Propanamide, N-[4-(2,4-dihydroxyphenyl)-2-thiazolyl]-2-methyl-

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 1998

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N-[4-(2,4-dihydroxyphenyl)-1,3-thiazol-2-yl]-2-methylpropanamide

EC Number:

807-621-3

Cas Number:

1428450-95-6

Molecular formula:

C13H14N2O3S

IUPAC Name:

N-[4-(2,4-dihydroxyphenyl)-1,3-thiazol-2-yl]-2-methylpropanamide

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V., Horst, The Netherlands
- Age at study initiation: 7 weeks
- Weight at study initiation: 212 - 256 g (males), 140 - 160 g (females)
- Housing: Animals were housed in groups of 3 or 4 in Makrolon type-4 cages with wire mesh tops and standard softwood bedding including paper enrichment.
- Diet: Pelleted standard HarlanTeklad 2914C rat / mouse maintenance diet, ad libitum
- Water: Community tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
From: 29 Nov 2012
To: 28 Feb 2013

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: PEG 300

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The dose formulations were prepared weekly. The test substance was weighed into a glass beaker on a tared balance and the vehicle added. The mixtures were stirred using a magnetic stirrer. Homogeneity of the test substance in the vehicle was maintained during the daily administration period using a magnetic stirrer.

VEHICLE
- Batch no.: BCBJ2013V

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The dose formulations were analyzed using an HPLC method provided by the Sponsor. Concentration and homogeneity of dose formulations were determined in samples taken after experimental start as well as during Weeks 3 and 12. On every timepoint, samples were taken from the top, the middle and the bottom of the vessel. Stability of the formulation was determined 4 hours and 8 days after experimental start.
The linearity of the analytical systems used for sample analyses was demonstrated with a good relationship between peak areas measured and calibration solution concentrations. All calibration points used met the acceptance limit of ± 20 % variation from the calibration curve derived by linear regression analysis. The coefficients of determination (R²) calculated were found to exceed 0.99.
The test substance peak was assigned in sample chromatograms by comparison to that of calibration solutions. In blank sample chromatograms no peak appeared at the retention time of the test substance and, therefore, the absence of the test substance in the control samples (PEG 300) was confirmed.
The test substance concentrations in the dose formulations ranged from 91.0 % to 104.4 % with reference to the nominal and were within the accepted range of ± 20 %.
The homogeneous distribution of the test substance in the preparations was confirmed because single results found did not deviate more than 4.2 % from
the corresponding mean and met the specified acceptance criterion of ≤ 15 %.
In addition, the test substance was found to be stable in application formulations when stored eight days at room temperature (20 ± 5 °C); recoveries met the variation limit of 10 % from the time-zero (homogeneity) mean value.

Duration of treatment / exposure:

90 days

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on a previous dose range finding toxicity study in Wistar rats, Harlan Laboratories study D58360 (non-GLP).

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily on days 1 to 3 (once daily immediately after treatment and again once daily thereafter), thereafter once weekly

BODY WEIGHT: Yes
- Time schedule for examinations: once weekly

FOOD CONSUMPTION:
- Time schedule: weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once during week 13
- Dose groups that were examined: control and high dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: once during week 13
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes
- How many animals: all

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: once during week 13
- Animals fasted: Yes
- How many animals: all

URINALYSIS: Yes
- Time schedule for collection of urine: once during week 13
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During week 13, relevant parameters from a modified Irwin screentest were evaluated in all animals in place of the usual weekly behavioral observation.
- Dose groups that were examined: all groups
- Battery of functions tested: sensory activity / grip strength / motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Examination of Lymph nodes (mesenteric and mandibular), Mammary gland area, Ovaries, Pancreas, Pituitary gland, Prostate gland and seminal vesicles incl. coagulating glands, Rectum, Salivary glands (mandibular, sublingual), Sciatic nerve, Skin, Spinal cord (cervical, midthoracic, lumbar), Spleen, Stomach, Testes (fixed in Bouin's solution), Thymus, Thyroid (incl. parathyroid gland, if possible), Trachea, Urinary bladder (filled w/formalin at necropsy), Uterus (incl. oviducts, cervix and vagina) and all gross lesions
- Weight determination of Ovaries, Spleen, Testes (fixed in Bouin's solution), Thymus and Uterus (incl. oviducts, cervix and vagina).
- Additionaly, Nasal cavity, Pharynx, Skeletal muscle, Tongue and Ureters were collected.
HISTOPATHOLOGY: Yes
- Examination of Lymph nodes (mesenteric and mandibular), Mammary gland area, Nasal cavity, Ovaries, Pancreas, Pharynx, Pituitary gland, Prostate gland and seminal vesicles incl. coagulating glands, Rectum, Salivary glands (mandibular, sublingual), Sciatic nerve, Skeletal muscle, Skin, Spinal cord (cervical, midthoracic, lumbar), Spleen, Stomach, Testes (fixed in Bouin's solution), Thymus, Thyroid (incl. parathyroid gland, if possible), Tingue, Trachea, Ureters, Urinary bladder (filled w/formalin at necropsy), Uterus (incl. oviducts, cervix and vagina) taken from animals of the control and high dose group and of all animals died before the end of the study period as well as all gross lesions of all groups.

Statistics:

Statistical analyses of the ophthalmoscopic findings, grip strength, locomotor activity, body weight, macroscopical findings, organ weights and ratios, as well as clinical laboratory data were made. The Dunnett-test (many to one t-test) based on a pooled variance estimate will be applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex. The Steel-test (many-one rank test) will be applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution. The Fisher's exact-test was also applied.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 800 mg/kg bw/day, transient clinical signs were noted at low incidence in a small number of male rats.
These findings were not considered to be systemic findings of toxicological relevance.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

one male of the mid dose group and two males of the high dose group died/were killed before the end of the study period (not test substance-related)

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

statistical significant reduced body weight and body weight gain in high dose males; slightly but not statistically significant reduced body weight and body weight gain in mid dose males

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

significantly reduced during almost the entire study period in high dose males

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

significantly elevated mean relative and absolute reticulocyte counts, increased mean corpuscular volume and relative count for high-fluorescent reticulocytes in high dose males and females

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

significantly higher bilirubin (mid dose females; high dose males/females), aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase (high dose males/females); significantly higher cholesterol and phospholipid levels (high dose females)

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

significantly increased urobilinogen were noted in high dose males; significantly increased urine production in high dose females

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

reduced grip strength in high dose males, reducedlocomotor activity in mid and high dose males

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

significantly higher: relative liver weights (low dose males, mid dose females, high dose males/females), relative kidney weights (low, mid, high dose males); significantly lower: relative thymus weights and thymus/brain weight ratios (high dose females)

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

MORTALITY
There were no deaths associated with systemic toxicity at any dose level.
All males of the control group and all males treated with 50 mg/kg bw/day survived until scheduled necropsy. All control and test substance-treated females survived until scheduled necropsy.
One male (no. 30) treated with 200 mg/kg bw/day was found dead on day 38 of treatment; death was considered to result from urogenital lesions of undetermined origin. At 800 mg/kg bw/day, one male (no. 37) was found dead on day 2 of treatment and a second male(no. 35) was killed for ethical reasons on day 29 of treatment, after a marked loss of body weight. The cause of death in these latter cases could not be definitively established but both were considered likely to be the result of misdosing.

CLINICAL SIGNS
Salivation was frequently noted immediately after dosing in both sexes treated with 200 mg/kg bw/day and at 800 mg/kg bw/day. This was considered to be a typical reaction to an unpleasant tasting test substance and was not considered to be an indication of systemic toxicity.
At 50 mg/kg bw/day and 200 mg/kg bw/day, there were no signs in males during weekly detailed clinical observations (weeks 1 - 12 of treatment). At 800 mg/kg bw/day, transient ruffled fur, swaying gait, Straub tail, breathing noises and unilateral ptosis were noted in a small number of males during the weekly detailed clinical observations performed during weeks 1 - 12). Neither the frequency nor type of findings were considered to be indicative of systemic toxicity.
There were no signs in any test substance-treated female during weekly detailed clinical observations (weeks 1 - 12 of treatment).

BODY WEIGHT AND WEIGHT GAIN
In males treated at 800 mg/kg bw/day, statistically significant reductions of the mean body weight (p<0.01) and mean body weight gain were noted from day 8 onwards. Males treated with 200 mg/kg bw/day had slightly lower mean body weight and mean body weight gain values, but the differences did not attain statistical significance. The mean body weight and the mean body weight gain of the male rats treated with 50 mg/kg bw/day compared favorably with those of the control males.
The test substance-treated females were unaffected.

FOOD CONSUMPTION
The mean daily food consumption values of the males treated with 800 mg/kg bw/day were significantly lower from days 1 - 15 and days 85 - 91 (p<0.01), and from days 29 - 85 (p<0.05). The mean relative food consumption of these males was significantly reduced (p<0.05) during days 1 - 8 and remained marginally lower during the entire treatment period.
In females treated with 800 mg/kg bw/day, the mean daily food consumption was significantly reduced (p<0.05) during days 1 - 8, but otherwise exceeded the food consumption of the control females during the remaining treatment period. The relative mean daily food consumption was significantly lower during days 1 - 8 (p<0.05), but significantly higher during days 43 - 50 (p<0.01), days 50 - 57 (p<0.05), days 71 - 85 (p<0.01) and days 85 - 91 (p<0.05).
At 50 mg/kg bw/day and 200 mg/kg bw/day, the mean absolute and relative food consumption of males and females were unaffected.

OPHTHALMOSCOPIC EXAMINATION
After 13 weeks, there were no test substance-related ophthalmoscopic changes in males or females treated at 800 mg/kg bw/day. The observed findings included unilateral corneal opacity or unilateral persistent pupillary membrane in single males. Females were without ophthalmoscopic findings.

HAEMATOLOGY
Males and females treated with 800 mg/kg bw/day had significantly elevated mean relative and absolute reticulocyte counts (mostly p<0.01) that exceeded the upper limit of the historical control data. Significantly elevated differences that did not exceed the historical control data but were considered to be related to the test substance included increased mean corpuscular volume (males, p<0.05 and females, p<0.01), and relative count for high-fluorescent reticulocytes (males and females, both p<0.05). The marginal increase in the mean relative hematocrit (females, p<0.05), mean absolute neutrophil count (males, p<0.01) and the mean corpuscular hemoglobin (females, p<0.05) remained within the range of the historical control data and considered to be unrelated to the treatment with the test substance.
The hematology parameters of the males and females treated with 200 mg/kg bw/day were unaffected. At 50 mg/kg bw/day, the mean red cell count was significantly reduced (p<0.05) in males, but in the absence of a dose response relationship or a similar finding in females was considered to be incidental. All other parameters were generally similar to those of the controls, were dose unrelated or were not supported by concomitant changes in related parameters.

CLINICAL CHEMISTRY
Test substance-related changes that were noted in the males and females treated with 800 mg/kg bw/day included significantly elevated total bilirubin (both p<0.01), increased activities of aspartate aminotransferase (p<0.01 in males and p<0.05 in females), alanine aminotransferase (both p<0.01) and alkaline phosphatase (both p<0.01). The increased enzyme activities were considered to be indicative of metabolic adaptation of the liver and the elevated total bilirubin may be a late effect of increased red cell elimination. Therefore, these findings were considered to be non-adverse effects.
Significantly higher cholesterol and phospholipid levels were noted in females only (both p<0.01); both exceeded the historical control levels. There were significant differences in several electrolytes as well, all of which remained within the limits of the historical control data: chloride was elevated in males (p<0.01) and calcium was elevated in males (p<0.01) and in females (p<0.05). In males only, the total protein was significantly lower (p<0.01) largely due to the significantly lower (p<0.01) globulin and which resulted in a significantly increased albumin/globulin ratio (p<0.01). The mean urea level was significantly reduced (p<0.05) in females treated with 800 mg/kg bw/day, but this finding is generally not associated with systemic toxicity and therefore considered to be incidental.
Total bilirubin was also elevated in the males and females treated with 200 mg/kg bw/day, but only the latter value attained statistical significance (p<0.01) and exceeded the historical control values. Males showed significantly (but dose-unrelated) increased sodium levels (p<0.01) and significantly elevated chloride levels (p<0.01). Males had significantly (but dose-unrelated) increased albumin level (p<0.05), significantly lower globulin level (p<0.01) and significantly increased albumin/globulin ratio (p<0.01).
At 50 mg/kg bw/day, significantly (but dose-unrelated) increased mean protein and albumin (both p<0.05) levels were noted in females.
All other parameters were generally similar to those of the controls, were dose unrelated or were not supported by concomitant changes in related parameters.

URINALYSIS
In animals treated with 200 and 800 mg/kg bw/day, significantly increased bilirubin levels (both p<0.01) were noted. In the absence of concomitant changes in related parameters, this finding was considered to be of no toxicological relevance and a typical difference that resulted from normal excretion of bilirubin. In males treated with 50 mg/kg bw/day, significantly increased bilirubin levels (p<0.01) were also noted, but this difference remained within the range of the historical control data.
At 800 mg/kg bw/day only, significantly increased pH (p<0.01) and urobilinogen (p<0.05) were noted in males (the latter difference exceeded the historical control data), and significantly increased urine production (p<0.01) was noted in females.
All other parameters were generally similar to those of the controls, were dose unrelated or were not supported by concomitant changes in related parameters.

NEUROBEHAVIOUR
At 800 mg/kg bw/day, Straub tail was noted in one male and dyspnea was noted in two males during the functional observational battery performed at week 13 of treatment. These findings were considered to be typical background findings without toxicological relevance. There were no signs in any test substance-treated males treated with 50 mg/kg bw/day or 200 mg/kg bw/day and no signs in any test substance-treated female during the functional observational battery (week 13 of treatment).
The mean fore- and hind grip strength of the males treated with 800 mg/kg bw/day were significantly reduced when compared with the control males (p<0.01 and p<0.05, respectively). Males treated with 50 mg/kg bw/day and 200 mg/kg bw/day were unaffected. Test substance-treated females were unaffected.
In males treated with 800 mg/kg bw/day, there was a statistically significant reduction in the mean locomotor activity (p<0.05) during the first 10-minute measurement interval when compared with the controls. During the subsequent measurement intervals (10-minute duration for a total measurement time of 60 minutes), the locomotor activity remained lower than that of the controls. The total locomotor activity (over 60 minutes) of these males was significantly lower (p<0.01) than that of the control males. Males treated with 200 mg/kg bw/day had reduced locomotor activity during all measurement intervals. Although the individual differences did not attain statistical significance, the total locomotor activity was significantly lower (p<0.05) when compared with the controls. The locomotor activity of the males treated with 50 mg/kg bw/day was generally similar to the control males. Minor differences were considered to be within the range of normal biological variability.
The mean locomotor activity of females was unaffected at all dose levels.

ORGAN WEIGHTS
In males treated with 800 mg/kg bw/day, significantly reduced mean absolute adrenal weights (p<0.05) were noted when compared with the controls. Females treated with 800 mg/kg bw/day had significantly elevated mean absolute liver weights (p<0.01), decreased mean absolute thymus weights (p<0.05), increased mean absolute kidney weights (p<0.05) and increased mean absolute ovary weights (p<0.05).
Females at 200 mg/kg bw/day showed significantly increased mean absolute liver weights (p<0.01) and kidney weights (p<0.05) when compared with the controls. The mean absolute organ weights of the males compared favorably with those of the control males.
At 50 mg/kg bw/day, the mean absolute liver weights of males and females were significantly higher (both p<0.01) than those of the respective controls.
However, there were marked differences in the mean terminal body weights of males (particularly at 800 mg/kg bw/day) that were not reflected in the terminal body weights of the females. Hence, the relative organ weight ratios were considered to more clearly illustrate test substance-related changes.
At 800 mg/kg bw/day, significantly elevated mean brain-to-body weight ratios (p<0.01) were noted in males, as were significantly elevated mean heart-to-body weight ratios (p<0.05). Females also showed significantly increased ovary-to-body weight ratio (p<0.01). These differences were considered to be body weight effects. Test substance-related changes noted at 800 mg/kg bw/day included significantly elevated mean liver-to-body weight ratios in males and females (both p<0.01), significantly decreased thymus-to-body weight and thymus-to-brain weight ratios in females (p<0.05) and significantly increased kidney-to-body weight ratio in males and females (both p<0.01). The increased relative liver weights were considered to be due to normal metabolic adaptation of the liver and the decreased relative thymus weights were considered likely to be the result of chemical stress. Therefore, these findings were considered to be non-adverse effects.
At 200 mg/kg bw/day, significantly increased kidney-to-body weight ratios were noted in males (p<0.01) and significantly increased liver-to-body weights were noted in females (p<0.01).
Males treated with 50 mg/kg bw/day had significantly increased liver-to-body weight ratios (p<0.01) and significantly increased kidney-to-body weight ratios (p<0.05). Females were unaffected.
Body weight-related changes included significantly reduced mean heart-to-brain weight ratios in males treated with 200 and 800 mg/kg bw/day (both p<0.05), and significantly elevated mean ovary-to-brain weight ratio in females at 800 mg/kg bw/day (p<0.05). Females at 800 mg/kg bw/day had significantly increased liver-to-brain weight ratio (p<0.01) and kidney-to-brain weight ratio (p<0.05), as well as significantly reduced thymus-to-brain weight ratio (p<0.05). Males had significantly decreased adrenal-to-brain weight ratio (p<0.05) and slightly increased (but statistically insignificant) thymus-to-brain weight ratios. At 50 and 200 mg/kg bw/day, significantly elevated mean liver-to-brain weight ratios (both p<0.01) were noted in females, whereas significantly increased kidney-to-brain weight ratios (p<0.05) were noted in females at 50 mg/kg bw/day.
None of the organ weight differences were associated with morphological changes and therefore, the differences in organ weight were considered to be without adverse toxicological relevance.

GROSS PATHOLOGY
There were no treatment-related macroscopical findings.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no treatment-related microscopical findings.
The findings recorded in this study were considered to be within the normal range of background alterations that is seen in untreated animals of this age and strain. In particular, a small increase in incidence ofalveolar macrophages in the lungs of high dose group males and females was considered unrelated to treatment because the lesions were extremely low in severity and because this is a common incidental finding in control rats of this strain and age in gavage studies.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

Target system / organ toxicity

Key result

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Mean male body weights [g]

		Negative Control	50 mg/kg bw/day	200 mg/kg bw/day	800 mg/kg bw/day
Day 1	Mean SD N	257.7 14.5 10	250.5 11.1 10	254.7 11.7 10	252.7 10.1 10
Day 8	Mean SD N	288.9 17.9 10	278.9 10.4 10	282.3 17.5 10	262.1** 13.5 9
Day 15	Mean SD N	307.5 18.6 10	301.6 12.3 10	300.7 19.9 10	280.2** 16.2 9
Day 22	Mean SD N	328.5 19.5 10	320.3 14.0 10	317.9 22.8 10	296.6** 21.3 9
Day 29	Mean SD N	339.5 25.0 10	333.5 14.5 10	329.4 23.7 10	300.3** 24.7 9
Day 36	Mean SD N	354.8 21.6 10	347.6 16.4 10	340.7 27.5 10	313.4** 25.2 8
Day 43	Mean SD N	370.1 22.5 10	362.8 18.3 10	355.5 32.0 9	320.4** 29.7 8
Day 50	Mean SD N	381.1 22.7 10	376.4 18.5 10	364.9 37.1 9	324.4** 38.1 8
Day 57	Mean SD N	392.7 24.0 10	385.5 20.5 10	376.0 36.4 9	338.5** 33.3 8
Day 64	Mean SD N	401.1 24.8 10	392.2 21.9 10	381.6 35.4 9	347.2** 35.3 8
Day 71	Mean SD N	409.0 25.2 10	402.1 24.8 10	387.0 35.3 9	350.0** 37.4 8
Day 78	Mean SD N	412.1 24.5 10	408.6 25.4 10	392.8 36.6 9	353.2** 39.2 8
Day 85	Mean SD N	419.0 25.4 10	415.1 25.7 10	399.0 38.6 9	355.7** 41.4 8
Day 91	Mean SD N	424.1 24.0 10	417.4 26.0 10	403.9 35.6 9	355.9** 41.2 8

Table 2: Mean female body weights [g]

		Negative Control	50 mg/kg bw/day	200 mg/kg bw/day	800 mg/kg bw/day
Day 1	Mean SD N	165.2 5.0 10	165.0 5.9 10	165.9 5.9 10	165.3 5.4 10
Day 8	Mean SD N	179.5 7.8 10	181.9 6.6 10	183.5 11.6 10	178.3 8.7 10
Day 15	Mean SD N	189.8 6.5 10	193.1 7.6 10	195.8 11.9 10	188.9 8.5 10
Day 22	Mean SD N	196.3 8.6 10	199.1 7.5 10	204.0 11.5 10	197.0 9.2 10
Day 29	Mean SD N	202.2 9.1 10	205.7 9.2 10	211.8 10.5 10	206.5 8.1 10
Day 36	Mean SD N	206.9 9.2 10	211.0 10.1 10	216.6 13.8 10	211.9 9.0 10
Day 43	Mean SD N	213.1 9.3 10	217.5 11.8 10	222.0 16.1 10	216.1 9.7 10
Day 50	Mean SD N	217.3 11.7 10	221.4 12.0 10	225.9 13.4 10	216.2 9.7 10
Day 57	Mean SD N	220.5 11.4 10	223.2 13.4 10	229.4 12.4 10	223.1 8.2 10
Day 64	Mean SD N	222.1 11.9 10	226.7 12.0 10	233.2 16.4 10	227.5 9.4 10
Day 71	Mean SD N	226.7 11.5 10	231.0 14.5 10	236.2 17.0 10	226.9 10.6 10
Day 78	Mean SD N	229.3 14.8 10	234.2 13.8 10	239.5 15.0 10	231.8 11.5 10
Day 85	Mean SD N	229.9 13.0 10	234.6 15.1 10	241.8 14.7 10	233.1 13.0 10
Day 91	Mean SD N	232.8 11.6 10	238.9 14.9 10	244.2 17.2 10	233.5 10.6 10

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, a no-observed-effect-level (NOEL) could not be established
for W 630 (Sample ID: 26480), but 50 mg/kg body weight/day was considered to be the noobserved-
adverse-effect-level (NOAEL).

Executive summary:

In this subchronic toxicity study, W 630 (Sample ID: 26480) was administered daily by oral

gavage to SPF-bred Wistar rats of both sexes at dose levels of

Group 1: 0 mg/kg body weight/day

Group 2: 50 mg/kg body weight/day

Group 3: 200 mg/kg body weight/day

Group 4: 800 mg/kg body weight/day

for a period of 91/92 days. A control group was treated similarly with the vehicle, PEG 300, only.

The groups comprised 10 animals per sex which were sacrificed after 91/92 days of treatment.

Clinical signs, outside cage observation, food consumption and body weights were recorded

periodically during the acclimatization and treatment periods. Ophthalmoscopy, clinical

laboratory diagnostics, functional observational battery, locomotor activity and grip strength

were performed during week 13. Samples of bone marrow suspensions were evaluated for the

presence of micronuclei.