Registration Dossier

Administrative data

Description of key information

3-L-Menthoxypropane-1,2-diol (Takasago Coolact 10) when administered in diet for 28 days to male and female Sprague-Dawley (Crl:CD®BR) rats at dose levels of 250, 500, 1000, 2000 and 5000 mg/kg bw/day, revealed a lowest observed adverse effect level (LOAEL) at 250 mg/kg bw/day for male and female rats (based on adverse effects observed in clinical chemistry and organ weights observed at this dose level).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
5 July 1991 to 30 August 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Remarks:
According to the US FDA principles of GLP
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD®BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 44 days old
- Weight at study initiation: 223-255 g for males, 147-173 g for females
- Housing: Two/cage by sex in stainless-steel, hanging, wire-mesh cages until randomisation. After randomisation animals were housed individually.
- Diet: Rodent Chow was provided ad libitum
- Water: Tap water; ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 20.5 - 22.8°C
- Relative humidity: 57 - 74%
- Air changes: 10 air changes/hour
- Photoperiod: 12 hours light/12 hours dark

IN-LIFE DATES: From: Aug. 01, 1991 to Aug. 30, 1991
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS
The test substance for each level was weighed by placing feeding into an appropriate-size glass beaker, making a pocket in the feed, and weighing the test material into the pocket feed. Each dietary level was blended in a blender with approximately 200 g of feed for approximately 2 minutes to ensure an apparent homogeneous mix. Based on test level, multiple premixes were carried out. Each of the five levels of premixes were placed in a small mixer and mixed for 15 minutes.

DIET PREPARATION
- Rate of preparation of diet: Fresh diets were prepared weekly
- Diet was thoroughly mixed with the appropriate amount of the test substance.
- Storage temperature of food: The feed was stored frozen intermittently.

VEHICLE
No vehicle for the test substance was used in the study. The test substance was mixed thoroughly with the diet prior to oral administration.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
STABILITY
Stability analysis of the test material in the diet was conducted at Days 3, 7, and 10 (stored at room temperature), Day 14 (stored frozen), Day 7 (Days 1-4 freezer, Days 5 - 7 room temperature), for low and high-dose levels. At Week 4, stability analysis was conducted again for Days 0, 3, and 4 at room temperature.Stability determinations indicated that there was a slight decrease in the amount of test material in the diet remaining after 7 days when stored at room temperature. Diet samples for Group 2 (250 mg/kg bw/day) were within 25% after 7 and 10 days, when stored at room temperature. However, diet samples for Group 6 (5000 mg/kg/day) were within 45% of the target after 7 and 10 days at room temperature. Analysis following 4 days in the freezer and 3 days at room temperature indicated that the 250 and 5000 mg/kg bw/day diet formulations were within 10% of the target concentration. Diet samples for Group 2 (250 mg/kg bw/day) were within 1% of target after 3 and 4 days during Week 4, when stored at room temperature. However, diet samples for Group 6 (5000 mg/kg bw/day were within 6% of target after 3 and 4 days during Week 4, when stored at room temperature.

HOMOGENEITY
Homogeneity of the test material in the diet was determined prior to the initiation of dosing and at Week 4 from duplicate samples taken from the top, middle and bottom of a full-size batch of the low- and high dose levels.
Homogeneity analysis of samples from the top, middle, and bottom of the blender indicated that the test material and diets were mixed homogeneously (within 10% of the target and each other). Routine analyses at each weekly preparation indicated that all levels were within 7% of the target level.

ROUTINE ANALYSIS
Routine analyses were performed on one of the duplicate samples collected from the middle of each batch at all weeks of study. Analyses were performed by Gas chromatography.
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
Daily in feed
Dose / conc.:
250 mg/kg diet
Remarks:
nominal
Dose / conc.:
500 mg/kg diet
Remarks:
nominal
Dose / conc.:
1 000 mg/kg diet
Remarks:
nominal
Dose / conc.:
2 000 mg/kg diet
Remarks:
nominal
Dose / conc.:
5 000 mg/kg diet
Remarks:
nominal
No. of animals per sex per dose:
5 rats/sex/dose
Control animals:
yes, plain diet
Details on study design:
- Rationale for animal assignment: The animals were assigned to different groups by weight randomization using computer program designed to ensure homogeneity of body weights.
Positive control:
None
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations checked: Mortality, moribundity, obvious indications of toxic and pharmacologic effects.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Gross signs of toxic and pharmacologic effects were performed weekly at each weighing interval.

BODY WEIGHT: Yes
- Time schedule for examinations: Prior to study initiation and weekly thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes, individual food consumption recorded twice weekly and reported weekly.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to initiation of the in-life phase and during Week 4 of the study. Ophthalmoscopic examinations were performed using an indirect ophthalmoscope with 1% Mydriacyl to dilate the pupil.
- Dose groups that were examined: All animals examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At terminal euthanasia during Week 5, blood was collected from the orbital sinus.
- Anaesthetic used for blood collection: Yes (CO2/O2)
- Animals fasted: Yes, overnight
- How many animals: Samples collected for all surviving animals
- Parameters checked: Cell morphology, corrected leukocyte count (COR WBC), erythrocyte (RBC), haematocrit (HCT), haemoglobin (HGB), leukocyte count (WBC), leukocyte differential, platelet count (PLATELET).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At terminal euthanasia during Week 5, blood was collected from the orbital sinus
- Animals fasted: Yes
- How many animals: Samples collected for all surviving animals
- Parameters checked: Alanine aminotransferase (ALT), albumin, aspartate aminotransferase (AST), blood urea nitrogen (BUN), calcium, chloride, creatinine, globulin, glucose, inorganic phosphorus, potassium, sodium, total bilirubin, and total protein.

URINALYSIS: Yes
- Time schedule for collection of urine: At terminal euthanasia during Week 5, approximately 10 mL of urine was collected from each rat overnight prior to euthanasia. These samples were weighed and stored frozen (-70°C) for possible future analysis of test material.
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- Parameters checked: Appearance, glucose, pH, protein, ketones, microscopic examination of sediment, occult blood, specific gravity and urine volume.

NEUROBEHAVIOURAL EXAMINATION: Not examined
Sacrifice and pathology:
GROSS PATHOLOGY: Yes: Necropsies were performed on all surviving animals following 29 days of compound administration.
Each necropsy included examination of the following: external surfaces of the body, all orifices, carcass, external surface of the brain (at necropsy); the external surface of the spinal cord and cut surfaces of the brain and spinal cord were examined at the time of the tissue trimming, nasal cavity and paranasal sinuses, thoracic, abdominal, and pelvic cavities and their viscera, cervical tissues and organs, cranial cavity.
Organ weights were collected from the brain with stem, heart, kidneys, liver and stomach.
The following tissues from each animal were preserved in 10% neutral-buffered formalin: adrenals, aorta, bone marrow (femur), brain with brainstem, cervical spinal brainstem, colon, cecum, rectum, duodenum, jejunum, ileum, esophagus, eyes, heart, kidneys, lesions, liver, lumbar spinal cord, lungs, mammary gland, mesenteric lymph node, mid-thoracic spinal cord, ovaries, pancreas, pituitary glands, sciatic nerve, spleen, stomach, testes with epididymides, thymus, thyroid/parathyroids, trachea, urinary bladder and uterus.

HISTOPATHOLOGY: Yes: Gross lesions, brain with stem, and stomach were embedded in paraffin, sectioned, stained with haematoxylin and eosin, and examined microscopically from all animals in the control and high-dose groups. Liver and kidneys were embedded in paraffin, sectioned, stained with haematoxylin and eosin, and examined microscopically from all animals in all groups.
Statistics:
Absolute body weight (Weeks 1, 2, 3, 4, and 5), absolute body weight gains (Weeks 1, 2, 3, and 4), total body weight gains (Weeks 1 - 4), absolute food consumption values (Weeks 1, 2, 3, and 4), total food consumption (Weeks 1 - 4), clinical pathology data (except for cell morphology gradings and urinalysis), organ weight data (absolute and body weight ratios only) of the control group were compared statistically to the data from the same sex of the treated groups.
Statistical analyses were performed as listed: Levene’s Test of homogeneity of Variances (Log 10 Transformation, Square Transformations, Square Root Transformations, Reciprocal Transformations, Angular (Arcsine) Transformation, Rank Transformation, ANOVA, if significant then Dunnett’s Control vs. treatment comparisons (for equal variances, for unequal variances, if heterogeneous).
If variances of untransformed data were heterogeneous, a series of transformations was performed in an effort to achieve variance homogeneity. When the series of transformations was not successful in achieving variance homogeneity, analyses were performed on rank transformed data. Control versus compound-treated group mean comparisons were evaluated at the 5% two-tailed probability level.
Clinical signs:
no effects observed
Description (incidence and severity):
Observations of clinical signs were few and did not appear to be treatment-related.
Mortality:
no mortality observed
Description (incidence):
All animals survived until terminal euthansia.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related decreases were noted in the mean body weights and body weight changes of the 2000 and 5000 mg/kg bw/day males and 5000 mg/kg bw/day females. Statistically significant decreases in mean body weights were noted for the 5000 mg/kg bw/day males and females for Weeks 2-5 when compared to the concurrent control group.
There were significantly decreased mean total body weight change values for the 2000 mg/kg bw/day males at Weeks 1 - 4 and 5000 mg/kg bw/day males at Weeks 1, 2, and 4 and Weeks 1 - 4 when compared to the control group.
The mean body weight changes for the 5000 mg/kg/day females was significantly decreased at Week 1 and Weeks 1 - 4 when compared to the concurrent control groups.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The mean weekly and total food consumption values of the 5000 mg/kg bw/day males were decreased when compared to corresponding control values, with statistical significance occurring for the total food consumption value. The mean weekly food consumption for 5000 mg/kg bw/day females decreased at Week 1 compared to the concurrent control group. However, because of the food spilled by the 5000 mg/kg bw/day females, total food consumption could not be calculated.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No treatment-related changes were noted in the ophthalmological examinations.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No significant differences were noted in haematologic measures between treated and control groups. The mean erythrocyte, haemoglobin, and haematocrit values were decreased in the 5000 mg/kg bw/day males, but this decrease was primarily the result of abnormally low values noted in all three parameters of in one animal of the group.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The treatment-related decreases were noted for the mean glucose values of all test material dose group males, with statistical significance occurring for all test groups at or above 500 mg/kg/day.
Mean blood urea nitrogen values were significantly decreased for the 250, 1000, 2000, and the 5000 mg/kg bw/day males when compared to corresponding control values; no comparable significant decreases were observed in treated females.
The mean creatinine values were significantly decreased for 5000 mg/kg bw/day males when compared to corresponding control values.
The mean sodium value was significantly increased for 2000 mg/kg bw/day males when compared to corresponding control values.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No marked differences were observed between control and treated groups for the urine analysis tests performed.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related increase were noted in the absolute and relative liver weights of all test material dosed animals, with statistical significance occurring for all values except for the mean absolute and relative liver weights of the 250 mg/kg/day males, absolute liver weights of the 1000 mg/kg/day females, and relative liver weights of the 250 mg/kg/day females.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Gross lesions were noted in the liver, kidney, spleen stomach, and skin. Treatment-related liver lesions consisted of pale areas or enlarged appearance of the liver, which was noted primarily in the 5000 mg/kg/day males and females.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related histomorphologic alterations were noted in liver sections from test material males dosed with greater than 500 mg/kg/day and females dosed with either 2000 mg/kg/day or 5000 mg/kg/day. Diffuse hepatocellular enlargement occurred in a dose-related manner, with minimal to slight change in 1000 mg/kg/day males and 2000 mg/kg/day females and moderate enlargement in nine 5000 mg/kg/day animals. In addition to hepatocellular enlargement, eosinophilic cytoplasmic inclusions were noted in liver sections from males dosed with 250, 1000, 2000, or 5000 mg/kg/day, with the greatest incidence and severity occurring in 5000 mg/kg/day males.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
SEX DIFFERENCE: The sex difference in toxicity, as noted in the clinical and anatomic pathology, was not apparent.
Key result
Dose descriptor:
LOAEL
Effect level:
250 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
organ weights and organ / body weight ratios
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg diet
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
not specified
Conclusions:
3-L-Menthoxypropane-1,2-diol (Takasago Coolact 10) when administered in diet for 28 days to male and female Sprague-Dawley (Crl:CD®BR) rats at dose levels of 250, 500, 1000, 2000 and 5000 mg/kg bw/day, revealed a lowest observed adverse effect level (LOAEL) at 250 mg/kg bw/day for male and female rats (based on adverse effects observed in clinical chemistry and organ weights observed at this dose level).
Executive summary:

The 28 day subacute oral toxicity study of 3-L-Menthoxypropane-1,2-diol ( Takasago Coolact 10) was performed by following methods similar to the OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents).

Male and female Sprague-Dawley (Crl:CD®BR) rats with body weight range 223-255 g (males) and 147-173 g (females) were used in the study. The animals were housed two/cage by sex in stainless-steel, hanging, wire-mesh cages until randomization. After randomisation animals were housed individually.

The animals were maintained under controlled environmental conditions (temperature: 20.5 - 22.8°C, relative humidity: 57 - 7%, air changes: 10 times per hour and 12 h artificial light /12 h dark). Certified rodent chow (with the exception that animals were fasted before blood and urine sampling prior to scheduled termination) and tap water were provided ad libitum.

The animals were administered daily with the test material at dose levels of 0 (control), 250, 500, 1000, 2000 and 5000 mg/kg bw/day corresponding to control (Group 1) and Group 2 to Group 6 respectively in diet for 28 days. Plain diet was provided to the control animals. Five animals/sex/dose were used in the study.

The rats were observed twice daily for mortality and moribundity. Cage side observations were conducted daily for obvious indications of toxic and pharmacologic effects. Detailed physical examinations of each animal for gross signs of toxic and pharmacologic effects were performed weekly at each weighing interval. Individual body weights and physical examinations were recorded prior to initiation and weekly thereafter. Individual food consumption was recorded twice weekly and reported weekly.

Ophthalmoscopic examination was conducted for all animals prior to initiation of the in-life phase and during Week 4 of the study. Clinical pathology evaluations (haematology, clinical chemistry, and urinalysis) were performed and samples were taken from all surviving animals at terminal euthanasia (Week 5). Urine samples were collected overnight from all surviving animals prior to termination. Blood and urine samples were weighed and stored frozen at approximately -70 °C for possible future analysis of the test material.

Necropsies were performed on all surviving animals following 29 days of compound administration. Terminal body weights were recorded prior to necropsy. At termination, animals were exsanguinated under sodium pentobarbital anaesthesia. Animals were necropsied in a sequential manner so that one rat/sex/group was necropsied before repeating the sequence. Gross pathology (all surviving animals) and histopathology (in the control and high-dose groups) was also conducted.

All animals survived until terminal euthanasia. Observations of clinical signs were few and did not appear to be treatment-related. The only treatment-related clinical observation was few or no faeces, which occurred in 5000 mg/kg bw/day during Weeks 1and 2. Mean body weights and total body weight gains were decreased in 2000 and 5000 mg/kg bw/day males and 5000 mg/kg bw/day females, with statistical significance noted in 2000 (total body weight gain only) and 5000 mg/kg bw/day males and 5000 mg/kg bw/day females when compared to the concurrent control groups. Mean total food consumption was significantly decreased in the 5000 mg/kg bw/day males. Mean total food consumption in the Group 6 females could not be evaluated as a result of spillage.

No treatment-related changes were noted in the ophthalmological examinations.

In clinical pathology (haematology, clinical chemistry, and urinalysis) a dose-related decrease in serum glucose was observed in all the treatment group males (statistically significant in 500, 1000, 2000 and 5000 mg/kg bw/day). However, mean glucose levels were comparable in all female groups. Mean blood urea nitrogen values were significantly decreased for  250, 1000, 2000 and 5000 mg/kg bw/day males when compared to corresponding control values; no comparable significant decreases were observed in treated females. The mean creatinine value was significantly decreased for 5000 mg/kg bw/day females, and the mean sodium value was significantly increased for 2000 mg/kg bw/day males when compared to corresponding control values.

Gross and anatomic pathology revealed treatment-related changes in the liver. At gross necropsy, pale areas or enlarged appearance was noted primarily in the livers of 5000 mg/kg bw/day animals. There were significant dose-related increases in the liver and liver/body weight ratios in the males and females. The mean liver and liver/body weight ratios of the males were increased significantly in 500, 1000, 2000 and 5000 mg/kg bw/day. The mean liver and liver body weight ratios of the females were increased significantly in all groups except for the liver/body weight ratio in 250 mg/kg bw/day and liver weight in 1000 mg/kg bw/day.

Accompanying the increased liver weights was diffuse hepatocellular enlargement in 500, 1000, 2000 and 5000 mg/kg bw/day males and 2000 and 5000 mg/kg bw/day females and eosinophilic cytoplasmic inclusions in 500, 1000, 2000 and 5000 mg/kg bw/day males. The diffuse hepatocellular enlargement was dose related with minimal to slight changes noted in 500 mg/kg bw/day males and 2000 mg/kg bw/day females and moderate enlargement in nine 5000 mg/kg bw/day rats (males/females). Eosinophilic cytoplasmic inclusions were noted in the liver from 500, 1000, 2000 and 5000 mg/kg bw/day males, with the greatest incidence and severity noted in 5000 mg/kg bw/day males. No eosinophilic cytoplasmic Inclusions were noted in the female groups.

Therefore, 3-L-Menthoxypropane-1,2-diol (Takasago Coolact 10) when administered by oral gavage daily for 28 days to male and female Sprague-Dawley (Crl:CD®BR) rats at dose levels of 250, 500, 1000, 2000 and 5000 mg/kg bw/day, revealed a lowest observed adverse effect level (LOAEL) at 250 mg/kg bw/day for male and female rats (based on adverse effects observed in clinical chemistry and organ weights observed at this dose level).

This repeated dose oral (28 days) toxicity study is regarded as acceptable, and satisfies the guideline requirements of OECD 407 method (Repeated Dose 28-Day Oral Toxicity in Rodents).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
250 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
A study conducted under GLP conditions using methodology equivalent to that of a standardised guideline is available. The quality of the database is therefore considered to be good.
System:
hepatobiliary
Organ:
liver

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

The 28 day subacute oral toxicity study of 3-L-Menthoxypropane-1,2-diol (Takasago Coolact 10) was performed by following methods similar to the OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents).

About 44 day old male and female Sprague-Dawley (Crl:CD®BR) rats with body weight range 223-255 g (males) and 147-173 g (females) were used in the study. The animals were housed two/cage by sex in stainless-steel, hanging, wire-mesh cages until randomization. After randomization animals were housed individually.

The animals were maintained under controlled environmental conditions (temperature: 20.5-22.8 °C, relative humidity: 57-7%, air changes: 10 times per hour and 12 h artificial light /12 h dark). Certified rodent chow (with the exception that animals were fasted before blood and urine sampling prior to scheduled termination) and tap water were provided ad libitum.

The animals were administered daily with the test substance at dose levels of 0 (control), 250, 500, 1000, 2000 and 5000 mg/kg bw/day corresponding to control (Group 1) and Group 2 to Group 6 respectively in diet for 28 days. Plain diet was provided to the control animals. Five animals/sex/dose were used in the study.

The rats were observed twice daily for mortality and moribundity. Cage side observations were conducted daily for obvious indications of toxic and pharmacologic effects. Detailed physical examinations of each animal for gross signs of toxic and pharmacologic effects were performed weekly at each weighing interval. Individual body weights and physical examinations were recorded prior to initiation and weekly thereafter. Individual food consumption was recorded twice weekly and reported weekly.

Ophthalmoscopic examination was conducted for all animals prior to initiation of the in-life phase and during Week 4 of the study. Clinical pathology evaluations (haematology, clinical chemistry, and urinalysis) were performed and samples were taken from all surviving animals at terminal euthanasia (Week 5). Urine samples were collected overnight from all surviving animals prior to termination. Blood and urine samples were weighed and stored frozen at approximately -70 °C for possible future analysis of the test substance.

Necropsies were performed on all surviving animals following 29 days of compound administration. Terminal body weights were recorded prior to necropsy. At termination, animals were exsanguinated under sodium pentobarbital anaesthesia. Animals were necropsied in a sequential manner so that one rat/sex/group was necropsied before repeating the sequence. Gross pathology (all surviving animals) and histopathology (in the control and high-dose groups) was also conducted.

All animals survived until terminal euthanasia. Observations of clinical signs were few and did not appear to be treatment-related. The only treatment-related clinical observation was few or no faeces, which occurred in 5000 mg/kg bw/day during Weeks 1and 2. Mean body weights and total body weight gains were decreased in 2000 and 5000 mg/kg bw/day males and 5000 mg/kg bw/day females, with statistical significance noted in 2000 (total body weight gain only) and 5000 mg/kg bw/day males and 5000 mg/kg bw/day females when compared to the concurrent control groups. Mean total food consumption was significantly decreased in the 5000 mg/kg bw/day males. Mean total food consumption in the Group 6 females could not be evaluated as a result of spillage.

No treatment-related changes were noted in the ophthalmological examinations.

In clinical pathology (haematology, clinical chemistry, and urinalysis) a dose-related decrease in serum glucose was observed in all the treatment group males (statistically significant in 500, 1000, 2000 and 5000 mg/kg bw/day). However, mean glucose levels were comparable in all female groups. Mean blood urea nitrogen values were significantly decreased for  250, 1000, 2000 and 5000 mg/kg bw/day males when compared to corresponding control values; no comparable significant decreases were observed in treated females. The mean creatinine value was significantly decreased for 5000 mg/kg bw/day females, and the mean sodium value was significantly increased for 2000 mg/kg bw/day males when compared to corresponding control values.

Gross and anatomic pathology revealed treatment-related changes in the liver. At gross necropsy, pale areas or enlarged appearance was noted primarily in the livers of 5000 mg/kg bw/day animals. There were significant dose-related increases in the liver and liver/body weight ratios in the males and females. The mean liver and liver/body weight ratios of the males were increased significantly in 500, 1000, 2000 and 5000 mg/kg bw/day. The mean liver and liver body weight ratios of the females were increased significantly in all groups except for the liver/body weight ratio in 250 mg/kg bw/day and liver weight in 1000 mg/kg bw/day.

Accompanying the increased liver weights was diffuse hepatocellular enlargement in 500, 1000, 2000 and 5000 mg/kg bw/day males and 2000 and 5000 mg/kg bw/day females and eosinophilic cytoplasmic inclusions in 500, 1000, 2000 and 5000 mg/kg bw/day males. The diffuse hepatocellular enlargement was dose related with minimal to slight changes noted in 500 mg/kg bw/day males and 2000 mg/kg bw/day females and moderate enlargement in nine 5000 mg/kg bw/day rats (males/females). Eosinophilic cytoplasmic inclusions were noted in the liver from 500, 1000, 2000 and 5000 mg/kg bw/day males, with the greatest incidence and severity noted in 5000 mg/kg bw/day males. No eosinophilic cytoplasmic Inclusions were noted in the female groups.

Therefore, 3-L-Menthoxypropane-1,2-diol (Takasago Coolact 10) when administered by oral gavage daily for 28 days to male and female Sprague-Dawley (Crl:CD®BR) rats at dose levels of 250, 500, 1000, 2000 and 5000 mg/kg bw/day, revealed a lowest observed adverse effect level (LOAEL) at 250 mg/kg bw/day for male and female rats (based on adverse effects observed in clinical chemistry and organ weights observed at this dose level).

This repeated dose oral (28 days) toxicity study is regarded as acceptable, and satisfies the guideline requirements of OECD 407 method (Repeated Dose 28-Day Oral Toxicity in Rodents).

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, although effects were seen in the group dosed at levels of 250 mg/kg bw/day the registrant considers that the severity of these effects is such that the substance does not require classification with respect to this endpoint.