1,3-Oxathiolane-2-carboxylic acid, 5-hydroxy-, (1R,2S,5R)-5-methyl-2-(1-methylethyl)cyclohexyl ester, (2R,5R)-

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26.08.2004 to 14.12.2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was performed according to the OECD guideline 407 and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

OS Menthol Ester

IUPAC Name:

OS Menthol Ester

Constituent 2

Reference substance name:

(1R,2S,5R)-2-isopropyl-5-methylcyclohexyl (2R,5R)-5-hydroxy-1,3-oxathiolane-2-carboxylate

EC Number:

604-569-1

Cas Number:

147126-62-3

Molecular formula:

C14H24O4S

IUPAC Name:

(1R,2S,5R)-2-isopropyl-5-methylcyclohexyl (2R,5R)-5-hydroxy-1,3-oxathiolane-2-carboxylate

Constituent 3

Reference substance name:

(2R, 5R)-5-Hydroxy-[1,3]oxatbiolane-2-carboxylic acid, 2S-isopropyl-5R-methyl-1R-cyclohexyl ester

IUPAC Name:

(2R, 5R)-5-Hydroxy-[1,3]oxatbiolane-2-carboxylic acid, 2S-isopropyl-5R-methyl-1R-cyclohexyl ester

Test material form:

other: White powder

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Age at study initiation: 49-56 days (at study initiation / at test item administration)
- Weight at study initiation: males 252-319 g, females 175-209 g
- Fasting period before study: overnight
- Housing: individually
- Diet: Teklad Certified Rodent Diet #8728C ad libitum
- Water: ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25°C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Methyl cellulose

Details on oral exposure:

VEHICLE
- Concentration in water: 1 %
- Amount of vehicle (if gavage): 10 mL/kg
- Justification for choice of vehicle: The test system is an internationally accepted species for use in toxicity studies.
- Lot/batch no. (if required): 2489E

MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The Blank is the solvent blank and should be less than the MDL. The Calibration Verification Standard (CVS) is an independently prepared standard inDCM which should be between 75 and . 125 % recovery. The fit(~) of the calibration curve should be better than 0.99 and the Relative Percent
Difference of the duplicates should be less than 25%.

Duration of treatment / exposure:

28 consecutive days follwoed by a 14-day recovery period in a dedicated subset of rats.

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
62.5 mg/kg bw/day
Basis:
other: Nominal amount of 6.25 mg/ml of vehicle

Doses / concentrations 2

Remarks:

Doses / Concentrations:
125 mg/kg bw/day
Basis:
other: Nominal amount of 12.5 mg/ml of vehicle

Doses / concentrations 3

Remarks:

Doses / Concentrations:
250 mg/kg bw/day
Basis:
other: Nominal amount of 25.0 mg/ml of vehicle

No. of animals per sex per dose:

Each 5 males and 5 females in all 4 groups for necropsy after 28 days of treatment and each 5 males and 5 females in groups 1 (vehicle control) and group 4 (high dose) for a 14-day recovery period follwoing the treatment period.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses selected for this study were based on results of a range finding study in which dose levels ranging from 125 to 2000 mg/kg bw/day were tested with a treatment for up to 12 days.

Positive control:

None

Examinations

Observations and examinations performed and frequency:

- Moratlity: Twice daily
- Cage side observations/clinical signs: Twice daily
- Weighing: Day 0 (prior to dosing), Day 1 and weekly thereafter
- Food consumption: During acclimation and weekly thereafter until scheduled necropsy
- Water consumption: Monitored daily
- Ophthalmoscopy: During the last week of treatment
- Clinical pathology: At scheduled necropsy
- Organ weights: At necropsy

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All animals were eufuanized as per fue protocol schedule upon completion of the treatment and post-treatment periods by Isoflurane
overexposure, followed by exsanguination. Gross necropsy included an examination of the external surfaces of the body; all orifices; cranial
cavity; external surfaces of the brain and spinal cord; nasal cavity and paranasal sinuses; joints; thoracic, abdominal, and pelvic cavities and viscera.

HISTOPATHOLOGY: Yes

With the exception of the animal-identification, the tissues listed under Tissue Preservation from all Control and High Dose, Main Study and Recovery animalswere prepared for microscopic examination by embedding in paraffin wax, sectioning and staining with hematoxylin and eosin: Tissues of selected Low Dose and Mid Dose rats that were macroscopically abnormal were similarly processed.

Statistics:

In -life data was collected using the ·current Lab-Cat® In-Life module (Version 6.1; July 5/00). Necropsy data and organ weights were collected using the current Organ Weights/Necropsy module (V3.28; Apr. 23/99 - Innovative Programming Associates). Numerical data collected during the course of the study was subjected to calculation of group means and standard deviations. The data for males and females was separately analyzed for homogeneity of variance and for normality. Homogenous data was analyzed using the Analysis of Variance (at p=0.05) and the significance of intergroup differences was analyzed using Duncan's or other appropriate test. Heterogenous data was analyzed using the Kruskal-Wallis· test and the significance of intergroup differences between the control and treated groups were assessed using Dunn's or another appropriate test.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No mortality was observed. Three females at 250 mg/kg bw/day showed piloerection and lethargy at the end of treatment week 1.

Mortality:

mortality observed, treatment-related

Description (incidence):

No mortality was observed. Three females at 250 mg/kg bw/day showed piloerection and lethargy at the end of treatment week 1.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In one male and 3 females at 250 mg/kg bw/day a transient weight loss without statistical significance.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No significant effects in test item treated groups.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

No effects were noted.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No effects in all test item treated groups.

Haematological findings:

no effects observed

Description (incidence and severity):

All data within histroical control ranges at the end of treatment and recovery period.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

A marginal increase group mean for alanine aminotransferase (AL T) in high dose females at end of treatment. It was considered minimal, and to be a result of hepatocellular leakage caused by "fatty liver" as observed at necropsy and histopathology.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No eefetcs at all dose levels.

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

No statistical significant changes noted in males. Increased liver weights and decreased spllen and thymus weights in females at 250 mg/kg bw/day. No differences at end of recovery.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Only standard background findings were noted.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

High dose: Lymphoid tissue, activity spleen reduced, local irritant stomach/forestomach mucosa. No relevant findings at low and mid dose.

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
Animals from all treatment and control groups survived the 28-day treatment period.

BODY WEIGHT AND WEIGHT GAIN
Despite the weight loss observed in one male and three females from the High Dose Group, there were no statistically significant differences (ANOV A; p=0.05)in body weights and food consumption between the control and test groups, and among the test groups for males and females analyzed separately during thetreatment and recovery periods.

GROSS PATHOLOGY
The most prominent gross findings in High Dose female animals were the presence of microulcers in the glandular portion of the stomach (four
animals), and fatty infiltration of livers (two animals).
Gross findings in animals from all other groups were unremarkable, or incidental. Findings in some animals which were considered incidental were as follows: lung haemorrhaging (terminal), hydronephrosis, enlarged uterine horns etc. These findings are common in laboratory rats and are not considered treatment-related.
At the end of recovery period, no microulceration or fatty livers or any other relevant findings were observed in High Dose female animals.

HISTOPATHOLOGY:
Some animals treated with the test article at 250.0 mg/kg/day exhibited weight loss, piloerection, lethargy, local irritation of the mucosa of the
stomach and hepatic steatosis. Female animals appeared more affected than males indicating that there may be a gender difference in response to
the treatment. Lack or lower intensity of these observations in the recovery animals was suggestive of the reversibility of the pathologic processes induced by the test article.

Effect levels

Dose descriptor:

NOAEL

Effect level:

125 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Body weights of rats

Dose (mg/kg bw/day)	Mean body weights (g)			Final weight relative to vehicle controls (%)
	Initial*	final	Change**
Males
0	X ± Y	X ± Y	X ± Y	-
Test item	X ± Y	X ± Y	X ± Y	X ± Y
Test item	X ± Y	X ± Y	X ± Y	X ± Y
Test item	X ± Y	X ± Y	X ± Y	X ± Y
Test item	X ± Y	X ± Y	X ± Y	X ± Y
Test item	X ± Y	X ± Y	X ± Y	X ± Y
Females
0	X ± Y	X ± Y	X ± Y	-
Test item	X ± Y	X ± Y	X ± Y	X ± Y
Test item	X ± Y	X ± Y	X ± Y	X ± Y
Test item	X ± Y	X ± Y	X ± Y	X ± Y
Test item	X ± Y	X ± Y	X ± Y	X ± Y
Test item	X ± Y	X ± Y	X ± Y	X ± Y

Applicant's summary and conclusion

Conclusions:

In this 4-week oral toxicity study in the rat a combined NOAEL of 125 mg/kg bw was found.

Executive summary:

In conclusion, analysis of all generated data, including clinical observations, clinical pathology, gross necropsy and histopathology revealed no test item related toxicity in rats treated orally at 62.5 and 125.0 mglkg/day for 28 days. Some animals treated with the test item at 250.0 mglkg/day exhibited weight loss, piloerection, lethargy, local irritation of the mucosa of the stomach due to local irritation and hepatic steatosis. Female animals appeared more affected than males indicating that there may be a gender difference in response to the treatment. Lack or lower intensity of these observations in the recovery animals was suggestive of the reversibility of the pathologic processes induced by the test item.