Allyl (cyclohexyloxy)acetate

Endpoint summary

Description of key information

In a subchronic feeding study according to OECD guideline 408 in rats, the no observed adverse effect level (NOAEL) of the test substance is considered to be 0.1%, corresponding to 67.6 mg/kg bw/day (males) and 75.7 mg/kg bw/day (females). 

 

In a supporting study (28-day feeding study in rats, according to OECD guideline 407), the NOAEL of the test substance was 0.03%, corresponding to 32.8 mg/kg bw/day (males) and 32.2 mg/kg bw/day (females) under the conditions of this study.

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

Remarks:

with special focus on reproductive organs and tissues

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

24 Feb - 29 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted: 3 Oct 2008

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

(Crl:CD(SD)), SPF

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: not specified
- Age at study initiation: 6 weeks
- Weight at study initiation: males: 191.8 - 209.5 g; females: 137.1 - 165.0 g
- Housing: individually in stainless steel wire mesh cages (260W×350D×210H mm)
- Diet: LabDiet® CERTIFIED RODENT DIET 5002, powder type; ad libitum
- Water: public tap water filtered and irradiated by ultraviolet light, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.2 - 24.4
- Humidity (%): 44.0 -58.8
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light):12/12

Route of administration:

oral: feed

Vehicle:

corn oil

Details on oral exposure:

DIET PREPARATION
The required amount of the test substance was weighed and mixed with required amount of corn oil using a vortex mixer. The required amount of powder feed, was mixed with the test substance formulation using a ball mill.
- Storage temperature of food: 4.3 - 6.3 °C

VEHICLE
- Concentration in vehicle: 10 mL of vehicle / 1 kg of powder feed
- Lot/batch no.: MKBV2080V, MKBZ9899V

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses of the dosing formulations were conducted using a Gas Chromatography (GC-2010series, Shimadzu Corp., Japan). Samples were taken three times from the middle of each dosing formulation and analyzed for verification of dose level concentration prior to dosing. As a result, the accuracies at 0.01, 0.03 and 0.1% were 112.60, 109.67 and 98.84%, respectively. These results were within the acceptable range (range: ±15% of nominal values).

Duration of treatment / exposure:

28 days

Frequency of treatment:

ad libitum, daily, 7 days a week

Dose / conc.:

0.01 other: %

Remarks:

corresponding to 11.5 and 10.7 mg/kg bw/day in males and females, respectively

Dose / conc.:

0.03 other: %

Remarks:

corresponding to 32.8 and 32.2 mg/kg bw/day in males and females, respectively

Dose / conc.:

0.1 other: %

Remarks:

corresponding to 108.6 and 96.0 mg/kg bw/day in males and females, respectively

No. of animals per sex per dose:

5 for main and recovery group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses were selected based on the results of a dose range finding study where a test substance-related decrease in body weight (about 20% in males and 10% in females), as well as a liver weight increase of about 15%, were observed in males and females in the 0.3% groups. Therefore, 0.1% was set as the high dose level. Mid and low dose levels were selected at 0.03 and 0.01%, respectively. The control animals received the standard basal powder feed with corn oil (vehicle).
- Post-exposure recovery period in satellite groups: 2 weeks

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily for clinical signs and twice daily for mortality/moribundity
- Cage side observations: clinical signs, mortality, moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were observed for detailed clinical signs prior to dosing and once weekly during the observation period; observation included: Skin, fur, eyes, mucous membrane, occurrence of secretion, excretion; autonomic activity (lacrimation, piloerection, pupil size, unusual respiratory pattern, etc.); changes in gait, posture and response to handling, and the presence of clonic or tonic movement; stereotypy (excessive grooming, repetitive circling, etc.) or bizarre behavior (selfmutilation, walking backward, etc.)

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded just prior to dosing on Day 1, once a week during the
dosing and recovery periods and on the day of necropsy. Body weight data recorded on the day of necropsy was not included in the evaluation of body weights since these data are body weights of fasting animals.

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

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 29 (main group) and Day 43 (recovery group)
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes; 18 h prior to necropsy
- How many animals: 5 per control and main groups
- Parameters checked: total erythrocyte count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), total leukocyte count (WBC), neutrophils (NEU), lymphocytes (LYM), monocytes (MONO), eosinophils (EOS), basophils (BASO), reticulocytes (Reti), prothrombin time (PT), activated partial thromboplastin time (APTT)


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 29 (main group) and Day 43 (recovery group)
- Animals fasted: Yes; 18 h prior to necropsy
- How many animals: 5 per control and main groups
- Parameters checked: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), blood urea nitrogen (BUN), creatinine (Crea), total bilirubin (T-Bili), total protein (TP), albumin (Alb), globulin (Glo), A/G ratio, glucose (Glu), total cholesterol (T-Chol), triglyceride (TG), phosphorus (P), sodium (Na), potassium (K), chloride (Cl), calcium (Ca), Triiodothyronine (T3), Total Thyroxine (T4)


URINALYSIS: Yes
- Time schedule for collection of urine: Fresh (3-hour) urine and 24-hour urine samples were collected from all animals of both sexes per group in the main group and from all animals in the recovery group at Week 4 and Week 6.
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Animals were fasted during the fresh urine collection, but were allowed free access to drinking water.
- Parameters checked: in fresh urine samples: pH, protein, glucose, ketone body, bilirubin, occult blood, color and turbidity, sediment; in 24-hour urine samples: urine volume, specific gravity

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Week 4 (main group) and Week 6 (recovery group)
- Dose groups that were examined: all main and recovery groups
- Battery of functions tested: Auditory, visual and proprioceptive stimuli (visual response, proprioceptive stimulus, auditory and pain responses, aerial righting reflex and hindlimb landing foot splaygrip strength); grip strength (using push-pull gauge for forelimbs and hindlimbs); motor activity (using activity monitor)

IMMUNOLOGY: No


OTHER:
Observation of estrus cycle:
The observation of estrus cycle for females was conducted on all females of the main group from Week 3 to Week 4 and of the recovery group from Week 5 to Week 6. For the examination of estrous cycle, the vaginal smear was conducted in the morning for approximately 14 days. Prepared smear of the vaginal mucosa was stained with Diff-Quick stain. Stained vaginal mucosa smear was examined using a light microscopy.

Sperm examination:
- Examination of caudal epididymal sperms:
Sperm motility was evaluated at necropsy with all males. The epididymides were weighed. The left caudal epididymis was pierced and placed in DPBS (Dulbecco’s Phosphate Buffered Saline) contacting 1% BSA (Bovine Serum Albumin). Sperm was incubated for approximately 3 to 10 min in the 1% BSA-DPBS culture media (approximately 37°C). Samples were placed on glass slides and the following parameters were analyzed using a sperm analyzer (HTM-TOX IVS, Hamilton Thorne Biosciences, U.S.A.):
MOT (motility, %), VAP (velocity of average path, μm/s), VSL (velocity straight line, μm/s), VCL (velocity curvilinear, μm/s), ALH (amplitude of lateral head displacement, μm), LIN (linearity, LIN = VSL / VCL × 100), STR (straightness, STR = VSL / VAP × 100), BCF (beat-cross frequency, Hz), Elongation (%), Area (μm sq), Rapid (rapid sperm, %), Medium (medium sperm, %), Slow (slow sperm, %), Static (static sperm, %)
Sperm sample was smeared on the glass slide, stained with Diff-Quick and examined for morphology using a light microscope (approximately 200 sperms/smear). Malformation was calculated as follows: Sperm malformation (%) = (Number of abnormal sperm/Number of total sperm (200)) × 100

- Testicular spermatid head counts:
Total sperm counting was evaluated at necropsy with all males. The right testis was weighed and was placed in 10 mL of distilled water, homogenized for one min and sonicated for three min. One drop of the homogenization-resistant sperm head solution was placed onto the sperm number Makler counting chamber and total sperm counts for each animal were determined using a microscope. The number of sperm per 1g of the right testis was calculated.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All animals were sacrificed by exsanguination from the abdominal aorta under isoflurane anesthesia for the main group on Day 29 and for the recovery group on Day 43. Complete gross postmortem examinations were performed on all animals including the external surface and internal organs. All grossly visible abnormalities were recorded.

ORGAN WEIGHTS: Yes
Paired organs were weighed together. Animals were fasted overnight prior to necropsy and body weights were recorded on the day of necropsy. Organs were weighed and organ-to-body weight ratios were calculated. Following organs were weighed: brain, heart, liver, kidney, testis, prostate (including seminal vesicle and coagulating glands), ovary, thyroid and parathyroid, thymus, spleen, adrenals, uterus

HISTOPATHOLOGY: Yes
The following organs and tissues were harvested and preserved in 10% neutral buffered formalin except for the testes, eyes and optic nerve which were fixed in Davidson fixative, and then preserved in 10% neutral buffered formalin:
brain, pituitary, thyroid, parathyroid, lung incl. bonchi, thymus, heart, trachea, liver, spleen, kidneys, adrenal, salivary gland (submandibular, sublingual and parotid glands), stomach, esophagus, duodenum, jejunum, ileum, cecum, colon, rectum, testis, epididymis, prostate, seminal vesicle and coagulating gland, ovary, vagina, uterus incl. cervix, submandibular lymph node, mesenteric lymph node, bone marrow (femur and sternum), spinal cord, sciatic nerve, eye, urinary bladder, sternum incl. bone marrow, mammary gland (inguinal), skin (inguinal), skeletal muscle (tight)

Histopathological evaluations of the samples were performed as follows:
- Organs or tissues from all animals of the control and high dose groups
- Organs with macroscopic lesions in the low dose group

Statistics:

Statistical analysis was performed using SAS Program (version 9.3, SAS Institute Inc., U.S.A.). Main groups: the data of body weight, food consumption, functional observations (hindlimb landing foot splay, grip strength and motor activity), urine volume, hematology, clinical chemistry, estrus cycle, examination of sperm (except for sperm malformation) and organ weights were analyzed utilizing Bartlett’s test for homogeneity of variance (significance level: 0.05). One-way analysis of variance (ANOVA) was employed on homogeneous data; then, if significant, Dunnett’s test was employed for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed). Kruskal-Wallis test was employed on heterogeneous data; then, if significant, Steel test was employed for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed). Main group of sperm malformation: Kruskal-Wallis test was employed on heterogeneous data; then, if significant, Steel test was employed for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed). Recovery group: the data of body weight, food consumption, functional observations (hindlimb landing foot splay, grip strength and motor activity), urine volume, hematology, clinical chemistry, estrus cycle, examination of sperm and organ weights were analyzed utilizing Folded-F test for homogeneity of variance (significance level: 0.05). Student t-test was employed on homogeneous data (significance level: 0.05) or Aspin-Welch t-test was employed for heterogeneous data (significance level: 0.05) for verifying significance (significance levels: 0.05 and 0.01, two-tailed).

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Crust formation in left neck was observed in one male in the 0.1% group from Day 20 to Day 22. However, it was not considered to be a test substance-related effect since it was observed in only one animal incidentally. No detailed clinical abnormalities were observed in any dose group.

Mortality:

no mortality observed

Description (incidence):

All animals survived the duration of the study. There were no effects on mortality.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

During the dosing period, there were no statistically significant differences in body weights in females in the 0.01 and 0.03% groups when compared to the control groups. There were statistically significant decreases in body weights of males in the 0.01% group at Week 4, of males in the 0.03% group at Weeks 3 and 4, and of males in the 0.1% group from Week 1 to Week 4 and of females in the 0.1% group at Week 4 when compared to the control groups. During the recovery period, there were statistically significantly decreases in body weights of males in the 0.1% group when compared to the control group.
There were no statistically significant differences in body weight gain in females in the 0.01, 0.03 and 0.1% groups when compared to the control group. There were statistically significant decreases in body weight gain in males in the 0.01% group at Weeks 3 and 4, in males in the 0.03% group at Week 3, and in males in the 0.1% group from Week 1 to Week 4 when compared to the control group.
The mean body weights of males in the 0.1% group at the end of dosing period (Week 4) and of recovery period (Week 6) were 15.0% and 10.8% lower than those of the control groups, respectively. The decrease in body weights was correlated with the reduced food consumption in males in the 0.1% group. At the end of recovery period, a recovery of changes in body weight and food consumption was observed.

The decreased body weights and food consumption especially in males in the 0.1% group was considered as test substance-related adverse effect.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There was no statistically significant difference in food consumption in males and females in the 0.01% groups and in females in the 0.03% group when compared to the control groups. There were statistically significant decreases in food consumption in males in the 0.03% group at Weeks 1, 3 and 4, and in males from Week 1 to Week 4 and in females at Weeks 1 and 4 in the 0.1% groups.
During the dosing period, there were no statistically significant differences in the relative food consumption in both sexes in the 0.01% groups and in females in the 0.03% group when compared to the control groups. There were statistically significant decreases in the relative food consumption in males in the 0.03% group at Weeks 0 and 1, and in males at Week 1 and in females at Week 4 in the 0.1% groups when compared to the control groups. During the recovery period, there were statistically significant increases in the relative food consumption in males in the 0.1% group at Week 5 (recovery Week 1) when compared to the control group. At the end of recovery period, food consumption changes were observed to showa tendency of recovery. The changes in food consumption were correlated with the decrease in the mean body weights and considered as test substance-related changes.

The mean total test substance intakes at 0.01, 0.03 and 0.1% were 11.5, 32.8 and 108.6 mg/kg/day for males, and 10.7, 32.2 and 96.0 mg/kg/day for females, respectively.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Test substance-related changes were not observed in males and females in the 0.01, 0.03 and 0.1% groups.
All differences in hematology parameters were judged to be produced due to biological variation or were considered to be unrelated to dosing of the test substance based on their small magnitude or because those changes were not observed in the main groups.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Test substance-related changes were not observed in males and females in the 0.01, 0.03 and 0.1% groups.
All differences in clinical chemistry parameters were judged to be produced due to biological variation or were considered to be unrelated to dosing of test substance because of inconsistency in both sexes and related parameters, and due to small magnitude or because those changes were not observed in the main groups.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Test substance-related changes were not observed in males and females in the 0.01, 0.03 and 0.1% groups.
All differences in urinalysis were judged to be biological variations or considered to be unrelated changes with dosing of the test substance based on their small magnitude or because the individual data showed minor variations within the historical reference range.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Test substance-related changes were not observed in males and females in the 0.01, 0.03 and 0.1% groups.
All differences in hindlimb landing foot splay, grip strength, ambulatory counts and vertical counts were considered to be changes unrelated to dosing based on their small magnitude or because those changes were not observed in the main groups.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Test substance-related changes were not observed in males and females in the 0.01, 0.03 and 0.1% groups.
The other organ weight changes, which were statistically significant, were not considered to be toxicologically important because of small magnitude, lack of morphological alteration or because those changes were not observed in the main groups.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Test substance-related changes were not observed in males and females in the 0.01, 0.03 and 0.1 % groups. All macroscopic findings were considered to be incidental and not related to the test substance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Test substance-related changes were not observed in males and females in the 0.1% groups. All other microscopic findings in various organs and tissues were considered to be incidental, spontaneous, and of no toxicological significance.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Observations of estrus cycle:
Test substance-related changes were not observed in females in the 0.01, 0.03 and 0.1% groups. No statistically significant differences in estrus cycle were noted in females in the 0.01, 0.03 and 0.1% groups when compared to the control group.

Examination of Sperm:
Test substance-related changes were not observed in males in the 0.01, 0.03 and 0.1% groups. All differences in sperm parameters were judged to be produced due to biological variation or were considered to be unrelated to dosing of the test substance based on small magnitude.

Key result

Dose descriptor:

NOAEL

Effect level:

32.8 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

Key result

Critical effects observed:

no

Conclusions:

Based on the results of this study, the NOAEL for systemic toxicity was 32.8 mg/kg bw/day (males) and 32.2 mg/kg bw/day (females), corresponding to 0.03% in diet. The NOAEL was based on body weight effects in males and females at 0.1% in diet (equivalent to 108.6 and 96.0 mg/kg bw/day in males and females, respectively).

Executive summary:

In a subacute feeding study according to OECD guideline 407 and GLP, doses of 0.01, 0.03 and 0.1% (corresponding to 11.5, 32.8 and 108.6 mg/kg bw/day in male animals and 10.7, 32.2 and 96 mg/kg bw/day in female animals) were given to 5 Sprague Dawley rats per sex per dose over a period of 28 days. A recovery group consisting of 5 control and high dose animals per sex was continued for 14 days without treatment.

Doses were selected based on the results of a dose range finding study where an adverse test substance-related decrease in body weights compared to the control (approx. 21% in males and 14% in females at the end of the study period), as well as a liver weight increase of about 15%, were observed in males and females in the 0.3% groups. Therefore, 0.1% was set as the high dose level. Mid and low dose levels were selected at 0.03 and 0.01%, respectively. The control animals received the standard basal powder feed with corn oil (vehicle).

There were statistically significant decreases in food consumption in males in the 0.03% group at weeks 1, 3 and 4, and in males from week 1 to week 4 and in females at weeks 1 and 4 in the 0.1% group. There were statistically significant decreases in the relative food consumption in males in the 0.03% group at Weeks 0 and 1, and in males at week 1 and in females at week 4 in the 0.1% groups when compared to the control groups. At the end of recovery period, food consumption changes were observed to show a tendency of recovery. The changes in food consumption were correlated with the decrease in the mean body weights and considered as test substance-related changes.

There were statistically significant decreases in body weights of males in the 0.01% group at Week 4, of males in the 0.03% group at Weeks 3 and 4, and of males in the 0.1% group from Week 1 to Week 4 and of females in the 0.1% group at Week 4 when compared to the control groups. During the recovery period, there were statistically significant decreases in body weights of males in the 0.1% group when compared to the control group.

There were statistically significant decreases in body weight gain in males in the 0.01% group at Weeks 3 and 4, in males in the 0.03% group at Week 3, and in males in the 0.1% group from Week 1 to Week 4 when compared to the control group.

The mean body weights of males in the 0.1% group at the end of dosing period (Week 4) and of recovery period (Week 6) were 15.0% and 10.8% lower than those of the control groups, respectively. The decrease in body weights was correlated with the reduced food consumption in males in the 0.1% group. At the end of recovery period, a slight recovery of changes in body weight and food consumption was observed. However, as body weights were still more than 10% decreased compared to the control after the recovery period, it was considered as test substance-related adverse effect.

Based on the results of this study, the no-observed-adverse-effect level (NOAEL) for systemic toxicity was 0.03% in diet (equivalent to 32.8 and 32.2 mg/kg bw/day in males and females, respectively). Additionally, no adverse effects on reproductive organs, estrus cycle and sperm parameters were observed.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-04-29 to 2022-02-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Regulation on Test Methods for Chemical Substances” Notification No. 2020-28, National Institute of Environmental Research, Republic of Korea

Version / remarks:

2020-08-19

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998-09-21, adopted 2018-06-25

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

(Crl:CD(SD)), SPF

Details on species / strain selection:

Sprague-Dawley rats are commonly used in toxicity studies, having a large historical control data base.

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: 6 weeks
- Weight at study initiation: 186.5 to 214.2 g (males) and 147.8 to 180.1 g (females)
- Fasting period before study: No
- Housing: Singly in stainless wire mesh cages, 260Wx350Dx210H (mm)
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: At least 7 days

DETAILS OF FOOD AND WATER QUALITY: The certificate of feed analysis was provided by the manufacturer, LabDiet®. The results of feed analysis met the allowable standard of this facility. Samples of drinking water are analyzed for specified microorganisms once a month and all environmental contaminants once a year according to the Regulation of Quality Criteria for Potable Water and Test (Ministry of Environment Ordinance No. 792, Revision Dec. 26, 2018). The results of water analysis met the allowable standard of this facility.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5–25.7
- Humidity (%): 42.8–83.0
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From day 1 to day 91 or from day 1 to day 119 (recovery groups)

Route of administration:

oral: feed

Details on route of administration:

The oral (dietary) route was selected to assess the toxicity by oral (dietary) exposure of the test substance.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of the test substance was weighed and placed in a bottle. The required amount of vehicle (required amount: 10 mL of vehicle for 1 kg of powder feed), corn oil, was added and mixed using a vortex mixer until dissolved. The required amount of powder feed, except for the amount of the test substance, was weighed. The required amount of the test substance formulation and a small amount of powder feed were mixed in a bottle. Then, the mixture was placed in a ball mill and residual powder feed was added and mixed for approximately 5 – 10 minutes to yield the desired concentration. For the control group, the required amounts of corn oil and powder feed were weighed on an electronic balance and mixed using the ball mill for approximately 5 – 10 minutes.

DIET PREPARATION
- Rate of preparation of diet: Every 7-14 days
- Mixing appropriate amounts with: Powder feed rodent chow (LabDiet® CERTIFIED RODENT DIET 5002, powder type)
- Storage temperature of food: At approx. 4°C for 14 days or at room temperature for 7 days

VEHICLE
- Justification for use and choice of vehicle: Solubility properties
- Concentration in vehicle: 10% (for a final concentration of 0.1%), 3% (for a final concentration of 0.03%) and 1% (for a final concentration of 0.01%)
- Batch No: MKCH1635, MKCK6411

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses for homogeneity and stability: Homogeneity and stability were analyzed in a separate study (Study No: B16569).Tthe 0.01% and 0.3% dosing formulations were confirmed to be homogenous and stable for 7 days at room temperature and for 14 days under refrigeration.

Verification of dose level concentrations: Dose level concentration were analyzed in a separate study (Study No: B16569) using Gas Chromatography. Samples were taken three times from the middle of each dosing formulation and analyzed for verification of dose level concentration prior to dosing and at Week 13 . As a result, the accuracies at 0.01, 0.03 and 0.1% were 106.10, 102.13 and 97.72% prior to dosing and 109.20, 103.67 and 100.80% at week 13, respectively. These results were within the acceptable range (range: +/-15% of nominal values).

Duration of treatment / exposure:

90 days (all dose and control groups) or 118 days (recovery groups)

Frequency of treatment:

Continously

Dose / conc.:

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

Remarks:

Determined for females, corresponding to 0.1% of the test substance in feed

Dose / conc.:

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

Remarks:

Determined for males, corresponding to 0.1% of the test substance in feed

Dose / conc.:

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

Remarks:

Determined for females, corresponding to 0.03 % of the test substance in feed

Dose / conc.:

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

Remarks:

Determined for males, corresponding to 0.03 % of the test substance in feed

Dose / conc.:

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

Remarks:

Determined for females, corresponding to 0.01% of the test substance in feed

Dose / conc.:

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

Remarks:

Determined for males, corresponding to 0.01% of the test substance in feed

No. of animals per sex per dose:

10 animals + 5 recovery animals/sex/group in the control and high dose groups and 10 animals/sex/group in the low and mid dose groups

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In dose range finding study of the OECD 407 (Study No.: B16567), the body weight was significantly reduced at 0.3 % (21 % in males, 14 % in females). Also the body weight gain showed a significant decrease of more than 40 %. Therefore, 0.1 % was selected as the high dose level for the main study of the OECD 407 (Study No.: B16568). This 4-week repeated dose toxicity study showed a significant decrease of body weight (15%) along with a significant decrease of the body weight gain of more than 30 %. This was regarded as an early indication of toxicity. Taken together the DRF and the main study, higher doses than 0.1 % were not considered suitable for the longer exposure time of the OECD 408 and the high dose level for the 90-Day repeated dose toxicity study was therefore set to 0.1 %. Mid and low dose levels were selected at 0.03 and 0.01%, respectively. For detailed explanation on dose selection, please refer to the dose selection statement attached to this endpoint study record.
- Fasting period before blood sampling for clinical biochemistry: Approximately 18 hours
- Rationale for selecting satellite groups: Recovery groups (control group and high dose group) were were kept without treatment for 28 days after the last dosing day to examine the reversibility of potential effects.
- Post-exposure recovery period in satellite groups: 28 days

Positive control:

Not included

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily (for clinical signs) and twice daily (for mortality and moribundity)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to dosing and once weekly
- Detailed clinical observations checked in table 1 were included.

BODY WEIGHT: Yes
- Time schedule for examinations: Prior to dosing on Day 1, once a week during the dosing and recovery periods and on the day of necropsy. Body weight data recorded on the day of necropsy was not included in the evaluation of body weights since these data are body weights of fasting animals.

FOOD CONSUMPTION AND COMPOUND INTAKE
- 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: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to dosing (all animals) and at Week 13 (all animals in the control and high dose groups of the main group)
- Dose groups that were examined: Control and high dose group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On the day of necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked in table 2+3 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On the day of necropsy
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked in table 4 were examined.

PLASMA/SERUM HORMONES/LIPIDS: Yes
- Time of blood sample collection: On the day of necropsy
- Animals fasted: Yes
- How many animals: All animals

URINALYSIS: Yes
- Time schedule for collection of urine: At week 13 (all animals of the main group) and at recovery week 4 (recovery groups)
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: While fresh urine (3-hour) was collected, feeding and dosing were not performed.
- Parameters checked in table 5 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: On the main group animals at Weeks 12–13 and on the recovery group animals at Recovery Weeks 3–4.
- Dose groups that were examined: All dose groups and the control group were examined.
- Battery of functions tested: Sensory activity / grip strength / motor activity

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 6)

HISTOPATHOLOGY: Yes (see table 7)

Optional endpoint(s):

Optional endpoints: Yes

OBSERVATION OF ESTRUS CYCLE
Observation of the estrus cycle stage for all females in the main and recovery group were conducted in the morning on the day of necropsy. Prepared smears of the vaginal mucosa were stained with Diff Quick stain. Stained vaginal mucosa smears were examined using light microscopy.

EXAMINATION OF SPERM
Examination of sperm was evaluated for all surviving males in the main group and all males in the recovery group. The left epididymis was weighed.

EXAMINATION OF SPERM CAUDAL EPIDIDYMAL SPERM
The epididymides were weighed. At necropsy, the left caudal epididymis was quickly incised, weighed and placed in DPBS (Dulbecco’s phosphate buffered saline) containing 1% BSA (Bovine Serum Albumin). Sperm was incubated for approximately 3 to 10 minutes in 1% BSA-DPBS culture medium (at approximately 37°C). Samples were diluted to the appropriate concentrations, placed on glass slides and evaluated for sperm motility using a sperm analyzer. Parameters for sperm motility are shown in table 8. Sperm samples were smeared on the glass slides, stained with Diff-Quick and examined for morphology using a light microscope. The number of abnormal sperm was counted from approximately 200 sperms per smear.

EXAMINATION OF CAUDAL EPIDIDYMAL SPERMATID HEAD COUNTS
The left epididymis used for sperm motility test was wiped off all external particulate matter and refrigerated for each animal. The tail of the epididymis was incised, weighed, and the membrane was removed. The epididymis were placed in 8 mL of distilled water and homogenized. The total sperm counts for each animal was determined by counting homogenization-resistant sperm heads using a microscope. The number of sperm per 1 g of the left epididymis was calculated.

Statistics:

Main groups: the data of body weight, food consumption, functional observations (hindlimb landing foot splay, grip strength and motor activity), urine volume, hematology, clinical chemistry, examination of sperm (except for sperm malformation) and organ weights were analyzed utilizing Bartlett’s test for homogeneity of variance (significance level: 0.05). One-way analysis of variance (ANOVA) was employed on homogeneous data; then, if significant, Dunnett’s test was employed for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed). Kruskal-Wallis test was employed on heterogeneous data; then, if significant, Steel’s test was employed for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed).
Main group of sperm malformation: Kruskal-Wallis test was employed on heterogeneous data; then, if significant, Steel’s test was employed for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed).

Recovery group: the data of body weight, food consumption, functional observations (hindlimb landing foot splay, grip strength and motor activity), urine volume, hematology, clinical chemistry, examination of sperm and organ weights were analyzed utilizing Folded-F test for homogeneity of variance (significance level: 0.05). Student’s t-test was employed on homogeneous data (significance level: 0.05) or Aspin-Welch t-test was employed for heterogeneous data (significance level: 0.05) for verifying significance (significance levels: 0.05 and 0.01, two-tailed).

The analyses are considered to be appropriate.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

0.1% group: One male animal with overgrown teeth (day 50), and one male with loss of teeth on day 116 of the recovery period.

0.03% group: One female animal showing loss of teeth (day 26).

Loss of teeth was not considered to be a test substance-related effect since it was observed in only one animal of each sex and thus judged to be incidentally.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

0.1% group: Statistically significant decreases in body weights of males from Week 1 to Week 13 and of females from Week 5 to Week 13 (except for Week 7) when compared to the control group. Neither during nor at the end of the recovery period, there were statistically significant differences in body weights in males and females when compared to the control groups.

Statistically significant decreases in body weight gain in males from Week 1 to Week 4 (except for Week 3) and Week 7.

The mean body weights at the end of the dosing period (Week 13) and after the recovery period (Week 17) were 14.2% and 7.6% (males) or 7.9% and 1.1% (females) lower than those of the control groups, respectively.

The decrease in body weights was correlated with the reduced food consumption in males and females in the 0.1% group. At the end of the recovery period, body weight and food consumption changes were observed to have the tendency of recovering.

0.03% group: Statistically significant decreases in body weight gain in females at Week 11 when compared to the control group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

0.1% group: Statistically significant decreases in food consumption in males at week 1-13 and in females from week 2-11 (except for week 3). Statistically significant decrease in relative food consumption at week 1, 2, 4, 5 and 7 (males) and at weeks 5 and 9 (females).

0.03% group: Statistically significant decreases in food consumption in males at weeks 1, 2, 4 and 7 and 8. Statistically significant decreases in relative food consumption in males at weeks 1, 2, 4 and 7 and in females at week 5.

0.01% group: Statistically significant decreases in relative food consumption in females at week 9.

During the recovery period, there were statistically significant increases in the relative food consumption in males in the 0.1% group at week 15 (recovery week 2) when compared to the control group. At the end of the recovery period, food consumption changes had a tendency of recovering.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Description (incidence and severity):

All differences in hematology parameters were of small magnitude and even if statistically significant they were judged to be produced due to biological variation and are within the historical control range. In addition, the variations were not dose-depended or only observed in the recovery group.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

All differences in clinical chemistry parameters were of small magnitude and even if statistically significant they were judged to represent normal biological variations and were within the historical control range and/or without any histopathological correlate. In addition, the variations were neither observed in both sexes nor confirmed in related parameters. Furthermore, some changes were observed in the recovery group only.

Endocrine findings:

no effects observed

Urinalysis findings:

no effects observed

Description (incidence and severity):

Differences in urinalysis were of small magnitude thus judged to be of biological variation, considered to be unrelated to dosing of the test substance or because the individual data showed minor variations being within the historical reference range.

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

If organ weight changes were noted, there were of small magnitude or within the historical control range. In addition, the variations were neither accompanied by morphological alteration (no histopathological changes) nor confirmed by related parameters. Furthermore, some changes were considered secondary effects caused by decreased body weights only.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

All other microscopic findings in various organs and tissues were considered to be incidental, spontaneous, and of no toxicological significance.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

No effects on estrus cyclicity and sperm parameters were observed. All differences in sperm parameters were judged to be produced due to biological variation or were considered to be unrelated to dosing of the test substance based on small magnitude.

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

Key result

Dose descriptor:

NOAEL

Effect level:

>= 67.6 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Body weight reduction exceeded 10% at 67.6 mg/kg bw/day, but was not accompanied by other toxicologically relevant effects. Body weights partially recovered during the recovery period and thus, 67.6 mg/kg bw/day was considered as NOAEL for male animals.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 75.7 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Body weights were reducted at 75.7 mg/kg bw/day, but were not accompanied by other toxicologically relevant effects. Body weights fully recovered during the recovery period and thus, 75.7 mg/kg bw/day was considered as NOAEL for female animals.

Key result

Critical effects observed:

no

Conclusions:

In a subchronic oral feeding study according to OECD guideline 408, the no observed adverse effect level (NOAEL) of the test substance was 0.1%, corresponding to 67.6 mg/kg bw/day (males) and 75.7 mg/kg bw/day (females) under the conditions of this study.

Executive summary:

This study according to OECD guideline 408 and GLP was conducted to assess the cumulative toxicity of the test substance when administered orally (dietary) to 6-week-old Sprague-Dawley

(Crl:CD(SD)) rats for 90 days.

 

A total of 4 groups were designated as follows:

Three animal groups designated as Groups 2, 3 and 4 were treated at dose levels of 0.01, 0.03 and 0.1 % (7.0, 20.1 and 67.6 mg/kg/day for males, and 7.9, 23.5 and 75.7 mg/kg/day for females, respectively) along with a control group, Group 1 (the standard basal powder feed with corn oil), each consisting of 10 males and 10 females for main study. Five animals were added to each group for the recovery groups, control (G1) and high dose (G4) groups. Evaluated parameters included clinical signs, detailed clinical sign observations, body weight, food consumption, functional observations, ophthalmological examination, urinalysis, hematology, clinical chemistry, observation of estrus cycle, examination of sperm, gross post mortem examination, organ weights and histopathological evaluations of selected tissues.

 

All animals survived the duration of the study. There were no effects on mortality. Based on the results of this study, treatment with the test substance at 0.1% resulted in statistically significant reduced body weights in both sexes at the end of the dosing period, with body weight reduction exceeding 10% in the male in the 0.1% group, as well as in reduced body weight gains. These body weight changes correlated with statistically significant reduced food consumption in males and females and were not accompanied by other toxicologically relevant effects. Both sexes showed an increased body weight gain during the recovery phase leading to a complete (for females) or partial (for males) recovery

of the reduced body weights at the end of the recovery period when compared to respective

control groups. Therefore, the effects on body weights were considered as test substance-related

non adverse effects. No test substance-related toxic effects were noted in the results of clinical signs, detailed examinations of clinical signs, functional observations, urinalysis, hematology, clinical chemistry, observation of estrus cycle, examination of sperm, organ weights and necropsy. Histopathologically, there were no test substance-related effects in males and females in the

0.1% groups. Based on the results, the no observed adverse effect level (NOAEL) of the test substance was 0.1% in male and female rats (corresponding to and 67.6 mg/kg/day for males and 75.7 mg/kg/day for females) under the conditions of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

67.6 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable study (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.6, of Regulation (EC) No 1907/2006.

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

Subchronic oral toxicity, rat, RL1

This study according to OECD guideline 408 and GLP was conducted to assess the cumulative toxicity of the test substance when administered orally (dietary) to 6-week-old Sprague-Dawley (Crl:CD(SD)) rats for 90 days.

 

A total of 4 groups were designated as follows:

Three animal groups designated as Groups 2, 3 and 4 were treated at dose levels of 0.01, 0.03 and 0.1 % (7.0, 20.1 and 67.6 mg/kg/day for males, and 7.9, 23.5 and 75.7 mg/kg/day for females, respectively) along with a control group, Group 1 (the standard basal powder feed with corn oil), each consisting of 10 males and 10 females for main study. For detailed explanation on dose selection, please refer to the dose selection statement attached to the endpoint study record in section 7.5.1. Five animals were added to each group for the recovery groups, control (G1) and high dose (G4) groups. Evaluated parameters included clinical signs, detailed clinical sign observations, body weight, food consumption, functional observations, ophthalmological examination, urinalysis, hematology, clinical chemistry, observation of estrus cycle, examination of sperm, gross post mortem examination, organ weights and histopathological evaluations of selected tissues.

 

All animals survived the duration of the study. There were no effects on mortality. Based on the results of this study, treatment with the test substance at 0.1% resulted in statistically significant reduced body weights in both sexes at the end of the dosing period, with body weight reduction exceeding 10% in the male in the 0.1% group, as well as in reduced body weight gains. These body weight changes correlated with statistically significant reduced food consumption in males and females and were not accompanied by other toxicologically relevant effects. Both sexes showed an increased body weight gain during the recovery phase leading to a complete (for females) or partial (for males) recovery of the reduced body weights at the end of the recovery period when compared to respective control groups. Therefore, the effects on body weights were considered as test substance-related non adverse effects. No test substance-related toxic effects were noted in the results of clinical signs, detailed examinations of clinical signs, functional observations, urinalysis, hematology, clinical chemistry, observation of estrus cycle, examination of sperm, organ weights and necropsy. Histopathologically, there were no test substance-related effects in males and females in the 0.1% groups. Based on the results, the no observed adverse effect level (NOAEL) of the test substance was 0.1% in male and female rats (corresponding to and 67.6 mg/kg/day for males and 75.7 mg/kg/day for females) under the conditions of this study.

 

Subacute oral toxicity, rat, RL1

The test substance was tested in a 28-days repeated dose oral toxicity study according to OECD Guideline 407 and in compliance with GLP (2017). Sprague Dawley rats were treated with the test substance in the diet at dose levels of 0.01, 0.03 and 0.1% (equivalent to 11.5, 32.8 and 108.6 mg/kg/day for males, and 10.7, 32.2 and 96.0 mg/kg/day for females, respectively) for 28 days. 10 animals per sex and dose were allocated to the control and high dose group, 5 animals were allocated to the mid and low dose group. The control group received the vehicle corn oil. 5 rats per sex of the control and high dose group were designated to the recovery group and followed a 2–week treatment free period. The doses were selected on the basis of a repeated oral dose range-finding toxicity study in which a test substance-related decrease in body weight was observed in males and females in the 0.3% group. Evaluated parameters included clinical signs, detailed clinical sign observations, body weight, food consumption, functional observations, urinalysis, hematology, clinical chemistry, observation of estrus cycle, examination of sperm parameters, gross post mortem examination, organ weights and histopathological evaluations of selected tissues. All animals survived the duration of the study. There were no effects on mortality. Males and females in the 0.1% group showed a statistically significant decrease in body weights at Week 4 when compared to the control groups. During the recovery period, there were statistically significant decreases in body weights of males in the 0.1% group when compared to the control group. No test substance-related toxic effects were noted in the results of the other evaluated parameters. Necropsy revealed no test substance related findings in males and females in any dose group. No adverse effects were observed in reproductive organs and tissues, sperm parameters of males, estrus cycle of females and thyroid hormones. Based on the body weight effects observed in this study, the systemic NOAEL was 0.03 % in diet (equivalent to 32.8 and 32.2 mg/kg bw/day in males and females, respectively).

 

14-day oral DRF for a subsequent OECD 414, rat, RL2

Furthermore, a 14-days DRF for a subsequent OECD 414 study was included as supporting information. The purpose of this study was to determine the dose range of the test substance for prenatal developmental toxicity study by evaluating the effects on dams and embryo-fetal development when administered orally (dietary) to pregnant Sprague-Dawley (SD) rats from implantation to closure of the hard palate (from Day 5 to Day 19 of Gestation). The test doses were 0, 0.03, 0.1 and 0.3% (0, 27.2, 84.5 and 235.8 mg/kg/d). A decrease in body weight, body weight gain and food consumption was observed at 0.1% and 0.3% during the treatment period, with a significant toxicity seen at 0.3 %. Based on the dose range finding study results 0.2% was selected as the high dose of main study. The mid and low doses were selected to be 0.07 and 0.02%, respectively.

 

14-day oral DRF for a subsequent OECD 407, rat, RL2

Furthermore, a 14-days DRF for a subsequent OECD 407 study was included as supporting information. The purpose of this study was to determine the dose range of the test substance for a subsequent 28-day repeated dose toxicity study by evaluating the effects of the test substance on male and female Sprague-Dawley rats when given in feed for 14 days. The test doses were 0, 0.03, 0.1 and 0.3% (33.8, 106.6 and 257.2 mg/kg/day in males and 32.8, 103.0 and 286.0 mg/kg/day in females, respectively). In males and females in the 0.3% groups, the mean body weights and food consumption were decreased during the dosing period. The mean body weights of males and females at the end of dosing period (Day 14) in the 0.3% groups were 20.7% and 13.6% lower than control groups, respectively. In males and females in the 0.1% group, body weights were up to 8.8% (males) and 7.5% (females) lower than in the control group. Body weight effects at 0.3% were considered adverse test substance-related effects whereas  body weight effects at 0.1% were considered test substance-related but non-adverse. There were no test substance-related differences in hematology, clinical chemistry, organ weights and necropsy. Based on these results, the high dose level for the 4-week repeated dose toxicity study (OECD 407) was selected at 0.1%.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data the test item is not classified specific target organ toxicity (repeated exposure) according to Regulation (EC) No 1272/2008 (CLP), as amended for seventeenth time in Regulation (EU) No 2021/849.