substance continuously in the diet from Gestation Day 6 through 21. In the 1200 ppm group, lower mean male, female, and combined fetal weights were noted compared to the concurrent control group and the minimum mean value in the Charles River Ashland historical control data (version 2019.01). The effects on fetal weight at 1200 ppm would normally be considered to be adverse. However, these effects were considered to be secondary to the reduced food consumption of the dams, particularly as there were no other toxicological correlates, and are therefore considered to be not adverse. Intrauterine growth at 200 and 400 ppm and intrauterine survival, fetal morphology, and fetal anogenital distance at all exposure levels were unaffected by test substance administration.

In conclusion, a dosage level of 1200 ppm, equivalent to 75 mg/kg/day, was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity and embryo/fetal development.

 

 

OECD 421 Repro/Developmental Screening study:

An Oral (Dietary) Reproduction/Developmental Toxicity Screening Study of Lemonile in Rats was performed in compliance with GLP. The test article was assessed in rats, following OECD 421 guideline. Animals were administered with Lemonile continuously in the diet at 200, 400 and 1000 ppm. Males were exposed for 14 days prior to mating and continuing through the day of euthanasia. Females were exposed for 14 days prior to mating and continuing through Lactation Day 13.

Under the conditions of this screening study, there were no adverse effects indicative of systemic toxicity and no test substance-related effect on reproductive performance for F0 males and females at any exposure level; therefore, a concentration of 1000 ppm was considered to be the no-observed-adverse-effect level (NOAEL) for F0 systemic and reproductive toxicity of Lemonile when administered in the diet to male and female Crl:CD(SD) rats.

The 1000 ppm concentration level corresponded to actual consumption of 56 mg/kg/day for F0 males during the premating period and 55, 69, and 152 mg/kg/day for F0 females during the premating, gestation, and lactation periods, respectively.

Justification for classification or non-classification

Based on results on reproduction and developmental toxicity studies, Lemonile does not need to be classified according to CLP Regulation EC 1272/2008.

Additional information