Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
06 Feb 1996 to 07 Mar 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Version / remarks:
1981
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPP 82-2 (Repeated Dose Dermal Toxicity -21/28 Days)
Version / remarks:
1984
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Tif: RAIf, SPF strain
Sex:
male/female

Results and discussion

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

Achieved doses

Concentration analysis results: The mean test substance concentrations were 101.4%, 102.3%, 105.9% and 102.1% of nominal for formulations prepared at concentrations of 1, 10, 100 and 500 mg/mL, respectively predose. During administration concentrations were 116.0%, 115.4%, 113.3% and 109.9% of nominal for dose formulations prepared 10, 30, 125 and 500 mg/mL, respectively.

Homogeneity results: The test substance was homogeneously distributed in the vehicle and varied between ‑2% to +3% of the mean concentrations.

Stability results: The test substance was stable in the vehicle at room temperature over a period of 7 hours, covering the period of dosing.

 

Mortality: There were no mortalities.

 

Clinical observations: There were no treatment-related clinical signs and no local irritation at the application sites.

 

Body weight and weight gain: Weight gain of males at 1000 mg/kg was retarded during the first 2 weeks of treatment but was unaffected by treatment at other dose levels and in females at 1000 mg/kg.

 

Table 1: Intergroup comparison of body weights and body weight gain (g) – selected time points

 

Dose level of the test substance (mg/kg bw/day)

Males

Females

0

20

60

250

1000

0

20

60

250

1000

week 1

277.8

290.3

294.9*+

292.1

277.4

231.8

233.9

234.0

231.6

228.9

week 2

292.6

309.6*

311.4*

307.1

288.8

234.9

232.0

236.0

225.3

237.1

week 4

338.8

353.1

353.1

351.8

324.7

250.9

248.9

250.5

244.0

248.0

* p =< 0.05 (Wilcoxon), +/- p =< 0.01

 

Food consumption and compound intake: There were no treatment related effects on food consumption.

 

Haematology: There were no treatment-related effects on haematological profiles. All statistically significant values either showed no dose-relationship or recorded values were within historical ranges.

 

Clinical chemistry: Females at 250 and 1000 mg/kg had slightly raised plasma glucose levels and minimally raised alkaline phosphatase activities. Triglyceride levels were also slightly raised in females at 1000 mg/kg. Significantly elevated plasma K+ levels in all male groups are considered not to have been influenced by the test substance since control values were unusually low. Other differences from the controls in blood chemistry are considered not to be treatment related because they were too small to be of toxicological relevance (inorganic phosphate levels in males at 1000 mg/kg) or their occurrence showed no relationship to dose level.

 

Table 2: Intergroup comparison of treatment related clinical chemistry parameters

Parameter

Dose level of the test substance (mg/kg bw/day)

Male

Female

0

20

60

250

1000

0

20

60

250

1000

Glucose (mmol/L)

7.528

7.786

7.886

7.592

6.622

6.390

6.126

6.159

9.592*

9.739*+

Alk. phosphatase (u/L)

171.5

140.0

144.7

155.2

135.6

108.3

95.42

95.62

174.5*

139.8+

Triglycerides (mmol/L)

0.501

0.623

0.688

0.708

0.516

0.515

0.506

0.438

0.700

1.091*+

*p < 0.05 (Wilcoxon);+positive trend (p < 0.01 Jonckheere)

 

Sacrifice and pathology

Macroscopic findings: There were no treatment related macroscopic findings..

 

Organ weights: There were no treatment related effects on organ weights or ratios.

 

Microscopic findings: Morphological changes in the skin of the application site occurred at low incidence in both treated and control animals and are considered to have been induced by the treatment procedure rather than by the administration of the test substance. The liver, kidneys and adrenal glands were identified as target organs on the basis of histological findings. An increased incidence of minimal inflammatory cell infiltration and necrosis of single hepatocytes occurred in the liver of females at 250 and 1000 mg/kg. The elevated incidence of this lesion at 60 mg/kg is considered to fall within the limits of normal variation for this strain of rat. Effects on liver morphology were absent in the males. In the kidneys, minimal tubular hyaline change in males and minimal chronic tubular lesions in females were apparent at 1000 mg/kg. A single female at this dose level also showed minimal basophilic cell infiltration in the renal tubules. Lower dose levels were unaffected by these renal changes. In the adrenal glands, minimal inflammatory cell infiltration was evident in the cortex of females at 1000 mg/kg. No other groups were affected.

 

Table 3: Incidence of treatment-related histopathological findings

Organ/finding

Dose level of the test substance (mg/kg bw/day)

Male

Female

0

20

60

250

1000

0

20

60

250

1000

Adrenal glands:

cortical fatty change

inflammatory cell infiltration

 

4

0

 

4

0

 

3

0

 

3

0

 

4

0

 

1

0

 

0

1

 

1

0

 

1

1

 

1

3

Kidneys:

 

 

 

 

 

 

 

 

 

 

chronic progressive nephropathy

0

0

0

0

1

0

1

0

0

0

glomerulosclerosis

0

1

0

0

0

0

0

0

0

0

chronic tubular lesion

1

0

1

1

1

1

2

2

1

4

nephrocalcinosis

1

0

0

1

0

3

5

5

5

5

renal tubular cast

0

0

1

0

1

0

1

0

1

0

hyaline change

1

2

1

1

3

0

0

0

0

0

calcification

0

1

0

0

0

0

0

0

0

0

basophilic proliferation

2

2

0

2

1

0

0

0

0

1

Liver:

 

 

 

 

 

 

 

 

 

 

inflammatory cell infiltration

3

4

4

3

2

2

2

4

5

5

intrahepatic bile duct cholang/fibrosis

1

0

0

1

1

0

0

0

0

0

extramedullary hematopoiesis

0

0

0

0

0

1

0

0

0

1

Kupffer cell hyperplasia

0

0

0

0

0

1

0

1

0

0

hepatocyte necrosis

0

0

0

0

0

2

0

2

3

4

 

 

 

 

 

 

 

 

 

 

 

 

Applicant's summary and conclusion

Conclusions:
The no-observed-effect-level (NOEL) was 250 mg/kg (males) and 60 mg/kg (females) based on the occurrence of renal tubular hyaline change and minimal growth retardation at 1000 mg/kg (males) and histological changes in the liver at 250 mg/kg (females). In view of the low order of toxicity by the percutaneous route, a 90-day dermal study is not required.
Executive summary:

In this repeated dose toxicity test, performed according to OECD 410 under GLP, groups of 5 male and 5 female Sprague-Dawley-derived rats (Tif: RAIf, SPF strain) were exposed dermally to the test material at dose levels of 0, 20, 60, 250 and 1000 mg/kg bw/day, by topical application under occlusive dressing for 6 hours/day, 5 days/week, for 4 weeks. The test substance was formulated as a suspension in aqueous 0.5% carboxymethylcellulose/0.1% Tween 80. Mortality and clinical signs were checked daily, individual body weights and food and water consumption were recorded weekly. Haematological and clinical chemistry investigations were performed on all animals towards the end of the treatment period. All animals were subjected to necropsy and post mortem examination, major organs were weighed and selected tissues examined histopathologically.

There were no mortalities, no clinical signs of an adverse reaction to treatment and no local irritation at the application site at any dose level. Weight gain of males at 1000 mg/kg was retarded during the first 2 weeks of treatment but was unaffected by treatment at other dose levels and in females at 1000 mg/kg. Food consumption was unaffected by treatment at all dose levels. There were no treatment related effects on haematology profiles. Females at 250 and 1000 mg/kg had slightly raised plasma glucose levels and minimally raised alkaline phosphatase activities. Triglyceride levels were also slightly raised in females at 1000 mg/kg. No macroscopic anomalies were detected at necropsy and all organ weights and ratios were unaffected by treatment. A treatment related increase in the incidence of minimal inflammatory cell infiltration and necrosis of single hepatocytes occurred in the liver of females at 250 and 1000 mg/kg. In the kidneys, minimal tubular hyaline change in males and minimal chronic tubular lesions in females were apparent at 1000 mg/kg. A single female at this dose level also showed minimal basophilic cell infiltration in the renal tubules. In the adrenal glands, minimal inflammatory cell infiltration was evident in the cortex of females at 1000 mg/kg.

The no-observed-effect-level (NOEL) was 250mg/kg (males) and 60mg/kg (females) based on the occurrence of renal tubular hyaline change and minimal growth retardation at 1000 mg/kg (males) and histological changes in the liver at 250 mg/kg (females). In view of the low order of toxicity by the percutaneous route, a 90-day dermal study is not required.