Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

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.

Data source

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

Materials and methods

Test guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
(2Z/6Z)-3,7-dimethylnona-2,6-dienenitrile
Molecular formula:
C11H17N
IUPAC Name:
(2Z/6Z)-3,7-dimethylnona-2,6-dienenitrile
Constituent 2
Chemical structure
Reference substance name:
(2Z/6E)-3,7-dimethylnona-2,6-dienenitrile
Molecular formula:
C11H17N
IUPAC Name:
(2Z/6E)-3,7-dimethylnona-2,6-dienenitrile
Constituent 3
Chemical structure
Reference substance name:
(2E/6Z)-3,7-dimethylnona-2,6-dienenitrile
Molecular formula:
C11H17N
IUPAC Name:
(2E/6Z)-3,7-dimethylnona-2,6-dienenitrile
Constituent 4
Chemical structure
Reference substance name:
(2E/6E)-3,7-dimethylnona-2,6-dienenitrile
Molecular formula:
C11H17N
IUPAC Name:
(2E/6E)-3,7-dimethylnona-2,6-dienenitrile
impurity 1
Chemical structure
Reference substance name:
(3E/6Z)-3,7-dimethylnona-3,6-dienenitrile
Molecular formula:
C11H17N
IUPAC Name:
(3E/6Z)-3,7-dimethylnona-3,6-dienenitrile
impurity 2
Chemical structure
Reference substance name:
(3E/6E)-3,7-dimethylnona-3,6-dienenitrile
Molecular formula:
C11H17N
IUPAC Name:
(3E/6E)-3,7-dimethylnona-3,6-dienenitrile
Test material form:
liquid
Specific details on test material used for the study:
Identification: Lemonile (CAS No. 61792-11-8)
Physical Description: Clear, colorless liquid

Test animals

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

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Details on results (P0)

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.

Effect levels (P0)

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

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

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

Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

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.

Effect levels (F1)

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

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
200 ppm
Treatment related:
no

Any other information on results incl. tables

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)

Applicant's summary and conclusion

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.