(2E)-2-methyl-3-(4-methylphenyl)prop-2-en-1-ol

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:

15-09-2011 to 24-10-2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Justification for type of information:

Information as to the availability of the in vivo study is provided in 'attached justification'.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: US EPA OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents. Office of Prevention, Pesticides and Toxic Substances (7101), EPA 712-C-00-366, (2000).

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(2E)-2-methyl-3-(4-methylphenyl)prop-2-en-1-ol

EC Number:

700-548-7

Cas Number:

56138-10-4

Molecular formula:

C11H14O

IUPAC Name:

(2E)-2-methyl-3-(4-methylphenyl)prop-2-en-1-ol

Details on test material:

- Physical state: Waxy solid at temperature < 23°C and liquid at temperature > 23°C
- Storage condition of test material: In refrigerator (2-8°C) in the dark under nitrogen
- Other: white waxy solid at temperature < 23°C and clear colourless liquid at temperature > 23°C

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

The species and strain was selected in accordance with the OECD TG 407 and the other relevant guidelines.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Recognised supplier (reported in the full study report)
- Females (if applicable) nulliparous and non-pregnant: Yes.
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: males 132 - 149 g and females 110 – 129 g; individuals were randomly allocated to treatment groups using a randomization procedure. All animals within ± 20% of the sex mean.
- Fasting period before study: None
- Housing: Macrolon cages with sawdust bedding and paper, changed at appropriate intervals; group housed (5 per group) by sex. During locomotor investigations were housed individually in polycarbonate cages without cage-enrichment or bedding material.
- Diet (e.g. ad libitum): pelleted rodent diet, certified (recognised supplier), ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days.

DETAILS OF FOOD AND WATER QUALITY: Feed: pelleted rodent diet, certified (recognised supplier) – batch numbers and certificates of analysis provided in the full study report. The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3 – actual: 19.4 – 21.5 °C
- Humidity (%): 55 ± 15 (or 40 to 70) – actual: 43 – 82%
- Air changes (per hr): 15 per hour
- Photoperiod (hrs dark / hrs light): 12 h light / 12 h dark

IN-LIFE DATES: From: 15-09-2011 to 24-10-2011

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was prepared at the appropriate concentrations as a suspension in Propylene Glycol. Formulations (w/w) were prepared daily within 6 hours prior to dosing. The stability and homogeneity of the test item formulations were determined. Results show the formulations to be stable for at least 6 hours. Formulations (w/w) were prepared daily within 6 hours prior to dosing, and were homogenized to visually acceptable levels. Adjustment was made for specific gravity of the test substance and vehicle. No correction was made for the purity of the test substance. The test substance was heated to a maximum of 37.0°C for a maximum duration of approximately 3 hours. Test substance weighed for dosing on Days 11 to 17 was heated to a maximum of 44.6°C for approximately 1 hour.

DIET PREPARATION
- Rate of preparation of diet (frequency): Not applicable.
- Mixing appropriate amounts with (Type of food): Not applicable.
- Storage temperature of food: Not applicable.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Applicant assessment indicates: Propylene Glycol was considered as appropriate based on test item solubility. The stability and homogeneity of the test item formulations were determined during the study. Results show the formulations to be homogeneous and stable in vehicle.
- Concentration in vehicle: Samples of the test item formulations were taken on at least two occasions and analyzed for concentration of test item (method of analysis provided in full study report). The results concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). No test substance was detected in the Group 1 formulations.
- Amount of vehicle (if gavage): Treatment volume was 5 mL/kg for control (negative, untreated group) and all treatment groups with applicable test item concentrations per group.
- Other: Dose-formulations were analysed during the study and were reported as within ± 10% applied limits.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- The homogeneity and stability: Samples of the test item formulations were taken on at least two occasions and analyzed for concentration of test item (method of analysis provided in full study report). The results concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). The mean recoveries of the procedural recovery samples fell within the criterion of 90-110%. It demonstrated that the analytical method was adequate for the determination of the test substance in the test samples.
- The analysis consisted of HPLC analysis with external calibration (within a dedicated formulation analysis report attached to the full study report). Samples in Propylene Glycol were accurately fortified with known amounts of test item in an end solution of 50/50 (v/v) acetonitrile/water. These were then subjected to analysis by HPLC-DAD analysis using external calibration, with linear regression to calibration standard. The analytical method was validated (details available within the full study report). The test item samples were subject to a known dilution factor (details available within the full study report) to place the end samples in the calibration range within 50/50 (v/v) acetonitrile/water.
- Mean concentrations of dose-formulations analysed during the study were within ± 10% applied limits confirming accurate test item/vehicle formulation.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

Low – Group

Doses / concentrations 3

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

Intermediate – Group

Doses / concentrations 4

Dose / conc.:

600 mg/kg bw/day (nominal)

Remarks:

High – Group

No. of animals per sex per dose:

5 per sex per dose (5 male / 5 female)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on the results of a previously conducted 5-day sighting study (Report number attached to and cited in the full study report). Dose levels in the 5-day sighting test were: Group 1: 500 mg/kg bw/day (in Propylene Glycol) Group 2: 1000 mg/kg bw/day. In the 5-day range finder (administered consecutively, for 5-days) in three females the following effects were determined. High dose females: indicated a single mortality, and demonstrated lethargy, flat/abnormal posture, uncoordinated movements, abnormal gait, laboured respiration, piloerection and/or salivation (all animals, primarily on Days 4 and 5). Slightly lower to normal bodyweight in survivors. Slightly low food consumption and Pelvic dilation of the kidneys (one animal). Reduced liver weight for one surviving animal (normal for the other animal). Kidney weights considered to be normal. At 500 mg/kg bw/day (in Propylene Glycol) females indicated: no mortality. Lethargy and/or flat posture (two animals on Day 3). Normal bodyweights, food consumption. No abnormalities at necropsy and organ weights were normal. Basis: other: nominal in vehicle (Propylene glycol).
- Rationale for animal assignment (if not random): Randomly assigned
- Post-exposure recovery period in satellite groups: None.
- Section schedule rationale: Random

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards, detailed clinical observations were made in all animals. Once prior to start of treatment and at weekly intervals, this was also performed outside the home cage in a standard arena. These clinical observations were conducted at least between 1 and 2 hours after dosing. Additional functional observations were made as ‘special evaluations’. Functional performance tests were also performed on all animals during Week 4, together with an assessment of sensory reactivity to different stimuli. Observations were conducted between 1 and 3 hours after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded prior to dosing on Day 1 and at weekly intervals thereafter. Body weights were also performed prior to termination.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Not applicable.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not applicable.
- Other: Food consumption was recorded for each cage group at weekly intervals throughout the study.

FOOD EFFICIENCY: Yes.
- Body weight gain % was determined.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: End of treatment period (day 28) for all test and control group individuals.
- Anaesthetic used for blood collection: Yes. Nitrous oxide/oxygen (recognised supplier)
- Animals fasted: Yes. Overnight (maximum 20 hours).
- How many animals: All animals
- Parameters checked: Hemoglobin (Hb), Erythrocyte count (RBC), Hematocrit (Hct), Erythrocyte indices – including: mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), Total leukocyte count (WBC), Differential leukocyte count – including: neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas), Platelet count (PLT), Reticulocyte count (Retic). Additionally: Prothrombin time (CT) was assessed and Activated partial thromboplastin time (APTT) was assessed.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: End of treatment period (day 28) for all test and control group individuals.
- Animals fasted: Yes. Overnight (maximum 20 hours).
- How many animals: All animals
- Parameters checked: Urea, Aspartate aminotransferase (ASAT), Glucose, Alanine aminotransferase (ALAT), Total protein (Tot.Prot.), Alkaline phosphatase (AP), Albumin, Creatinine (Creat), Total cholesterol (Chol), Sodium (Na+), Total bilirubin (Bili), Potassium (K+), Triglycerides (Tri), Chloride (Cl-), Bile acids, Calcium (Ca++), Inorganic phosphosphate (P)

URINALYSIS: No.

NEUROBEHAVIOURAL EXAMINATION: Yes. Was conducted as part of ‘special evaluations’
- Time schedule for examinations: Functional performance tests were also performed on all animals during Week 4, together with an assessment of sensory reactivity to different stimuli. Observations were conducted between 1 and 3 hours after dosing.
- Dose groups that were examined: All.
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

OTHER: Additional post-termination observations were made at necropsy.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- organs weighed: Adrenal glands, Spleen, Brain, Testes, Epididymides, Thymus, Heart, Uterus (including cervix), Kidneys, Prostate1, Liver, Seminal vesicles including coagulating glands1, Ovaries, Thyroid including parathyroid1.
Where marked 1: weighed when fixed for at least 24 hours.

HISTOPATHOLOGY: Yes
- Organs and tissues preserved in neutral buffered 10% formalin: Identification marks: not processed, Ovaries, Adrenal glands, (Pancreas), (Aorta), Peyer's patches [jejunum, ileum] if detectable, Brain, [cerebellum, mid-brain, cortex], (Pituitary gland), Caecum, (Preputial gland), Cervix, Prostate gland, (Clitoral gland), Rectum, Colon, (Salivary glands - mandibular, sublingual), Duodenum, Sciatic nerve, Epididymides *, Seminal vesicles including coagulating gland, Eyes (including optic nerve [if detectable] and harderian gland) *, Skeletal muscle (Skin), (Female mammary gland area), Spinal cord -cervical, midthoracic, lumbar, Femur including joint, Spleen, Heart, Sternum with bone marrow, Ileum, Stomach, Jejunum, Testes *, Kidneys, Thymus (Larynx), Thyroid including parathyroid [if detectable], (Lacrimal gland, exorbital), (Tongue), Liver, Trachea, Lung, infused with formalin, Urinary bladder, Lymph nodes - mandibular, mesenteric, Uterus, (Nasopharynx), Vagina, (Oesophagus), All gross lesions
Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

The following slides were examined by a pathologist:
- all tissues collected at the scheduled sacrifice from all group 1 and 4 animals,
- all tissues from animal no. 3 which was found dead prior to scheduled blood sampling,
- stomach (both sexes) and spleen and kidneys (males) from group 2 and 3 animals,
- all gross lesions.

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one ttest) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex. (Ref. 1)
- The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution. (Ref. 2)
- The exact Fisher-test was applied to frequency data. (Ref. 3)

Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test (Ref. 4) to determine intergroup differences followed by the Wilcoxon test (Ref. 5) to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances.

1 C.W. Dunnett, A Multiple Comparison Procedure for Comparing Several Treatments with a Control, J. Amer. Stat. Assoc. 50, 1096-1121 (1955).
2 R.G. Miller, Simultaneous Statistical Inference, Springer Verlag, New York (1981).
3 R.A. Fisher, Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh (1950).
4 Kruskal W.H. and Wallis W.A., Use of ranks in one-criterion variance analysis. Journal of the American Statistical Association 47 (260): 583-621, December (1952).
5 Wilcoxon, F. Individual comparisons by ranking methods. Biometrics, 1, 80-83 (1945).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs of toxicity were noted during the observation period.
The incidence of breathing rales and laboured respiration shown by one male and one female respectively at 150 mg/kg occurred within the range of background findings to be expected for rat of this age and strain. These were considered signs of no toxicological significance.

Mortality:

no mortality observed

Description (incidence):

One control group male (No.3) exhibited mortality during the bleeding procedure on Day 28 of the study. This death was considered procedure related therefore is of no toxicological significance. There were no further unscheduled mortalities.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No treatment related effects on body weight development in test animals, which was similar to that of controls.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption before or after allowance for body weight was similar between treated and controls.

Food efficiency:

no effects observed

Description (incidence and severity):

No adverse effect on food consumption during the study. Body weight gains in test animals was similar to that of controls.

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

no effects observed

Ophthalmological findings:

not examined

Description (incidence and severity):

There were no toxicologically significant reported effects to the eyes (in life or post termination) in the parameters examined.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no toxicologically significant effects detected in the haematological parameters examined at the end of the treatment period.

At 600 mg/kg bw/day dose level: Females at 600 mg/kg showed a lower haemoglobin concentration and lower mean corpuscular haemoglobin concentration (MCHC) (means were inside the range considered normal for rats of this age and strain). In males, the higher mean platelet count was minor in nature (mean within normal range for rats of this age and strain)

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no toxicologically significant effects detected in the blood chemical parameters examined at the end of the treatment period.

At 600 mg/kg bw/day dose level: Statistically significant changes in clinical biochemistry parameters distinguished animals at 600 mg/kg from control animals Specifically: Higher albumin levels and lower inorganic phosphate levels in females. Higher glucose levels in males. This was not considered to be toxicologically relevant (all means were inside the range considered normal for rats of this age and strain).

At 600, 150 and 30 mg/kg bw/day dose levels: The higher potassium level in males at 600 mg/kg, the lower total protein level in males at 150 mg/kg and higher bile acid level in females at 30 mg/kg occurred in the absence of a dose-related trend and remained within the range considered normal for rats of this age and strain. Therefore was considered of no toxicological significance.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no treatment-related changes in the behavioural parameters or in sensory reactivity. There was no toxicologically relevant changes in functional performance.

Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals. The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with high activity in the first interval that decreased over the duration of the test
period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no toxicologically significant effects detected in the in the organ weights examined at the end of the treatment period.

At 600 mg/kg bw/day dose level: males showed a statistically significant higher liver to body weight ratio. Liver weights of females were also slightly higher, but not statistically significant (and the mean was within the range considered normal). Heart to body weight ratio was also slightly lower in these females (statistically significant, but within the range considered normal). This was considered of no toxicological significance as there was no morphological correlates and observations were slight in nature.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

There were no toxicologically significant effects detected in the macro-pathology parameters examined at the end of the treatment period.

At 600 mg/kg bw/day dose level: One male at 600 mg/kg (no. 16) showed an irregular surface of the forestomach.

Microscopic examination of the stomach revealed hyperplasia and hyperkeratosis of the non-glandular stomach in all animals at 600 mg/kg, and at lower incidence at 150 mg/kg. This corresponded to an irregular surface of the forestomach of one male at 600 mg/kg. The isolated reddish foci on the
glandular mucosa of this animal correlated with minimal congestion. These findings were considered to reflect irritant properties of the test substance. Given the generally mild nature of these local stomach effects, these were considered not to be of toxicological relevance.

The incidence of all other macroscopic incidental findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be of no toxicological relevance, and included pelvic dilation of the kidneys, red discolouration of the thymus, mesenteric and mandibular lymph nodes, reduced size of the epididymides or preputial glands, enlargement of the mandibular lymph node, red foci on the thymus or glandular mucosa, fluid in the uterus, watery-clear cysts on the kidneys, and an isolated yellowish nodule on the right lateral lobe of the liver.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There were no toxicologically significant effects detected in the in the histopathology examinations at the end of the treatment period.

At 600 mg/kg bw/day dose level: Stomach: Diffuse slight to moderate hyperplasia and minimal to slight hyperkeratosis of the nonglandular stomach (all animals at 600 mg/kg). Spleen: slightly increased severity of hematopoiesis in males at 600 mg/kg (up to moderate degree). Kidneys: slightly increased severity of hyaline droplet formation in males at 600 mg/kg (up to moderate degree).
At 150 mg/kg bw/day dose level: Stomach: Minimal hyperplasia of the non-glandular stomach was also present in 4 of 5 males and 2 of 5 females at 150 mg/kg. Minimal hyperkeratosis was present 1 of 5 males and 1 of 5 females at 150 mg/kg.

Microscopic examination of the stomach revealed hyperplasia and hyperkeratosis of the non-glandular stomach in all animals at 600 mg/kg, and at lower incidence at 150 mg/kg. This corresponded to an irregular surface of the forestomach of one male at 600 mg/kg. The isolated reddish foci on the
glandular mucosa of this animal correlated with minimal congestion. These findings were considered to reflect irritant properties of the test substance. Given the generally mild nature of these local stomach effects, these were considered not to be of toxicological relevance.

The slightly increased severity of splenic hematopoiesis in males at 600 mg/kg and the lower haemoglobin and mean corpuscular haemoglobin concentration in females of the same dose group were not correlated on sex level. Also, these changes were slight in nature and were considered not indicative of an adverse effect on red cell turn over.

The slightly increased severity of hyaline droplet formation in males at 600 mg/kg occurred in the absence of any degenerative changes in the kidneys, or changes in clinical biochemistry parameters indicative of renal dysfunction. Also given the slight nature of this finding, this was considered to be of no toxicological relevance.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

There were no treatment-related macroscopic abnormalities detected. There were no microscopic findings that were considered to be related to treatment with the test item.

Other effects:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

>= 600 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to adverse toxic effects at highest dose / concentration tested

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for males and females is considered to be 600 mg/kg body weight per day.

Executive summary:

The study was performed according the requirements of OECD TG 407, EU method B.7 and US EPA OPPTS 870.3050 guidelines under GLP conditions. Following a previously conducted 5-day sighting study, the systemic toxic potential of the test item was assessed orally in a 28 day oral gavage study in Wistar: Crl:WI(Han) rats. Three groups, each comprising five male and five female rats, received test item at doses of 30, 150 or 600 mg/kg/day. A control group of five males and five females was dosed with vehicle alone (Propylene Glycol). Chemical analyses of formulations were conducted once during the study to assess accuracy, homogeneity and stability over 6 hours. Formulation analyses confirmed that formulations of test substance in propylene glycol were prepared accurately and homogenously, and were stable over at least 6 hours. The following parameters were evaluated: clinical signs daily; functional observation tests in week 4; body weight and food consumption weekly; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues. In-life examinations (clinical appearance, functional observations, body weight and food consumption) did not reveal any abnormalities up to 600 mg/kg. Minor treatment-related microscopic findings were recorded in the stomach (both sexes), spleen (males) and kidneys (males) of Group 4 animals and in the stomachs (both sexes) of Group 3 animals. Stomach alterations consisted of diffuse minimal to moderate hyperplasia and minimal to slight hyperkeratosis in the non-glandular stomach of both sexes at 150 mg/kg and above. In addition, in 600 mg/kg males, there was an increased severity of two commonly observed microscopic findings hyaline droplets in the tubular epithelium of the kidney and hematopoiesis in the spleen. Based on the histopathological assessment, the no effect level for both sexes was 30 mg/kg, but none of these findings were considered to be detrimental to organ system function or vitality of the animals. No toxicologically relevant changes were noted in any of the other parameters examined in this study up to 600 mg/kg. Under the conditions of this study, the No-Observed-Adverse-Effect-Level (NOAEL) was regarded to be 600 mg/kg/day for males/females.