Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 15 May 2019 to 11 June 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
under GLP conditions
Justification for type of information:
Following an ECHA decision CCH-D-2114394631-45-01/F on EC:263-214-5 (3,7-dimethylnona-2,6-dienenitrile), it was requested to conduct additional toxicological studies:
- In vitro gene mutation study in mammalian cells, OECD 476;
- Screening for reproductive/developmental toxicity in rats, oral route, OECD 421,
- Sub-chronic toxicity study (90-day), oral route, in rats, OECD 408,
- Pre-natal developmental toxicity study, oral route, rats or rabbits, OECD 414,
- Identification of degradation products.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
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:
Batch No.: PE00231703
Expiration Date: 07 Feb 2021
Physical Description: Clear, colorless liquid
Purity: 99.7%

Test animals

Species:
rat
Strain:
Sprague-Dawley
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
- Source: Charles River Laboratories. Raleigh, NC
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 200 to 320 g (males)/150 to 250 g (females)
- Fasting period before study: Animals were fasted overnight prior to blood collection for clinical pathology evaluations and prior to necropsy.
- Housing: On arrival, animals were group housed (3 animals of the same sex) until randomization.
Following randomization, animals were group housed (2 animals of the same sex and same dosing group together) in solid-bottom cages containing appropriate bedding and equipped with an automatic watering valve.
Animals were separated during designated procedures/activities. Each cage was clearly labeled with a color-coded cage card indicating study number, group number, cage number, dosage level, animal number(s), and sex. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals. The animal facilities at Charles River Ashland are accredited by AAALAC International.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 7 days.

DETAILS OF FOOD AND WATER QUALITY: 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 was considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

Water: 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 was performed, and results of these analyses are on file at the Testing Facility. It was considered that there were no known contaminants in the water that could interfere with the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C to 26°C
- Humidity (%): 30% to 70%
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air (no air recirculation)
- Photoperiod (hrs dark / hrs light): 12 hour light / 12 hour dark cycle

IN-LIFE DATES: From: To: 16 May 2019 - 30 August 2019

Administration / exposure

Route of administration:
oral: feed
Details on route of administration:
The route of administration will be oral (dietary), since the oral route is the most likely route of human exposure.
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
For the control group (Group 1), an appropriate amount of the basal diet, PMI Nutrition International, LLC Certified Rodent LabDiet 5002 meal, was weighed out approximately weekly and placed in a labeled bag. The control group diets were stored in a freezer set to maintain -20°C until use.

DIET PREPARATION
Test substance dietary formulations were prepared at appropriate concentrations to meet dose level requirements. The test substance was added to PMI Nutrition International, LLC Certified Rodent LabDiet 5002 meal on a weight/weight basis and prepared per SOP T2-011. The prepared test diets were not adjusted for purity. The dietary formulations were prepared approximately weekly, split into aliquots for daily dosing, and stored in a sealed container in a freezer set to maintain -20°C until use.


VEHICLE
Not need (dietary study).
Analytical verification of doses or concentrations:
yes
Remarks:
Testing Facility Study No. 01179013
Details on analytical verification of doses or concentrations:
Analytical Method:
Analyses were performed by a gas chromatography method 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 each 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 value at each sampling location 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:
Stability analyses performed previously demonstrated that the test substance is stable in the diet when prepared and stored in a sealed container for at least 10 days in a freezer at approximately -20°C.
Duration of treatment / exposure:
Basal diet and dietary admixes containing test substance were administered ad libitum for at least 90 consecutive days.
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm
Dose / conc.:
125 ppm
Remarks:
Equivalent to 7 mg/kg bw/day in males and 8 mg/kg bw/day in females
Dose / conc.:
500 ppm
Remarks:
Equivalent to 27 mg/kg bw/day in males and 32 mg/kg bw/day in females
Dose / conc.:
2 000 ppm
Remarks:
Equivalent to 111 mg/kg bw/day in males and 128 mg/kg bw/day in females
No. of animals per sex per dose:
10
Control animals:
yes
Details on study design:
The route of administration will be oral, as requested by ECHA.

The Sprague Dawley rat was chosen as the animal model for this study as it is an accepted rodent species for preclinical toxicity testing by regulatory agencies. The total number of animals to be used in this study is considered to be the minimum required to properly characterize the effects of the test substance. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives. 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.

It is anticipated that the high-dosage level will show toxic effects but not produce an incidence of fatalities that would prevent a meaningful evaluation.
Though priority was given to detecting a dose-related trend, it was expected that the low-dosage level would be a no-observed-adverse-effect concentration (NOAEC). Based on the results of a 2-week range-finding dietary study, palatability of the test substance in the diet (decreased food consumption) resulted in mild to severe lower body weight gain at dietary concentrations of 5000 and 10,000 ppm. Minimal effects in body weight and food consumption were noted at 500 ppm. Therefore, dietary concentrations of 0, 125, 500 and 2000 ppm were selected by the Study Director for this study.

In an Oral Dietary Dose Range Finder Study, 5 animals/sex/group were administered the test substance continuously in the diet for 14 consecutive days at level of 500, 1000, 5000 and 10000 ppm.
The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, food consumption, gross necropsy findings, and organ weights.
Average achieved dose (compound consumption) during the treatment period was 30, 55, 254, and 452 mg/kg/day for males and 30, 55, 243, and 443 mg/kg/day for females in the 500, 1000, 5000, and 10,000 ppm groups, respectively.

All animals survived to the scheduled necropsy. There were no test substance-related clinical observations. There were no test substance-related macroscopic findings.

Test substance-related lower body weights and body weight gains and/or body weight losses were noted in the 500 and 1000 ppm group females and the 5000 and 10,000 ppm group males and females. These effects were considered adverse at 5000 and 10,000 ppm for males and at all levels for females and correlated with lower mean food consumption.

Test substance-related lower food consumption was noted in males and females in all treated groups. Lower mean food consumption was considered adverse at 1000, 5000, and 10,000 ppm in males and females.

Test substance-related slightly higher mean testis to final body weights were noted in the 5000 and 10,000 ppm group males. Test substance-related higher mean liver to final body weights were noted in the 5000 and 10,000 ppm group males and females. There were no effects on mean absolute liver weights for males or females, therefore, these effects were considered to be secondary to the direct effect of the test substance on body weight and not directly associated with exposure to the test substance. Test substance-related statistically significant lower mean absolute (and relative to final body weight) prostate with seminal vesicle weights were noted in the 5000 and 10,000 ppm group males. In addition, test substance-related lower mean absolute (and relative to final body weight) thymus weights were noted in the 10,000 ppm group males and females. Lower mean absolute spleen weights were also noted in the 5000 and 10,000 ppm group males and females.

Based on the results of this Dose Range Finder Study, oral administration of lemonile via the diet to Crl:CD(SD) rats at dosage levels of 500, 1000, 5000, and 10,000 ppm for 14 days resulted in adverse body weight effects in females in all treated groups and in males in the 5000 and 10,000 ppm groups. These body weight effects correlated with test substance-related lower mean food consumption in males and females in the 1000, 5000 and 10,000 ppm groups. Additionally, test substance-related organ weight effects were noted in the 5000 and 10,000 ppm group males and females. Therefore, the NOAEL for lemonile administered orally in the diet to Crl:CD(SD) rats for 14 consecutive days was 500 ppm for males and was less than 500 ppm for females. This is equivalent to 30 mg/kg/day and less than 30 mg/kg/day for males and females respectively.

Examinations

Observations and examinations performed and frequency:
The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, food consumption, ophthalmology, clinical pathology parameters (hematology, coagulation, serum chemistry, and urinalysis), thyroid hormones (total T3, total T4, and TSH), gross necropsy findings, organ weights, and histopathologic examinations.

CAGE SIDE OBSERVATIONS: Cage side observations were performed daily, beginning on Day 1 and lasting throughout the dosing period. During social housing, some observations (e.g., fecal observations) may not have been attributable to an individual animal.

DETAILED CLINICAL OBSERVATIONS: The animals were removed from the cage, and a detailed clinical observation was performed on Day 1 (prior to dosing), weekly (± 2 days) during the study period, and on the day of the scheduled necropsy.

BODY WEIGHT: Yes / No / Not specified
Animals were weighed individually starting on Day 1 (prior to dosing) and daily during the study period, on the day prior to the scheduled necropsy, and on the day of the scheduled necropsy. A fasted weight was recorded on the day of the scheduled necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. Food consumption was quantitatively measured daily starting on Day 1 and continued throughout the dosing period. The weekly averages were reported .

FOOD EFFICIENCY:
Yes. Food efficiency (body weight gained as a percentage of food consumed) was calculated and reported. The weekly averages were reported.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

OPHTHALMOSCOPIC EXAMINATION: Yes. Ocular examinations were conducted by a board certified veterinary ophthalmologist during the pretreatment period (Day -9) and near the end of the dosing period (Day 89). An indirect ophthalmoscope and slit lamp biomicroscope were used to examine the ocular structures. Prior to examination an appropriate mydriatic agent was used for pupillary dilation.

HAEMATOLOGY: Yes
Animals were fasted overnight prior to blood collection. Urine was collected overnight using metabolism cages. Blood samples for hematology and serum chemistry were collected from the jugular vein. Blood samples for coagulation parameters were collected by necropsy personnel from the inferior vena cava at the time of euthanasia from animals euthanized via carbon dioxide inhalation.

K2EDTA was used for the anticoagulant on samples collected for hematology. Sodium citrate was used for samples collected for clotting determinations. Samples for serum chemistry were collected without anticoagulants.
Blood samples were analyzed for the parameters: Differential leukocyte counta, Erythrocyte count, Total hemoglobin, Hematocrit, Mean corpuscular hemoglobin, Mean corpuscular volume, Mean corpuscular hemoglobin concentration, Platelet count, Red cell distribution width, Reticulocyte count, Total leukocyte count.

Blood samples were processed for plasma, and the plasma was analyzed for the parameters specified: Activated partial thromboplastin time, Fibrinogen, Prothrombin time, Sample Quality.

CLINICAL CHEMISTRY: Yes
Blood samples were processed for serum, and the serum was analyzed for the parameters specified: Alanine aminotransferase, Albumin, A/G ratio (calculated), Alkaline phosphatase, Aspartate aminotransferase, Bile acids, Calcium, Chloride, Creatinine, Gamma glutamyltransferase, Globulin (calculated), Glucose, High density lipoprotein cholesterol, Low density lipoprotein cholesterol, Phosphorus, Potassium, Sodium, Sorbitol dehydrogenase, Total bilirubin, Total cholesterol, Total protein, Triglycerides, Urea nitrogen, Appearance.

URINALYSIS: Yes
Urine samples were processed and analyzed for the parameters specified: Bilirubin, Color and clarity, Glucose, Ketones, Occult blood, pH, Protein, Specific gravity, Volume.

NEUROBEHAVIOURAL EXAMINATION: Yes / No / Not specified
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

IMMUNOLOGY: No

OTHER:
Thyroid Hormone Assessment:
Blood was collected via a jugular vein of all animals without anticoagulant. Samples were collected for 4 Groups at days 92 and 93.
Blood samples were maintained at room temperature during collection and allowed to clot at room temperature for at least 30 minutes prior to centrifugation. All samples were centrifuged (approximately 3000 rpm; approximately 2056 x g) for approximately 10 minutes at
approximately 4°C within 2 hours of collection. Following centrifugation, approximately 150 μL serum was placed in the first aliquot to be used for total T3 and T4 hormone analysis and all remaining serum (at least 250 μL) was placed in the second aliquot to be used for TSH analyses. Serum was placed into 2 aliquots to be used for TSH, T3, and T4 analyses. Samples were stored in a freezer set to maintain a target of -55°C to -85°C. The serum samples to be analyzed for total T3 and T4 were transferred to the Bioanalytical Chemistry Department and serum samples to be analyzed for TSH were transferred to the ADME/DMPK department.

Analyses of serum samples to determine total T3 and T4 concentrations were conducted using a validated UPHLC/MS/MS assay (Method No. LM No. 099.764A.RS).4 Analyses of serum samples to determine TSH concentrations were conducted using a qualified radioimmunoassay.


Sacrifice and pathology:
Animals were euthanized by carbon dioxide inhalation followed by exsanguination. Animals were fasted overnight before the scheduled necropsies.
Animals were subjected to a complete necropsy examination, which included evaluation of all external surfaces and orifices; the cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues. A veterinary pathologist, or other suitably qualified person, was available.

The organs identified were weighed at necropsy for all scheduled euthanasia animals: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries with oviducts, Pituitary, Prostate with seminal vesicles and coagulating glands, Spleen, Testes, Thymus, Thyroid with parathyroids, Uterus.

Representative samples of the tissues identified were collected from all animals and preserved in 10% neutral buffered formalin, unless otherwise indicated: Adrenal glands, Aorta, Bone with marrow, Femur (with joint), Sternum, Bone marrow smear (from femur), Brain, Cervix, Epididymides, Eyes with optic nerves, Gastrointestinal tract, Esophagus, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum, Harderian glands, Heart, Kidneys, Larynx, Liver (sections of 2 lobes), Lungs (including bronchi, fixed by inflation withfixative) Lymph nodes, Axillary, Mandibular, Mesenteric, Nasal cavitye, Ovaries with oviductsf, Pancreas, Peripheral nerve (sciatic), Peyer’s patches, Pharynx, Pituitary, Prostate with coagulating glands, Salivary glands (mandilar), Seminal vesicles, Skeletal muscle (quadriceps), Skin with mammary glandg, Spinal cord (cervical, thoracic, lumbar), Spleen, Testes, Thymus, Thyroid (with parathyroids), Tongue, Trachea, Urinary bladder, Uterus, Vagina, Gross lesions (when possible).


Histology
Tissue trimming was performed at the Testing Facility. Tissues identified above (except bone marrow smears) from animals in the control and high-dose groups at the terminal necropsy were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin. In addition, gross lesions were prepared from all animals in the low- and mid-dose groups at the terminal necropsy.

Histopathology
Pathological evaluation was performed by a board-certified veterinary pathologist. Tissues identified in Text Table 12 for microscopic examination were evaluated from all animals in the control and high-dose groups at the terminal necropsy. In addition, gross lesions were examined microscopically from all animals in the low- and mid-dose groups at the terminal necropsy.
Statistics:
Any data collected during the predose period were not tabulated, summarized or statistically analyzed. All statistical analyses were performed within the respective study phase, unless otherwise noted. Numerical data collected on scheduled occasions was summarized and statistically analyzed as indicated below according to sex and occasion. For food utilization (efficiency), achieved dosage levels (compound consumption), TSH, T3, and T4 data, each mean was presented with the standard deviation (S.D.) and/or the number of animals or cages (N) used to calculate the mean. 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.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
There were no test substance-related clinical observations. All clinical observations in the test substance-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain.
Mortality:
no mortality observed
Description (incidence):
All animals survived until scheduled necropsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related, lower mean body weights were noted in the 500 and 2000 ppm group males and 2000 ppm group females. Mean body weights in the 500 and 2000 ppm group males and 2000 ppm group females were significantly lower than the control group during the entire study and during Weeks 1–13 in the 500 ppm group. During the last week of dosing, mean body weights were 11.7%, 14.8%, and 17.0% lower in the 500 ppm group males and 2000 ppm group males and females, respectively, compared to the control group.

Mean body weight gains in the 2000 ppm group males and females (and less frequently, the 500 ppm group males) were occasionally statistically significantly lower than the control group throughout the study. Mean cumulative body weight gains (Day 1–91) were 277.7 g, 256.0 g, and 79.9 g in the 500 ppm group males and 2000 ppm group males and females, respectively, compared to the control group males (345.3 g) and females (127.5 g).

There were no other test substance-related effects on body weight. However, some statistically significant differences were observed when the control and test substance-treated groups were compared. These differences were not noted in a dose-related manner and differences were small in magnitude. Therefore, they were considered not test substance-related.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Test substance-related lower mean food consumption was noted in test substance-treated males and females.

There was a dose-related decrease in food consumption for males and females in all test substance-treated groups. Significantly lower food consumption was noted as early as Week 1 in the 500 ppm group (males only) and 2000 ppm group males and females and continued throughout the course of the study (occasionally significantly significant). Lower food consumption reached statistical significance in the 125 ppm group males during 1 weekly interval (Week 5) and females during 3 weekly intervals (Weeks 5, 8, and 10). Mean weekly food consumption over the course of the study was 10.6% and 7.5% lower than controls for the 500 ppm group males and females, respectively. Mean weekly food consumption over the course of the study was 13.7% and 16.8% lower than controls for the 2000 ppm group males and females, respectively.
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No ophthalmic lesions indicative of ocular toxicity were observed in any of the test substance-treated groups. All findings observed were typical in prevalence and appearance for laboratory rats of this age and strain.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related and dose-dependent lower mean absolute reticulocyte counts on Day 92/93 were noted in all test-substance treated male groups compared to the control group (statistically significant in the 2000 ppm group only). These effects were considered nonadverse and may have been secondary to decreased food consumption and decreased body weights. All other differences in hematology parameters when the control test substance-treated groups were compared, regardless of statistical significance, were within historical control ranges and were consistent with biological variation or lack of a dose-response and were considered unrelated to test substance administration.

Coagulation parameters were unaffected by test substance administration. However, a statistically significant difference in prothrombin time (PT) for 2000 ppm group females was observed when the control and test substance-treated groups were compared. The magnitude of change was small and the mean value was within the range of historical control data. Therefore, the small change in PT for 2000 ppm group females was not considered to be test substancerelated but was considered due to normal biological variability.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related and dose-dependent higher mean chloride concentrations were noted on Day 92/93 for males and females in all test substance-treated groups compared to the control group (statistically significantly in all groups except the 125 ppm group males). These effects were considered nonadverse and may have been secondary to decreased food consumption and decreased body weights. All other differences in serum chemistry parameters, regardless of statistical significance, were
consistent with biological variation or lack of a dose-response and considered unrelated to test substance administration.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Urinalysis parameters were unaffected by test substance administration. However, some statistically significant differences were observed in male urine pH when the control and test substance-treated groups were compared. The magnitude of change was small and mean values were within the range of historical control data. Therefore, the small changes in urine pH were not considered to be test substance-related but were considered due to normal biological variability.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Statistically significantly lower mean spleen weights (absolute and/or relative to terminal body and brain weights) were noted in the 125, 500, and 2000 ppm group males and the 2000 ppm group females. Mean values were lower (both statistically significant and/or non-significant trends) compared to the 0 ppm group males but were within the range of historical control data and had no microscopic correlates. Given the changes across all test substance-treated groups and the decreases present in all of the absolute and relative to terminal body and brain weights, these findings were considered likely test substance-related but may additionally have been impacted by lower terminal body weights.
Some organ weight differences were statistically significant when compared to the control group but were considered to be a result of a test substance-related effect on terminal body weight. There were dose-dependent lower mean terminal body weights noted in the 500 and 2000 ppm group males and females. Specifically, in the 500 and/or 2000 ppm group males, mean absolute weights of the epididymis, pituitary gland, prostate/seminal vesicles, thyroid/parathyroid glands,
heart, and kidney and mean weights relative to brain weights of the pituitary gland and heart were lower comparted to controls, and mean weight relative to terminal body weight of the brain, liver, and testes were higher compared to controls. In the 2000 ppm group females, mean absolute weights of the heart, kidney, and liver mean weights relative to brain weights of the heart and kidney were lower comparted to controls, and mean weight relative to terminal body weight of the brain, thyroid/parathyroid glands, kidney, and liver were higher compared to controls.

There were no other test substance-related effects on organ weights.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test substance-related macroscopic findings at the scheduled necropsy. All macroscopic findings noted were considered to be spontaneous and/or incidental in nature and unrelated to test substance administration.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test substance-related histologic changes. All 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:
effects observed, non-treatment-related
Description (incidence and severity):
Thyroid Hormone:
Statistically significant lower mean total T4 concentrations were noted on Day 92/93 for males in all test substance-treated groups compared to the concurrent control group. Similar changes were not noted in females. Mean T3, T4 and TSH values for males and females were within Charles River Ashland historical control ranges (version ASH_RS_RE-H_2019.02). Based on the lack of effect in females, the lack of correlating microscopic findings in the thymus, the lack of a dose-proportional decrease in T4 concentrations compared to an approximately 16-fold increase in compound consumption and the similarity to historical control values, the effect on T4 concentration in males was not considered to be test substance-related but was considered to be the result of biological variation.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
>= 128 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Key result
Dose descriptor:
NOAEL
Effect level:
>= 111 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: Effects related to issues of palatability

Target system / organ toxicity

Key result
Critical effects observed:
no
System:
other:

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, dietary administration of Lemonile® to Crl:CD(SD) rats at concentrations of 125, 500, and 2000 ppm for at least 90 days resulted in lower body weights, lower body weight gains/loss, and decreased food consumption for males and females at 2000 ppm. These effects of food consumption and bodyweight gain are considered likely to be related to issues of palatability as this is often observed with fragrance substances and are therefore considered not adverse. Other test substance-related effects noted in reticulocyte counts, blood chloride concentration, and spleen weights were considered nonadverse and may have been secondary to decreased food consumption and decreased body weights. Therefore, the no-observed-adverse effect level (NOAEL) was considered to be 2000 ppm (equivalent to 111 and 128 mg/kg/day for males and females, respectively).
Executive summary:

The objective of this study was to evaluate the potential toxicity of the test substance when administered via diet to Sprague Dawley rats for at least 90 consecutive days.


 


The study design was as follows:


 











































 Group number    Treatment 

 Diatery Concentration*   


(ppm)


    Number of animals
 MalesFemales
 1Basal diet10  10
 2 Lemonile 125 10 10
3 Lemonile 500 10 10
 4 Lemonile 200010  10

 


 


*No correction factor was used. The test substance was present as a fixed dietary admix concentration without adjustment for body weight.


 


Animals were administered the test substance continuously in the diet for at least 90 days.


The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, food consumption, ophthalmology, clinical pathology parameters (hematology, coagulation, serum chemistry, and urinalysis), thyroid hormones (total T3, total T4, and TSH), gross necropsy findings, organ weights, and histopathologic examinations.


 


Average compound consumption during the treatment period was 7, 27, and 111 mg/kg/day and 8, 32, and 128 mg/kg/day in males and females in the 125, 500, and 2000 ppm groups, respectively.


 


All animals survived to the scheduled necropsy. There were no test substance-related clinical observations or effects on coagulation or urinalysis parameters. There were no test substancerelated ophthalmic, macroscopic, or microscopic findings.


 


Body weights in the 2000 ppm group males and females and 500 ppm group males were significantly lower than the control group during the entire study and during Weeks 1–13 in the 500 ppm group (generally statistically significant). During the last week of dosing, body weights were 11.7%, 14.8%, and 17.0% lower in the 500 ppm group males and 2000 ppm group males and females, respectively, compared to the control group.


 


Body weight gains in the 2000 ppm group males and females (and less frequently, the 500 ppm group males) were occasionally statistically significantly lower than the control group throughout the study.


 


There was a dose-related decrease in food consumption for males and females in all test substance-treated groups. Significantly lower food consumption was noted as early as Week 1 in the 500 ppm group (males only) and 2000 ppm group males and females and continued throughout the course of the study (occasionally statistically significant).


These effects of food consumption and bodyweight gain are considered likely to be related to issues of palatability as this is often observed with fragrance substances and are therefore considered not adverse.


Test substance-related, nonadverse, lower absolute reticulocyte counts were noted in all test substance-treated male groups compared to the control group. Additionally, higher chloride concentrations were noted in all test substance-treated group males and females compared to the control group. Test substance-related lower spleen weights were noted in the 125, 500, and 2000 ppm group males and 2000 ppm group females compared to the control group. These effects were considered nonadverse and may have been secondary to decreased food consumption and decreased body weights.


 


Based on the results of this study, dietary administration of Lemonile® to Crl:CD(SD) rats at concentrations of 125, 500, and 2000 ppm for at least 90 days resulted in lower body weights, lower body weight gains/loss, and decreased food consumption for males and females at 2000 ppm. These effects of food consumption and bodyweight gain are considered likely to be related to issues of palatability as this is often observed with fragrance substances and are therefore considered not adverse.


Other test substance-related effects noted in reticulocyte counts, blood chloride concentration, and spleen weights were considered nonadverse and may have been secondary to decreased food consumption and decreased body weights. Therefore, the no-observed-adverse effect level (NOAEL) was considered to be 2000 ppm (equivalent to 111 and 128 mg/kg/day for males and females, respectively).