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Diss Factsheets

Toxicological information

Repeated dose toxicity: dermal

Currently viewing:

Administrative data

Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 Sep - 19 Dec 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report date:
1991

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.28 (Sub-Chronic Dermal Toxicity Test: 90-Day Repeated Dermal Dose Study Using Rodent Species)
Version / remarks:
adopted in 1988
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Version / remarks:
adopted in 1981
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OPP 82-3 (Subchronic Dermal Toxicity 90 Days)
Version / remarks:
adopted in 1984
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Chlorocresol
EC Number:
200-431-6
EC Name:
Chlorocresol
Cas Number:
59-50-7
Molecular formula:
C7H7ClO
IUPAC Name:
4-chloro-3-methylphenol
Details on test material:
Batch No.: 280

Test animals

Species:
rat
Strain:
other: Wistar, Bor:WISW(SPF Cpb)
Details on species / strain selection:
The species is recommended in test guidelines for subchronic toxicological studies. Additionally, animals of this strain have been employed for many years for toxicological studies at the testing laboratory. Historical data on their physiology and spontaneous alterations is available.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Winkelmann, Borchen, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approximately 8 - 10 weeks
- Weight at study initiation: 196 - 213 g (males), 165 - 186 g (females)
- Housing: individual in Makrolon® cages type II on low-dust wood granules (during study and acclimation period)
- Diet: fixed-formula standard diet: Altromin® 1324 pellets (Altromin GmbH, Lage, Germany), ad libitum with the exception of the urine collection period
- Water: tap water, ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY: The tap water was of drinking quality. The nutritive composition and contaminant content of the standard diet were routinely spot-checked and analysed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): approx. 55 (temporarily at approximately 45% and/or 65%)
- Air changes (per hr): approx. 10 (reduced by approximately 50% during maintenance work on 2 Saturdays in November)
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Type of coverage:
occlusive
Vehicle:
polyethylene glycol
Remarks:
PEG 400
Details on exposure:
TEST SITE
- Area of exposure: dorsum to the left of the spinal column (approximately 5 x 5 cm)
- Type of wrap if used: 20-cm-long and approx. 10-cm-wide strip of Fixomull Stretch
- Time intervals for shavings or clipplings: weekly

REMOVAL OF TEST SUBSTANCE
- Washing: The treated area was cleaned with the formulation vehicle and cellulose.
- Time after start of exposure: approximately 6 h

TEST MATERIAL
- Amount(s) applied: 1 mL
- Constant volume used: yes
- For solids, paste formed: yes/no

VEHICLE
- Justification for use and choice of vehicle: The test substance was analytically shown to be stable in the vehicle.
- Amount(s) applied: 1 mL
- Lot/batch no.: 8259111
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability and content of the test compound in the formulations were examined using GC analysis. Prior to study initiation the stability of the test substance in the vehicle (polyethylene glycol 400) was investigated in two samples (100.9 mg test substance/100 mL solution and 70.069 g test substance/ 100 mL solution in vehicle) over a storage period of 7 days at room temperature. According to these results the mixtures were stable for a period of 3 days and 7 days at the low and the high dose, respectively. The measurement result of the low dose formulation after 7 days could not be evaluated due to increased unit areas. Homogeneity was not investigated, as the substance in question was a solution. The content of the test substance in vehicle was determined four times in the course of investigations. During the first scheduled content check (week 2) the content in the application formulation 100 mg/kg clearly deviated from nominal values (68 mg/kg analysed). Additional investigations were performed in week 5 and 6 because of these results; they showed no significant deviations from nominal values. At the second scheduled content check (week 10) no significant deviations from nominal values were determined either. The check of the preparation formula and substance consumption gave no indication of the deviations observed during the first content check. Since, during the additional investigations and the 2nd content check, no significant deviations were recorded for the formulations prepared according to the same formula; this incorrect nominal content is probably due to incorrect sampling in the animal room or to an error during determination in the analytical laboratory. Since the additional content checks at other times revealed no significant deviations from nominal values, assessment of the study is not affected by the incorrect nominal content at the first check.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily, 5 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
20 mg/kg bw/day
Dose / conc.:
50 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
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 the results of a subacute dermal study over 4 weeks in male Wistar rats, in which the test substance was dermally applied at concentrations of 0, 40, 200, and 1000 mg/kg bw/day. No effects were observed in the rats treated with 40 and 200 mg/kg bw/day. Animals of the high dose group showed decreased body weight gain (approximately 50% reduction) and decreased feed and water intake (approximately 14 and 12% reduction). Skin reactions in this group included erythemas, oedemas, wounds and escharoses on the treated areas as well as leathery and thick skin areas. Additionally, the mean skinfold thickness was bigger than that of the control animals. One animal of the high dose group dose was sacrificed in moribund conditions on the 16th day of experiment. This animal and one further animal necropsied at the end of study showed ureterectasia and blood clots in the urinary bladder.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (once daily on weekends and holidays)
- Cage side observations included: mortality, clinical signs, unusual features

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a week

DERMAL IRRITATION: Yes
- Time schedule for examinations: at daily cage side and weekly detailed clinical observations

BODY WEIGHT: Yes
- Time schedule for examinations: before treatment, then once weekly and prior to necropsy

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
No

WATER CONSUMPTION: Yes
- Time schedule for examinations: before treatment, then once weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: 3 days prior to study initiation and at the end of study in week 12 (day 80)
- Dose groups that were examined: all animals of the control and highest dose group (500 mg/kg bw/day)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in week 5 or 6 and at study termination
- Anaesthetic used for blood collection: Yes (ether; at study termination); no (week 5 or 6)
- Animals fasted: No (at study termination); yes (week 5 or 6)
- How many animals: all animals of each group
- Parameters examined: differential blood count, erythrocyte morphology, erythrocyte count, haemoglobin concentration, haematocrit, leukocyte count, mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), mean corpuscular cell volume (MCV), thromboplastin time, thrombocyte count, reticulocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in week 5 or 6 and at study termination
- Animals fasted: No (at study termination); yes (week 5 or 6)
- How many animals: all animals of each group
- Parameters examined: albumin, glucose, cholesterol, urea, total bilirubin, creatinine, total protein, triglycerides, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), glutamate dehydrogenase (GLDH), electrolytes (inorganic phosphate, chloride, calcium, potassium, sodium)

URINALYSIS: Yes
- Time schedule for collection of urine: in week 5 or 6 and at study termination over a 16 h period (overnight)
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- Parameters examined: semi-quantitative: ketone body, pH value, blood, glucose, bilirubin, protein, urobilinogen, sediment; quantitative: total protein, volume, density, protein, creatinine

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
- Measurement of skinfold thickness in all animals of all groups on the day of the 20th and 60th treatment prior to application and after removal of the bandage.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All surviving animals were sacrificed at study termination and the organs/tissues were subjected to a detailed gross pathological examination. The brain, heart, liver, lungs, spleen, kidneys (in pairs), adrenals (in pairs), and testes (in pairs) were weighed before fixation.

HISTOPATHOLOGY: Yes
The following organs of all control and the high dose group animals were investigated: adrenals, aorta, femur with bone marrow, brain, colon, caecum, duodenum, ileum, jejunum, rectum, duodenum, epididymis, eyes with nervi optici, femoral muscle heart, kidneys, liver, lungs, lymph nodes, mammary glands with skin, oesophagus, ovaries, pancreas, pituitary, prostate, salivary glands, seminal vesicles, nervus ischiadicus, skin of back (treated and normal skin), spleen, spinal cord, stomach, sternum with bone marrow, testicles, thymus, thyroid gland, trachea, urinary bladder, uterus, vagina, and all tissues with grossly apparent lesions. In addition, livers, lungs and kidneys of all animals of the 20 and 100 mg/kg bw/day dose groups were examined.
Statistics:
Arithmetic group means and standard deviations for bw, food consumption, water-intake, blood and urine analysis and organ weights were determined. The values for the test collective were compared with the control collective by significance test (U-test) using H.B. Mann and D.R. Whitney´s method, or by Wilcoxon´s method on the significance level alpha = 5% and alpha = 1% (two-tailed). Differences with p-values ≤ 0.01 and ≤ 0.05 were considered statistically significant.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
FEMALES
0, 20, 100 and 500 mg/kg bw/day: restless behaviour (increased motility with leaping, throwing down, jumping up, running in circles, biting into cage cover and occlusion) in all animals during the first 10 treatment days; the symptoms decreased during day 11 and 13; behaviour was normal from day 14 onwards
Dermal irritation:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
MALES
500 mg/kg bw/day: statistically significantly decreased mean body weight during week 2

FEMALES
20 mg/kg bw/day: statistically significantly increased mean body weight during weeks 0 and 3
500 mg/kg bw/day: statistically significantly increased mean body weight during week 0
0, 20, 100 and 500 mg/kg bw/day: decreased mean body weight during week 10
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
MALES
20 mg/kg bw/day: statistically significantly decreased leucocytes at study termination
100 mg/kg bw/day: statistically significantly decreased mean corpuscular haemoglobin concentration (MCHC), thromboplastin time and monocytes in week 6; statistically significantly increased lymphocytes in week 6; statistically significantly increased haemoglobin and haematocrit at study termination; statistically significantly decreased thrombocyte count and segmented neutrophils at study termination
500 mg/kg bw/day: statistically significantly decreased monocytes

FEMALES
20 mg/kg bw/day: statistically significantly increased leucoctyes in week 6
100 mg/kg bw/day: statistically significantly decreased thromboplastin time in week 6; statistically significantly decreased eosinophils at study termination

Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
MALES
20 mg/kg bw/day: statistically significantly increased urea and total bilirubin in week 6;
100 mg/kg bw/day: statistically significantly increased total bilirubin and potassium in week 6; statistically significantly decreased calcium in week 6
500 mg/kg bw/day: statistically significantly decreased triglycerides and calcium in week 6; statistically significantly increased potassium in week 6; statistically significantly decreased total protein at study termination

FEMALES
20 mg/kg bw/day: statistically significantly increased cholesterol in week 6; statistically significantly decreased potassium at study termination
100 mg/kg bw/day: statistically significantly decreased calcium and sodium in week 6; statistically significantly decreased potassium and calcium at study termination
500 mg/kg bw/day: statistically significantly increased creatinine and cholesterol in week 6; statistically significantly decreased calcium in week 6; statistically significantly decreased potassium and calcium at study termination
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
MALES
100 mg/kg bw/day: statistically significantly decreased protein and creatinine in week 6; statistically significantly decreased creatinine at study termination
500 mg/kg bw/day: statistically significantly decreased volume, protein and creatinine in week 6

FEMALES
20 mg/kg bw/day: statistically significantly increased volume and protein in week 6
100 mg/kg bw/day: statistically significantly decreased density at study termination
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
MALES
100 mg/kg bw/day: statistically significantly decreased absolute and relative (to body weight) liver weight

FEMALES
100 mg/kg bw/day: statistically significantly decreased spleen weight relative to body weight
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
MALES
Control: reduced testes size in 2/10 animals
20 mg/kg bw/day: yellow areas in the epididymides in 1/20 animal
500 mg/kg bw/day: pallid kidney in 2/10 animals

FEMALES
500 mg/kg bw/day: pallid kidney in 1/10 animal
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Details on results:
CLINICAL SIGNS
Since unsettled behaviour occurred in females only and across all dose groups including control animals a treatment-related effect was excluded.

BODY WEIGHTS AND BODY WEIGHT GAIN
Statistically significant effects on the mean body weights of males and females at 20 or 500 mg/kg bw/day were isolated findings and not considered treatment-related. Additionally, the reduced mean body weights of all females of all dose and control groups in week 10 were considered to be not treatment-related.

FOOD AND WATER INTAKE
Some isolated and slight, but statistically significant, deviations occurred which were not considered treatment-related.

HAEMATOLOGICAL FINDINGS
Some values marked as statistically significant differed only slightly from the control values, were within the scattering range of historical controls and/or were not dose-dependently distributed and are thus of no biolocgical/toxicological relevance. Significantly decreased monocytes of the high dose males were also not considered treatment-related since the means and all individual values were within the scattering range.

CLINICAL BIOCHEMISTRY FINDINGS
Statistical significantly lower triglyceride values in males at 500 mg/kg bw/day was not considered to be toxicologically relevant since the difference with respect to control animals was only slight and occurred only at one investigation date and only in one sex. The lower protein content in the same group was within the scattering range of historical controls and the difference with respect to the control group was only slight. Thus, this effect was not attributed to the treatment. Further findings, i.e. isolated effects on total bilirubin and urea in males or on cholesterol and creatinine in females, are of no biological/toxicological relevance, as the differences were only slight and/or distributed dose-independently and fortuitously. With regard to electrolyte concentrations, decreased calcium concentrations were noted at 100 and 500 mg/kg bw/day in males at females at the 6 week blood collection and also in females at study termination. The differences with respect to the controls were slight and the distribution was not dose-correlated. As the other investigations did not produce findings correlating with this one, the differences in calcium concentrations are not considered to be of toxicological relevance. Differences in potassium and sodium concentrations are considered to be of no biological/toxicological relevance, because the differences were only slight and/or distributed dose-independently and fortuitously.

URINALYSIS FINDINGS
The differences in mid and high dose males regarding protein and creatinine in urine at the 6 week urine collection and at 100 mg/kg bw/day at study termination as well as isolated effects on urinary volume, density and protein in low and mid dose females are not considered to be of biological/toxicological relevance, because the differences were only slight, not dose-correlated and occurred in a few isolated cases at only one date and in only one sex.

GROSS PATHOLOGICAL FINDINGS
Yellow areas observed in the epididymis of 1/20 male at 20 mg/kg bw/day is not considered treatment-related since this is a finding that sometimes occurs spontaneously in animals of this age and is of no toxicological relevance. The pale kidney observed in one high dose female and two high dose males was not associated with any functional or histopathological finding or changes in the kidney weight. Thus, observations in the kidney were not considered treatment-related or adverse.

ORGAN WEIGHTS
The decreased absolute and relative liver weight in mals and absolute spleen weight in females at 100 mg/kg bw/day were not dose-correlated and not attributed to the treatment.

Effect levels

Dose descriptor:
NOAEL
Effect level:
>= 500 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

Table 1: Mean body weights (g)

Week

               Dose (mg/kg bw/day)

0

20

100

500

0

20

100

500

MALES

FEMALES

0

205

203

203

203

174

180*

178

180*

1

227

226

222

223

175

180

178

178

2

251

248

245

241*

181

185

181

184

3

260

259

251

247

183

189*

185

185

4

267

268

258

257

186

190

187

187

5

277

276

264

264

187

192

192

189

6

289

288

279

279

191

193

195

192

7

303

303

292

292

195

196

197

196

8

313

311

302

302

197

198

201

199

9

321

318

308

310

200

200

202

201

10

321

319

311

314

196

195

198

196

11

334

332

321

323

203

202

206

204

12

331

335

320

325

202

204

206

203

13

329

332

320

326

202

201

205

203

*p < 0.05; ** p < 0.01

Table 2: Haematological findings

Dose (mg/kg bw/day)

Week

LEU

HB

HCT

MCHC

THRO

HQUICK

LYM

SEGM

EOS

MONO

MALES

0

6

12.7

152

0.475

321

1127

29.6

87.0

9.6

0.8

2.7

20

6

11.8

153

0.479

320

1129

30.5

86.9

10.6

0.4

2.1

100

6

11.9

156

0.492

317*

1130

27.4*

91.2*

7.8

0.0

1.0*

500

6

11.6

153

0.480

319

1137

28.5

86.4

11.7

0.2

1.8

0

13

16.3

153

0.472

325

1202

26.9

72.3

22.3

0.5

4.8

20

13

13.5*

154

0.478

323

1151

27.3

81.0

13.4

0.8

4.9

100

13

14.2

162*

0.501**

323

1104*

27.6

82.1

13.1*

0.7

4.1

500

13

13.6

158

0.486

325

1137

27.4

81.9

14.4

1.2

2.5**

 

 

FEMALES

0

6

8.6

147

0.471

313

1198

27.8

88.3

8.7

1.0

2.0

20

6

10.3**

149

0.470

317

1094

27.5

84.9

12.7

0.7

1.6

100

6

9.6

148

0.467

316

1158

24.3**

89.2

9.2

0.2

1.4

500

6

8.7

150

0.475

316

1111

26.6

87.4

10.4

0.6

1.7

0

13

8.3

136

0.426

319

1080

25.5

82.0

13.1

1.6

3.4

20

13

9.5

137

0.429

319

1082

25.6

87.4

9.9

0.5

2.2

100

13

8.9

139

0.431

323

1035

24.5

86.0

9.6

0.2*

4.2

500

13

8.1

140

0.433

324

1050

26.1

84.3

12.3

0.6

2.9

LEU = Leucocytes(10E9/L), HB = Haemoglobin (g/L), HCT = Haematocrit (L/L), MCHC = Mean corpuscular haemoglobin concentration (g/L erythrocytes), THRO = Thrombocytes(10E9/L), HQUICK = Thromboplastin time (sec), LYM = Lymphocytes (%), SEGM = segmented neutrophils (%), EOS = Eosinophils (%), MONO = monocytes (%)

*p < 0.05; ** p < 0.01

Table 3: Clinical biochemistry findings

Dose (mg/kg bw/day)

Week

Creatinine (µmol/L)

Urea (mmol/L)

Bilirubin (µmol/L)

Protein (g/L)

Cholesterol (mmol/L)

Triglycerides (mmol/L)

Sodium (mmol/L)

Potassium (mmol/L)

Calcium (mmol/L)

MALES

0

6

43

6.61

1.8

61.8

2.05

1.04

144

4.5

2.65

20

6

41

7.30*

2.1*

61.3

1.99

0.90

143

4.6

2.67

100

6

44

6.93

2.1**

62.5

1.97

1.09

143

5.0**

2.46**

500

6

45

6.90

1.9

60.9

1.98

0.76**

143

5.0**

2.46**

0

13

53

7.65

2.0

64.7

1.62

1.00

144

5.1

2.55

20

13

49

7.53

2.1

63.2

1.68

0.96

145

5.0

2.56

100

13

44

7.50

1.9

62.1

1.71

0.96

144

5.1

2.53

500

13

46

7.08

2.0

61.7*

1.75

0.88

144

5.3

2.50

 

 

FEMALES

0

6

44

7.59

1.9

63.6

1.88

0.77

143

4.7

2.61

20

6

50

7.76

1.9

64.2

2.17*

0.97

142

4.7

2.62

100

6

47

7.76

1.8

64.1

2.09

0.89

142*

4.8

2.46**

500

6

48*

7.43

1.8

63.9

2.11*

0.85

142

4.7

2.42**

0

13

53

7.03

1.9

63.7

1.75

0.76

143

5.3

2.55

20

13

45

7.39

2.2

65.1

1.88

0.78

143

4.9**

2.55

100

13

41

6.97

2.1

63.8

1.83

0.75

143

5.1*

2.46**

500

13

52

6.98

2.1

63.1

1.77

0.77

143

4.9*

2.49*

*p < 0.05; ** p < 0.01

Table 4: Urinalysis findings

Dose (mg/kg bw/day)

Week

Volume (mL)

Density (g/L)

Protein (conc. × urinary volume) (mg)

Creatinine (conc. × urinary volume) (mg)

MALES

0

6

20

1011

8.6

57

20

6

20

1009

7.7

54

100

6

15

1014

5.9**

49**

500

6

15*

1015

6.3*

51*

0

13

14

1017

7.0

65

20

13

15

1016

8.0

66

100

13

11

1021

5.7

57*

500

13

15

1019

7.3

68

 

 

FEMALES

0

6

9

1016

1.1

36

20

6

14*

1012

1.5*

40

100

6

10

1018

1.3

39

500

6

7

1021

1.0

36

0

13

7

1024

2.2

35

20

13

11

1019

2.5

37

100

13

9

1017*

2.4

36

500

13

8

1021

2.3

34

*p < 0.05; ** p < 0.01

Table 5: Absolute and relative organ weights at study termination

Dose (mg/kg bw/day)

Liver

Spleen

MALES – Absolute (mg)

0

12263

608

20

11946

636

100

11106*

574

500

11651

614

 

FEMALES – Absolute (mg)

0

7354

534

20

7377

562

100

7089

607

500

7197

603

 

MALES – Relative to body weight (mg/100 g bw)

0

3728

184

20

3594

192

100

3469**

179

500

3572

189

 

FEMALES – Relative to body weight (mg/100 g bw)

0

3644

264

20

3666

280

100

3466

297*

500

3540

297

Applicant's summary and conclusion

Executive summary:

The potential toxicity of the test substance was assessed in a 90-day sub-chronic dermal toxicity study in the Wistar rat performed according to EU method B.28 and similar to OECD Guideline 411 as well as in compliance with GLP.10 male and female rats per sex and dose group received the test substance at concentrations of 0, 20, 100 and 500 mg/kg bw/day for 13 continuous weeks. The applications were performed 5 days per week for a duration of 6 h each. Observations for clinical signs and mortality were made twice daily (once daily on weekends and holidays). Individual body weights were determined before start of treatment, weekly thereafter and prior to necropsy. Food consumption and water-intake were recorded once per week. A detailed physical examination of all animals was performed before treatment and then weekly thereafter. Blood samples for haematology and clinical chemistry examination were collected in week 5 or 6 and week 13. Urine specimens were collected in 16 h intervals in week 5 or 6 and 13 after blood sampling. On treatment days 20 and 60 the thickness of the skin was determined on all animals. All surviving animals were sacrificed at termination and a gross pathological examination was performed. Organ weights of the brain, heart, testis (paired), liver, lung, spleen, kidneys (paired) and adrenals (paired) were determined. Histopathological examinations were performed with the tissues of all animals of the control and high dose group. In addition, ophthalmoscopic examinations were performed on all animals of the control and high dose group 3 days before the first treatment and on day 80. No mortalities occurred and no treatment-related clinical symptoms were noted during the study period. Body weight gain, food consumption and water intake, haematological and clinical chemistry examinations as well as urine analyses revealed no treatment-related effects. Absolute and relative organ weights, gross pathological and histopathological examinations showed no evidence of organ damage.Thus, under the conditions of the study, the NOAEL over a 3-month period of dermal administration with the test substance to the rat was ≥ 500 mg/kg bw/day in both sexes.