2-Propenamide, 3-(1,3-benzodioxol-5-yl)-N,N-diphenyl-, (2E)-

Endpoint summary

Description of key information

In a key subchronic oral feeding study in rats according to OECD guideline 408, no clinical, histopathological, ophthalmological, body weight, body weight gain, food consumption, food efficiency changes, functional observational battery or motor activity results attributable to the substance administration were observed up to the highest tested concentration. However, it cannot be excluded that the reduction in thymus weight in the mid and high dose group (females) and decreases in WBC and in the white blood cell subpopulations in the high dose group (males and females) might be substance-related. Thus, using an endpoint-specific BMR of 19% for decrease WBC, BMDL–BMDU 90% confidence intervals of 124–781 and 101–1470 mg/kg bw/day were calculated for males and females, respectively. The lowest BMDL is 101 mg/kg bw/day (females) 124 mg/kg bw/day (males).

 

In a supporting 14-day range finding study in rats, the results indicate that male and female rats tolerate a repeated high dose dietary exposure of equal to or greater than 14000 ppm (corresponding to approx. 1381 mg/kg bw/day in females and 1443 mg/kg bw/day in males) of the test substance in a study of longer duration.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2012-09-04 to 2013-07-01

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

other: US FDA Toxicological Principles for the Safety Assessment of Food Ingredients, Redbook 2000, IV.C. 4 a. Short-Term Toxicity Studies with Rodents (2003).

Version / remarks:

2003

Deviations:

yes

Remarks:

As this study was designed as range-finding study, not all parameters specified in the guideline were examined.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

2008

Deviations:

yes

Remarks:

As this study was designed as range-finding study, not all parameters specified in the guideline were examined.

Principles of method if other than guideline:

- Principle of test: Range-finding study to evaluate the palatability and general toxicity of the test item in rats following at least 14 days of dietary administration. This data was used, along with existing data, to select dose levels for a definitive study in rats.

As this study was designed as range-finding study, not all parameters specified in the applicable guidelines were examined.

GLP compliance:

no

Remarks:

This study was not performed in full compliance with GLP standards, but was conducted in a GLP-compliant facility.

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:Sprague-Dawley CD® IGS rats

Details on species / strain selection:

The Sprague-Dawley rat is the system of choice because, historically, it has been the preferred and most commonly used species for dietary toxicity tests.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 194-234 g (males), 183-211 g (females)
- Fasting period before study: No
- Housing: The animals were individually housed in suspended stainless steel caging with mesh floors which conform to the size recommendations in the most recent Guide for the Care and Use of Laboratory Animals (Natl. Res. Council, 2011).
- Diet: Ad libitum (2016CM Harlan Teklad Global Rodent Diet®)
- Water: Ad libitum (filtered tap water)
- Acclimation period: Approx. 7 days

DETAILS OF FOOD AND WATER QUALITY: Water analysis was conducted by Precision Analytical Services, Inc., Toms River, NJ and South Brunswick Municipal Water Supply, South Brunswick, NJ. There are no known contaminants reasonably expected to be present in the certified diet or in the drinking water at levels that would interfere with the results of this study. Therefore, no analyses other than those routinely performed by the feed supplier or those mentioned in this protocol were conducted. Routine analysis consisting of each lot of feed used in this study was received from Research Diets, Inc., New Brunswick, NJ. Water analysis was conducted on a regular basis and the records are kept on file at the laboratory.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 49-66
- Air changes (per hr): Not specified
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From day 0 to day 14

Route of administration:

oral: feed

Details on route of administration:

The dietary route of administration was used because it is recommended in the referenced guidelines, as human exposure may occur via this route.

Vehicle:

unchanged (no vehicle)

Remarks:

The tests substance was directly mixed with feed

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was added to 2016CM Harlan Teklad Global Rodent Diet® and thoroughly mixed in a high-speed mixer. Control diet was mixed under the same conditions as the diets prepared with the test substance.

DIET PREPARATION
- Rate of preparation of diet: Weekly
- Mixing appropriate amounts with: 2016CM Harlan Teklad Global Rodent Diet®
- Storage temperature of food: All diets were refrigerated until used or were presented to the test animals on the same day as diet preparation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sampling of Diets: The test substance and all prepared diets (at each concentration) were sampled. Stability of Test Substance: On study Days 0 and 10, a sample of the test (neat) substance was retained for stability.
Stability in Dietary Matrix: During the first week of the study, samples to verify the stability of the test and control substance in the dietary matrix were prepared.
Homogeneity: No confirmation of homogeneity was scheduled for this range-finding study.
Concentration Verification: Concentration verification was assessed as part of the stability analysis.

Based on the overall stability and concentration verification analysis, animals were considered to have received target dietary concentrations of the test substance within an acceptable margin of variation (±10% or less).

Duration of treatment / exposure:

14 days

Frequency of treatment:

Continuously (feed)

Dose / conc.:

1 381 mg/kg bw/day (actual dose received)

Remarks:

In females, corresponding to 14000 ppm in the diet

Dose / conc.:

378 mg/kg bw/day (actual dose received)

Remarks:

In females, corresponding to 3500 ppm in the diet

Dose / conc.:

13.8 mg/kg bw/day (actual dose received)

Remarks:

In females, corresponding to 140 ppm in the diet

Dose / conc.:

1 443 mg/kg bw/day (actual dose received)

Remarks:

In males, corresponding to 14000 ppm in the diet

Dose / conc.:

359 mg/kg bw/day (actual dose received)

Remarks:

In males, corresponding to 3500 ppm in the diet

Dose / conc.:

14.6 mg/kg bw/day (actual dose received)

Remarks:

In males, corresponding to 140 ppm in the diet

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Corresponding to 0 ppm in the diet

No. of animals per sex per dose:

5 animals per sex per group

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Dietary concentrations of 140, 3500, and 14000 ppm were selected to target approximate exposures of 0.014, 0.35, and 1.4% of the test substance in the diet, and were expected to result in approximate exposures of 250, 500, and 1000 mg/kg bw/day based upon a daily food consumption of 25 grams for a 350 gram rat per day. The highest dose corresponds to the recommended limit dose according to OECD guideline 407 and was chosen based on the assumption that the test substance is relatively non-toxic.
- Rationale for animal assignment: Animals were randomly assigned, stratified by body weight, to test groups.

Positive control:

No positive control was included in the study.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily for viability. Cage-side observations of all animals were performed daily during the study.
- Cage side observations included: All abnormal findings

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: On Day 0 (prior to first presentation of the test substance) and on Days 7 and 14
- Detailed clinical observations included: Potential signs noted included, but were not limited to changes in skin, fur, eyes, and mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern). Likewise, changes in gait, posture, and response to handling, as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling), or bizarre behavior (e.g., self-mutilation, walking backwards)

BODY WEIGHT: Yes
- Time schedule for examinations: Two times during acclimation, on Day 0 (prior to study start) and on test Days 7 and 14

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. All animals in the study were subjected to a gross necropsy, which included examination of the external surface of the body, all orifices, musculoskeletal system, and the cranial, thoracic, abdominal and pelvic cavities with their associated organs and tissues.

HISTOPATHOLOGY: No

Optional endpoint(s):

Optional endpoints: No

Statistics:

The laboratory calculated the mean and standard deviations for all quantitative data and performed statistical analysis of all data collected during the in-life phase of the study. The use of the word “significant” or “significantly” indicated a statistically significant difference between the control and the experimental groups. Significance was judged at p < 0.05. Male and female rats were evaluated separately.

Mean and standard deviations were calculated for all quantitative data. If warranted by sufficient group sizes, data within groups were evaluated for homogeneity of variances and normality by Bartlett's test (Bartlett, 1937). Where Bartlett's test indicated homogeneous variances, treated and control groups were compared using a one-way analysis of variance (ANOVA). When oneway analysis of variance was significant, a comparison of the treated groups to control by Dunnett's test (Dunnett, 1964, 1980) for multiple comparisons was performed. Where variances were considered significantly different by Bartlett's test, groups were compared using a non-parametric method (Kruskal-Wallis non-parametric analysis of variance [Kruskal and Wallis, 1952]). When non-parametric analysis of variance was significant, comparison of treated groups to control was performed using Dunn's test (Dunn, 1964). Statistical analysis was performed on all quantitative data for in-life parameters using ProvantisTM version 8.4.2.2, Tables and Statistics, Instem LSS, Staffordshire UK.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

3500 ppm: Slight to moderate alopecia on the left and right forepaw of 1/10 females (Day 14), which corresponded to a detailed clinical observation of hair loss in the same females (Day 14).

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Sex:

female

Remarks on result:

not determinable because of methodological limitations

Remarks:

This dose-range finding study was not designed to derive a NOAEL. As no adverse effects occured up to the highest dose of 1381 mg/kg bw/day (14000 ppm in diet), it was concluded that this dose is also appropriate for the main study.

Key result

Dose descriptor:

NOAEL

Sex:

male

Remarks on result:

not determinable because of methodological limitations

Remarks:

This dose-range finding study was not designed to derive a NOAEL. As no adverse effects occured up to the highest dose of 1443 mg/kg bw/day (14000 ppm in diet), it was concluded that this dose is also appropriate for the main study.

Conclusions:

In a 14-day range-finding feeding study in rats, the results indicate that male and female rats tolerate a repeated high dose dietary exposure of equal to or greater than 14000 ppm (corresponding to approx. 1381 mg/kg bw/day in females and 1443 mg/kg bw/day in males) of the test substance in a study of longer duration.

Executive summary:

Four groups of adult Crl: Sprague-Dawley® CD® IGS rats (5/sex/group) were maintained on diets containing 0, 140, 3500, or 14000 ppm of the test substance for 14 days, calculated to provide an average daily intake of 0, 14.6, 359, and 1443 mg/kg bw/day or 0, 13.8, 378, and 1381 mg/kg bw/day, for males and females, respectively. Results from the stability and concentration analyses of the test diets indicate that the test substance was stable during presentation and at the target concentrations in the diet for all intake levels. Stability was assessed on Days 0, 4, 7 and 10 after preparation at all concentrations. The animals were observed daily for viability, signs of gross toxicity, and behavioral changes, and on Days 0, 7 and 14 for a battery of detailed observations. Body weights were recorded two times during the acclimation period including just prior to test initiation (Day 0), and during the study on Days 7 and 14 prior to terminal sacrifice. Individual food consumption was also recorded to coincide with body weight measurements. Gross necropsies were performed on all animals. There were no test substance-related or other mortalities. There were no adverse clinical observations, body weight, body weight gain, food consumption, food efficiency, or gross findings that were considered a result of test substance administration.

 

Based on the conditions of this 14-day test and the limited toxicological endpoints evaluated, these results indicate that male and female rats should tolerate a repeated high dose dietary exposure of equal to or greater than 14000 ppm (corresponding to approx. 1381 mg/kg bw/day in females and 1443 mg/kg bw/day in males) of the test substance in a study of longer duration.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-11-12 to 2013-02-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted 1998-09-21

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: CRL Sprague-Dawley CD® IGS rats

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI
- Age at study initiation: seven to eight weeks
- Weight at study initiation: males: 223 - 261 g; females: 158 - 199 g
- Housing: individually housed in suspended stainless steel caging with mesh floors
- Diet (ad libitum, except during fasting prior to blood collection): 2016CM Harlan Teklad Global Rodent Diet®
- Water (ad libitum): filtered tap water
- Acclimation period: 6 days prior to testing

ENVIRONMENTAL CONDITIONS
- Temperature: 19 - 23 °C
- Relative humidity: 38 - 59 %
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly (diets for males and females at each dose level were made separately)
- Mixing appropriate amounts with (Type of food): test substance was added to 2016CM Harlan Teklad Global Rodent Diet® and thoroughly mixed in a high-speed mixer.
- Storage temperature of food: refrigerated

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test substance and dietary preparation were analysed by gas chromatography (GC). The test substance and prepared diets (at selected concentrations) were sampled in duplicate and samples were frozen until analysis. The following analysis were performed:

1) Stability of test substance:
At the initial, middle, and final diet preparation, a sample of the test (neat) substance was retained for stability and stored frozen.
Analytical results of the initial and final stability samples were used to establish the stability of the test substance under normal laboratory conditions for the duration of the study.

2) Stability in Dietary Matrix:
During the study, samples to evaluate the stability of the test substance in the diet were collected from the initial and final diet preparation, targeting the lowest and highest diet concentrations prepared over the duration of the study. Samples were collected at the first and last presentation of the diet and after 4, 7, and 10 days and frozen until analysed.
The samples were evaluated as compared to target dietary concentrations.

3) Homogeneity:
Samples to evaluate homogeneity of the test substance distribution were collected from the initial and final diet preparation, targeting the lowest and highest diet concentrations prepared over the duration of the study. Samples were taken from approximately the top, middle and bottom of the diet mixer. Basal diet control samples were collected from the middle of the mixer only. Chemical analysis verified the diets as homogeneous and of accurate concentration throughout
the study.

4) Concentration Verification:
Samples were collected from representative animal diets of the initial, middle and final diet preparations during which time samples were retained and stored frozen. Samples were analysed to verify the concentration of the test diets.

Results:
1) Stability of test substance:
The test substance was determined to be stable under the conditions of storage at the laboratory over the course of this study.
Results of the stability analysis of the neat the test item were 91.9 % of the target concentration on Day 0 (initial), 98.5 % on Day 39 and 97.2 % on Day 84 (final). The difference in the neat test substance concentration over the course of the study was of 5.8 % and the overall test substance stability was determined to be 105.8 %.

2) Stability in Dietary Matrix:
The stability results for the low dose male dietary samples were 105.1, 103.2, and 97.6 % of the targeted dietary concentration of 259 ppm for Days 4, 7, and 10, respectively. The stability results for the high dose male dietary samples were 96.2, 98.4 and 98.9 % of the targeted dietary concentration of 8898 ppm for Days 4, 7, and 10, respectively
Overall stability for the test substance in the dietary matrix from Days 0-10 was 113.6 % with a percent change of 13.6 % for the low dose samples and 99.2 % with a percent change of -0.8 % for the high dose samples. The test substance was considered to be stable in the dietary matrix and consistent with the 259 or 8898 ppm of the test item target concentrations, under the conditions of the study, for intervals up to 10 days.

3) Homogeneity:
Homogeneity analysis resulted in a relative standard deviation of 9.1 and 1.9 % for males of the 30 and 500 mg/kg bw/day dose groups, respectively. Average percent of target dietary concentrations in the top, middle, and bottom samples for Days 0 (30 mg/kg bw/day dose group) and 84 (500 mg/kg bw/day dose group) were 85.9 and 99.7 % of the target concentrations, 259 and 8898 ppm, for males of the 30 and 500 mg/kg bw/day dose groups, respectively. The test substance was considered to be homogenously distributed in the dietary matrix at all study concentrations, within an acceptable margin of variation.

4) Concentration Verification:
Concentration verification analysis samples were collected on the day of preparation for initial (Day 0), mid (Day 39) and final (Day 84). The Day 0 samples averaged 85.9, 100.6, and 88.6 % for the male preparations at 259, 867, and 4335 ppm, respectively, and 92.7, 96.9, and 89.5 % for the female preparations at 268, 894, and 4470 ppm, respectively. The Day 39 samples averaged 97.5, 94.9, and 88.6 % for the male preparations at 478, 1592, and 7962 ppm, respectively, and 95.2, 99.1, and 85.1 % for the female preparations at 395, 1317, and 6585 ppm, respectively. The Day 84 samples were taken from the 500 mg/kg bw/day dose group male diets reflecting the highest concentration of the test substance which averaged 99.7 % for the target concentration of 8898 ppm. The variability of test substance concentrations between diet preparations at all dietary levels, were considered to be within an acceptable margin of variation. Weekly diet preparations were considered to be consistent in test substance concentration at all dietary level throughout the study.

Duration of treatment / exposure:

at least 90 days

Frequency of treatment:

ad libitum

Dose / conc.:

492.2 mg/kg bw/day (actual dose received)

Remarks:

Corresponding to 500 mg/kg bw/day in females (nominal in diet)

Dose / conc.:

98.6 mg/kg bw/day (actual dose received)

Remarks:

Corresponding to 100 mg/kg bw/day in females (nominal in diet)

Dose / conc.:

29.4 mg/kg bw/day (actual dose received)

Remarks:

Corresponding to 30 mg/kg bw/day in females (nominal in diet)

Dose / conc.:

489.5 mg/kg bw/day (actual dose received)

Remarks:

Corresponding to 500 mg/kg bw/day in males (nominal in diet)

Dose / conc.:

97.5 mg/kg bw/day (actual dose received)

Remarks:

Corresponding to 100 mg/kg bw/day in males (nominal in diet)

Dose / conc.:

29.4 mg/kg bw/day (actual dose received)

Remarks:

Corresponding to 30 mg/kg bw/day in males (nominal in diet)

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

feed

No. of animals per sex per dose:

10 males / 10 females

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale:
The Sponsor, in consultation with the study director of the laboratory and based on a 14-day palatability study (Product Safety Lab; study number: 35137), selected target dose levels of 30, 100, and 500 mg/kg bw/day. In the palatability study four groups of adult Crl: Sprague-Dawley® CD® IGS rats (5/sex/group) were maintained on diets containing 0, 140, 3500, or 14,000 ppm of the test item for 14 days, calculated to provide an average daily intake of 0, 14.6, 359, and 1443 mg/kg bw/day or 0, 13.8, 378, and 1381 mg/kg bw/day, for males and females, respectively.
The animals were observed daily for viability, signs of gross toxicity, and behavioural changes, and on Days 0, 7 and 14 for a battery of detailed observations. Body weights were recorded two times during the acclimation period including just prior to test initiation (Day 0), and during the study on Days 7 and 14 prior to terminal sacrifice. Individual food consumption was also recorded to coincide with body weight measurements. Gross necropsies were performed on all animals.
Results:
There were no test substance-related or other mortalities. There were no adverse clinical observations, effects on body weight, body weight gain, food consumption, food efficiency, or gross findings that were considered a result of test substance administration.
Based on the conditions of this 14-day test and the limited toxicological endpoints evaluated, these results indicate that male and female rats tolerate a repeated high dose dietary exposure of equal to or greater than 14,000 ppm of the test item. However, due to the due to the more than 6 times longer exposure period in the present subchronic study, 500 mg/kg bw/day (nominal) was chosen as the top dose in this study.

Positive control:

no data

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily for viability and daily for cage-side observations

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the first treatment (day 0) and approximately weekly thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: twice during acclimation, prior to study (day 0) and weekly thereafter (intervals of 7 days ± 1) as well as immediately prior to sacrifice (fasted)

FOOD CONSUMPTION AND COMPOUND INTAKE :
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- mean daily body weight gain/ mean daily food consumption: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during the acclimation period and on day 88
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 12 (except coagulation samples: at terminal sacrifice)
- Anaesthetic used for blood collection: Yes, isoflurane anaesthesia
- Animals fasted: Yes, fasted overnight
- How many animals: all animals
- Parameters examined: erythrocyte count, haematocrit, mean corpuscular haemoglobin, absolute reticulocyte count, haemoglobin concentration, mean corpuscular volume, red cell distribution width, platelet count, total white blood cell, differential leukocyte count, prothrombin time and active partial thromboplastin time
Mean corpuscular haemoglobin concentration was calculated.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 12
- Animals fasted: Yes, fasted overnight
- How many animals: all animals
- Parameters examined: serum aspartate aminotransferase, sorbitol dehydrogenase, total bilirubin, blood creatinine, triglycerides, total serum protein, globulin, inorganic phosphorus, potassium, serum alanine aminotransferase, alkaline phosphatase, urea nitrogen, total cholesterol, fasting glucose, albumin, calcium, sodium, and chloride

URINALYSIS: Yes
- Time schedule for collection of urine: day before collection of samples for the clinical pathology evaluation
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: quality, colour, clarity, volume, pH, glucose, specific gravity, protein, ketone, bilirubin, blood, urobilinogen, and microscopic urine sediment examination

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once towards the end of the exposure period (Week 12)
- Dose groups that were examined: all surviving animals
- Battery of functions tested: motor activity, forelimb and hindlimb grip strength, foot splay measurements, excitability, autonomic function, gait and sensorimotor coordination (open field and manipulative evaluations), reactivity and sensitivity (elicited behaviour) and other abnormal clinical signs including but not limited to convulsions, tremors, unusual or bizarre behaviour, emaciation, dehydration, and general appearance.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
At terminal sacrifice, all animals were euthanized by exsanguination under isoflurane anaesthesia. All animals in the study were subjected to a full necropsy, which included examination of the external surface of the body, all orifices, and the thoracic, abdominal and cranial cavities and their contents.

ORGAN WEIGHTS:
The following tissues (of all animals sacrificed by design) were weighed wet as soon as possible after dissection to avoid drying: adrenals (combined), kidneys (combined), spleen, brain, liver, thymus, epididymides (combined), ovaries (combined), uterus with oviducts, heart, and testes (combined).

HISTOPATHOLOGY: Yes
The following organs and tissues from all animals were preserved in 10 % neutral buffered formalin and procured for possible histopathological examination: accessory genital organs (prostate and seminal vesicles), adrenals, all gross lesions, aorta, bone (femur), bone marrow (from femur & sternum), brain (3 sections including medulla/pons, cerebellar, and cerebral cortex), cecum, cervix, colon, duodenum, oesophagus, Harderian gland, heart, ileum with Peyer’s patches, jejunum, kidneys, larynx, liver, lungs, lymph node mandibular, lymph node mesenteric, mammary gland, nose and nasal turbinates, ovaries, pancreas, peripheral nerve (sciatic), pharynx, pituitary, rectum, salivary glands (sublingual, submandibular, and parotid), skeletal muscle, skin, spinal cord (3 levels: cervical, mid- thoracic, and lumbar), spleen, sternum, stomach, thymus, thyroid/parathyroid, trachea, urinary bladder, uterus and oviducts, and vagina.
The following organs and tissues from all animals were preserved in modified Davidson’s fixative and then stored in ethanol for possible future histopathological examination: epididymides (combined), eyes with optic nerve, and testes.

Histological examination was performed on the preserved organs and tissues of the animals from both the control and 500 mg/kg bw/day dose group and reproductive organs and gross lesions from animals in the 30 and 100 mg/kg bw/day dose groups were also examined and included:
30 mg/kg bw/day:
uteri from 2/10 (females)
100 mg/kg bw/day:
liver from 1/10 (males)
uteri from 4/10 (females)

The fixed tissues were trimmed, processed, embedded in paraffin, sectioned with a microtome, placed on glass microscope slides, stained with haematoxylin and eosin and examined by light microscopy.

Statistics:

Mean and standard deviations were calculated for all quantitative data. If warranted by sufficient group sizes, data within groups were evaluated for homogeneity of variances and normality by Bartlett’s test (Bartlett, 1937)*. Where Bartlett’s test indicates homogeneous variances, treated and control groups were compared using a one-way analysis of variance (ANOVA). When one-way analysis of variance was significant, a comparison of the treated groups to control by Dunnett’s test (Dunnett, 1964, 1980)* for multiple comparisons was performed. Where variances are considered significantly different by Bartlett’s test, groups were compared using a non-parametric method (Kruskal-Wallis non-parametric analysis of variance; Kruskal and Wallis, 1952)*. When non-parametric analysis of variance was significant, comparison of treated groups to control was performed using Dunn’s test (Dunn, 1964)*. Overall total movements was analysed by a Two-Way Repeated Measures ANOVA.
Preliminary statistical tests for data normality & homogeneity of variance was conducted (refer to table 1 in the field below). Differences between values were considered statistically significant at p<0.05. Males & females were analysed separately. For all data, the test item treated groups were compared with the control group by one-way ANOVA to assess whether differences might be attributable to consumption of diets formulated with the test item.
*References:
- Bartlett, MS. Properties of sufficiency and statistical tests. Proceedings of the Royal Statistical Society Series A, 160:268–282, 1937.
- Dunnett, CW. New tables for multiple comparisons with a control. Biometrics, 482-491, 1964.
- Dunnett, CW. Pairwise multiple comparisons in the unequal variance case. J. Amer. Statist. Assoc. 75:796-800, 1980.
- Kruskal, WH and WA Wallis. Use of ranks in one-criterion analysis of variance. J. Amer. Statist. Assoc. 47, 583-621, 1952.
- Dunn, OJ. Multiple contrasts using rank sums. Technometrics, 6:241-252, 1964.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

- no test substance-related or other mortalities during the course of the 90-day study.
- no clinical observations attributed to dietary administration of the test substance.

-all observations were considered incidental, often transient, and regarded as toxicologically insignificant.

- males:
In-life clinical signs included:
Control group: slight to moderate alopecia on the left/right forepaw/forelimb/flank or neck in 5/10 males (Days 14 - 94);
30 mg/kg bw/day: unilateral red/black ocular discharge in 1/10 males (Days 35 - 94); slight to moderate alopecia on the left/right forepaw/forelimb/flank or neck in 4/10 males (Days 28 - 94); a scab on the neck of 1/10 males (Days 21-27); broken upper incisor in 1/10 males (Days 35-48); malocclusion of the upper incisor in 1/10 males (Days 35-94).
500 mg/kg bw/day: slight to moderate alopecia on the left/right forepaw/forelimb/flank or neck in 1/10 males (Days 23 - 94)

Detailed clinical observations:
Control group: hair loss in 5/10 males (Day 14-91)
30 mg/kg bw/day: lacrimation in 1/10 males (Days 35-91); hair loss in 4/10 males (Day 28 - 91)
500 mg/kg bw/day: hair loss in 1/10 males (Day 28 - 91)

-females:
In-life clinical signs included:
30 mg/kg bw/day: Unilateral red ocular discharge in 1/10 females (Days 63-76); slight to moderate alopecia on the left/right forelimb of 1/10
females (Days 56-95); malocclusion of the upper incisors in 1/10 females (Days 56-95).
100 mg/kg bw/day: slight to moderate alopecia on the left/right forelimb of 1/10 females (Days 56-83)
500 mg/kg bw/day: slight to moderate alopecia on the left/right forelimb of 1/10 females (Days 70 - 95)

Detailed clinical observations:
30 mg/kg bw/day: lacrimation in 1/10 females (Days 63-70); hair loss in 1/10 females (Days 56-91)
100 mg/kg bw/day: hair loss in 1/10 females (Days 56-77)
500 mg/kg bw/day: hair loss in 1/10 females (Days 70-91)

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

- no statistical changes in body weight or body weight gain in male and female rats attributable to the administration of the test substance.

- the statistically significant decrease in body weight gain in the 500 mg/kg bw/day dose group females as compared to control group between Days 0-7 was considered spurious in nature and regarded as toxicologically insignificant
- males: mean weekly body weights and mean daily body weight gain for male rats in the treatment groups were comparable to the control group males throughout the study.
- females: mean weekly body weights for female rats in the treatment groups were comparable to the control group females throughout the study. Mean daily body weight gain for female rats in the treatment groups was generally comparable to the control group females throughout the study. A statistically significant decrease (p<0.05) was noted in the 500 mg/kg bw/day dose group on Days 0 - 7 as compared to control group females.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

- no statistically significant changes in food consumption or food efficiency attributable to the administration of the test substance.

- males: mean daily food consumption and mean food efficiency for male rats in the treatment groups were comparable to that of the control group males throughout the study.
- females: mean daily food consumption and mean food efficiency for female rats in the treatment groups were comparable to that of the control group males throughout the study.

- test item dietary intake was calculated to be 29.4, 97.5, and 489.5 mg/kg bw/day for males and 29.4, 98.6, and 492.2 mg/kg bw/day for females in
30, 100, 500 mg/kg bw/day dose groups, respectively, over the course of the study. Animals were considered to have received target dietary intakes for the study.

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

- all animals included in the study were normal upon ophthalmic exam on Day 88. Therefore, the test substance was not considered an ocular toxicant.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Decrease in the number of basophils (high-dose: 31% decrease, p < 0.05) in males and statistically nonsignificant decreases in total white blood cells (WBC) count, neutrophils, lymphocytes, eosinophils and large unstained cells in both sexes and in basophiles in females at the highest dose level. Furthermore, a statistically significant decrease in haemoglobin and haematocrit levels (p < 0.05, mid-dose) and a decrease in mean corpuscular haemoglobin concentration (p < 0.05, low-dose) was observed in females.

Although changes were not always statistically significant or dose-dependent, decreases in total WBC and white blood cell subpopulations might indicate a slight effect on the immune system (WBC maturation process) which might be treatment-related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

- statistically significant differences (p<0.05) in clinical chemistry parameters between test and control group were measured in female rats on Day 86. Differences were not dose dependent, were within the range of normal historical values for this age, strain and sex of rat and without direct histologic correlate and, therefore the changes were not considered an adverse result of test substance administration.

- males: no statistically significant differences in clinical chemistry parameters as compared to control group, respective to sex, on Day 86.
- females: statistically significant differences (p<0.05) in clinical chemistry parameters between test and control group, within the range of historical control
Values independent of dietary dose on Day 86 included a decrease in 500 mg/kg bw/day dose group alanine aminotransferase concentration and an increase in 30 mg/kg bw/day dose group potassium concentration.

- no other statistically significant differences at Day 86 clinical chemistry parameters as compared to control group, respective to sex.

Endocrine findings:

not examined

Urinalysis findings:

no effects observed

Description (incidence and severity):

- no statistically significant differences in urine parameters as compared to control group, respective to sex on Day 86.
- microscopic evaluation of the urine in treatment groups males and females revealed no notable changes from that of control group, respective to sex.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

- no changes in functional observational battery parameters on Days 79 (males) and 80 (females) attributed to dietary administration of the test substance.

- all observations were considered incidental, often transient, and regarded as toxicologically insignificant.

- males: in males, findings included:
control group: hair loss in 4/10 males
30 mg/kg bw/day: lacrimation in 1/10 males and hair loss in 2/10 males, and 1/10 Group 4 males.
500 mg/kg bw/day: hair loss in 1/10 males

- females: in females, findings included:
30 mg/kg bw/day: hair loss in 1/10 females and
500 mg/kg bw/day: hair loss in 1/10 females

- forelimb grip strength, hindlimb grip strength, and hindlimb foot splay were comparable to controls for both male and female test substance-treated groups.

- motor activity results (i.e., mean Total Movements) of the test groups of male and female rats (Days 79/80) did not differ significantly from the control groups.
- all groups of animals (including controls) exhibited a similar level of movement over all intervals.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

- statistically significant differences in organ weight and ratios measured on Day 94/95 in male and female rats administered the test item were either without histologic correlates and were not directly correlated with any other study parameters. However, as a decrease in WBC and white blood cell subpopulations were detected in the hematological investigations, the decreased thymus weights might be correlated to this finding.

- males: there were no statistically significant differences in mean absolute organ weights for the 100 and 500 mg/kg bw/day dose groups as compared to control group males. The 30 mg/kg bw/day dose group males had a statistically significant decrease in brain weight (p<0.05) as compared to control group males.
There were no statistically significant differences in mean organ-to-body/brain weight ratios for the 30 and 100 mg/kg bw/day dose groups as compared to control group males. The 500 mg/kg bw/day dose group males showed a dose-independent statistically significant increase in liver-to-body weight ratio (p<0.05) as compared to control group males, within the standard deviation range for the control group.

- females: there were no statistically significant differences in mean absolute organ weights for the 30 mg/kg bw/day dose group as compared to control group females. The 100 and 500 mg/kg bw/day dose group females had dose-independent statistically significant decreases in thymus weight (p<0.05) within the standard deviation range for control group, and decreased in the 500 mg/kg bw/day dose group brain weight (p<0.05) as compared to control group females.
Statistically significant differences in mean organ-to-body/brain weight ratios as compared to control group females included: dose-independent decreased thymus-to-body ratios (p<0.05) in the 100 mg/kg bw/day dose group females; dose-independent increased liver-to-body weight ratios (p<0.05) in the 100 and 500 mg/kg bw/day dose group females; increased mean kidney-to-brain weight ratios (p<0.01-0.05) in 30 mg/kg bw/day dose group females; increased mean liver-to-brain weight ratios (p<0.01-0.05) in 30, 100, and 500 mg/kg bw/day dose group females

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- no macroscopic findings related to test substance exposure for males and females on Days 94 and 95, respectively.

- males: an incidental macroscopic observation included adhesion of the right upper lobe of the liver to the diaphragm in 1/10 of the 100 mg/kg bw/day dose group males. Microscopically, this adhesion corresponded to focal fibrosis of the liver capsule as a spontaneous alteration.
- females: incidental macroscopic observations included a 5 x 3 x 1mm soft, cyst-like raised area on the serosal surface of rectum in 1/10 of 30 mg/kg bw/day dose group females; fluid-filled uteri in 2/10 of 30 mg/kg bw/day dose group females, 4/10 of 100 mg/kg bw/day dose group females, and 4/10 of 500 mg/kg bw/day dose group females. Microscopically, in many instances, fluid-filled uteri corresponded to lumenal dilation of the uterus that was attributable to variation in the oestrous cycle in individual animals and was not associated with test substance exposure.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

- no microscopic findings on Days 94 and 95 in male and female rats, respectively, attributable to the administration of the test item. All findings observed were considered incidental with no patterns/trends, and can be observed in the age and strain of rats used in this study.

- males: incidental microscopic observations included: renal tubular cell hyperplasia, within a minimal focus of chronic progressive nephropathy, unilaterally, in the kidney of 1/10 of the 500 mg/kg bw/day dose group males, which was considered an isolated occurrence in this age and strain of rat. Slight to moderate laryngeal inflammation was observed in 1/10 control group and 1/10 of the 500 mg/kg bw/day dose males, characterized by bilateral granulomatous/pyogranulomatous involvement of the caudal portion of the epiglottal cartilage with evidence of necrosis. This microscopic observation had morphological similarities to historical observations made in Fischer 344 rats, and was considered a spontaneous finding, not associated with test substance administration. Inflammatory cell infiltrates in the prostate gland, primarily of minimal degree, were reported in the control group and the 500 mg/kg bw/day dose group males. Chronic inflammation was characterized by minimal to slight mononuclear cell infiltrates (lymphocytes an macrophages) within the interstitium of 4/10 control group 1 and 4/10 of the 500 mg/kg bw/day dose group males. Minimal to slight acute inflammation was characterized by the presence of neutrophils within glandular lumens in 3/10 of the 500 mg/kgbw/day dose group 5 males.

- females: incidental microscopic observations included: slight to moderate laryngeal inflammation in 1/10 control group and in 2/10 of the 500 mg/kg bw/day dose group females, characterized by bilateral granulomatous/pyogranulomatous involvement of the caudal portion of the epiglottal cartilage with evidence of necrosis. This finding in females, similar to that in males, was unassociated with test substance administration.

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

BMD: See "Remarks on results"

Effect level:

101 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

haematology

organ weights and organ / body weight ratios

Remarks on result:

other: The BMDL was calculated using BMDL – BMDU 90% confidence intervals (101–1470 mg/kg bw/day) around the BMD for decrease in total WBC count calculated from a BMR of 19%. The lowest BMDL is 101 mg/kg bw/day.

Key result

Dose descriptor:

BMD: See "Remarks on results"

Effect level:

124 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

haematology

Remarks on result:

other: The BMDL was calculated using BMDL – BMDU 90% confidence intervals (124–781 mg/kg bw/day) around the BMD for decrease in total WBC count calculated from a BMR of 19%. The lowest BMDL is 124 mg/kg bw/day.

Key result

Critical effects observed:

no

Conclusions:

In a key subchronic oral feeding study in rats according to OECD guideline 408, no clinical, histopathological, ophthalmological, body weight, body weight gain, food consumption, food efficiency changes, functional observational battery or motor activity results attributable to the substance administration were observed up to the highest tested concentration. However, it cannot be excluded that the reduction in thymus weight in the mid and high dose group (females) and decreases in WBC and in the white blood cell subpopulations in the high dose group (males and females) might be substance-related. Thus, using an endpoint-specific BMR of 19% for decrease WBC, BMDL–BMDU 90% confidence intervals of 124–781 and 101–1470 mg/kg bw/day were calculated for males and females, respectively. The lowest BMDL is 101 mg/kg bw/day (females) 124 mg/kg bw/day (males).

Executive summary:

Four groups of adult Crl: Sprague-Dawley® CD® IGS rats (10/sex/group) were maintained on diets prepared to contain 0, 30, 100, or 500 mg/kg bw/day of the substance. Homogeneity, stability, and concentration analyses of the test diets indicate that the substance was homogeneously distributed, stable and was considered to meet target concentrations in the diet for all intake levels.

Body weight and food consumption measurements were collected throughout the study used to calculate the mean overall daily intake of substance. Rats were fed diets prepared on the basis of body weight and food consumption with target doses of 30, 100, and 500 mg/kg bw/day. The diets were calculated to provide an average daily intakes of 29.4, 97.5, and 489.5 mg/kg bw/day for males and 29.4, 98.6, and 492.2 mg/kg bw/day for females, respectively, over the course of the study (Days 0-91).

The animals were examined by focal illumination and indirect ophthalmoscopy prior to initiation and again at the end of the study (Day 88), observed for viability, signs of gross toxicity and behavioural changes at least once daily during the study and weekly for a battery of detailed clinical observations. Urine and blood samples were collected on Day 86 from all study animals for urinalysis, haematology and clinical chemistry determinations and additional blood samples were collected for coagulation assessments on Day 94/95, prior to necropsy. Gross necropsies and histological evaluation of selected organs and tissues were performed on all study animals.

There were no mortalities, clinical observations or findings in the functional observational battery or motor activity, ophthalmological or changes in clinical chemistry or urinanlysis, effects on body weight, body weight gain, food consumption or food efficiency, pathological or histopathological findings attributable to treatment with the substance. Hematological examinations showed a decrease in the number of basophils (high dose group) in males and statistically nonsignificant decreases in total white blood cells (WBC) count, neutrophils, lymphocytes, eosinophils and large unstained cells in both sexes and in basophiles in females at the highest dose level. Statistically significant differences in organ weight and ratios were either within the historical control range or without histologic correlates. However, as a decrease in WBC and white blood cell subpopulations was detected in the hematological investigations, the decreased thymus weights observed in females (mid and high dose group, no dose-related trend) might be correlated to this finding.

Under the conditions of the study and based on the effects observed on hematological parameters, a BMDL–BMDU 90% confidence intervals of 124–781 and 101–1470 mg/kg bw/day were calculated for males and females, respectively, using an endpoint-specific BMR of 19% for decrease WBC.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

BMDL10

101 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

RL 1

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Key, 90-day subchronic toxicity study, rat, RL1

 

In a subchronic oral feeding study according to OECD guideline 408 and GLP, four groups of adult Crl: Sprague-Dawley® CD® IGS rats (10/sex/group) were maintained on diets prepared to contain 0, 30, 100, or 500 mg/kg bw/day of the substance. Homogeneity, stability, and concentration analyses of the test diets indicate that the substance was homogeneously distributed, stable and was considered to meet target concentrations in the diet for all intake levels.

Body weight and food consumption measurements were collected throughout the study used to calculate the mean overall daily intake of substance. Rats were fed diets prepared on the basis of body weight and food consumption with target doses of 30, 100, and 500 mg/kg bw/day. The diets were calculated to provide an average daily intakes of 29.4, 97.5, and 489.5 mg/kg bw/day for males and 29.4, 98.6, and 492.2 mg/kg bw/day for females, respectively, over the course of the study (Days 0-91).

The animals were examined by focal illumination and indirect ophthalmoscopy prior to initiation and again at the end of the study (Day 88), observed for viability, signs of gross toxicity and behavioural changes at least once daily during the study and weekly for a battery of detailed clinical observations. Urine and blood samples were collected on Day 86 from all study animals for urinalysis, haematology and clinical chemistry determinations and additional blood samples were collected for coagulation assessments on Day 94/95, prior to necropsy. Gross necropsies and histological evaluation of selected organs and tissues were performed on all study animals.

There were no mortalities, clinical observations or findings in the functional observational battery or motor activity, ophthalmological or changes in clinical chemistry or urinanlysis, effects on body weight, body weight gain, food consumption or food efficiency, pathological or histopathological findings attributable to treatment with the substance. Hematological examinations showed a decrease in the number of basophils (high dose group) in males and statistically nonsignificant decreases in total white blood cells (WBC) count, neutrophils, lymphocytes, eosinophils and large unstained cells in both sexes and in basophiles in females at the highest dose level. Statistically significant differences in organ weight and ratios were either within the historical control range or without histologic correlates. However, as a decrease in WBC and white blood cell subpopulations was detected in the hematological investigations, the decreased thymus weights observed in females (mid and high dose group, no dose-related trend) might be correlated to this finding.

Under the conditions of the study and based on the effects observed on hematological parameters, a BMDL–BMDU 90% confidence intervals of 124–781 and 101–1470 mg/kg bw/day were calculated for males and females, respectively, using an endpoint-specific BMR of 19% for decrease WBC.

 

Supporting, 14-day palatability study, rat, RL2

Four groups of adult Crl: Sprague-Dawley® CD® IGS rats (5/sex/group) were maintained on diets containing 0, 140, 3500, or 14000 ppm of the test substance for 14 days, calculated to provide an average daily intake of 0, 14.6, 359, and 1443 mg/kg bw/day or 0, 13.8, 378, and 1381 mg/kg bw/day, for males and females, respectively. Results from the stability and concentration analyses of the test diets indicate that the test substance was stable during presentation and at the target concentrations in the diet for all intake levels. Stability was assessed on Days 0, 4, 7 and 10 after preparation at all concentrations. The animals were observed daily for viability, signs of gross toxicity, and behavioral changes, and on Days 0, 7 and 14 for a battery of detailed observations. Body weights were recorded two times during the acclimation period including just prior to test initiation (Day 0), and during the study on Days 7 and 14 prior to terminal sacrifice. Individual food consumption was also recorded to coincide with body weight measurements. Gross necropsies were performed on all animals. There were no test substance-related or other mortalities. There were no adverse clinical observations, body weight, body weight gain, food consumption, food efficiency, or gross findings that were considered a result of test substance administration.

Based on the conditions of this 14-day test and the limited toxicological endpoints evaluated, these results indicate that male and female rats should tolerate a repeated high dose dietary exposure of equal to or greater than 14000 ppm (corresponding to approx. 1381 mg/kg bw/day in females and 1443 mg/kg bw/day in males) of the test substance in a study of longer duration.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on repeated dose toxicity, the test item is not classified and labelled for Specific target organ toxicity (repeated exposure) (STOT-RE) according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighteenth time in Regulation (EU) 2022/692.