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Toxicological information

Repeated dose toxicity: dermal

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Administrative data

Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Peer reviewed data base

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Unnamed
Year:
2008
Report Date:
2008
Reference Type:
secondary source
Title:
Unnamed
Year:
1990

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EPA OPPTS 870.3250 (Subchronic Dermal Toxicity 90 Days)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under test conditions: Stable in distilled water over a four-week period, in the dark at room temperature. Stable in pyrogen-free sterile water over 14 days in the dark at room temperature.

Test animals

Species:
rat
Strain:
other: CD(SD)BR (VAF plus)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, England
- Age at study initiation: not stated
- Weight at study initiation: the rats were in a weight range of 230 to 276 g (males) and 197 to 234 g (females) at the start of the dosing period

Administration / exposure

Type of coverage:
semiocclusive
Vehicle:
water
Details on exposure:
- Area covered: Not reported.
- Occlusion: Application sites were covered with a semi-occlusive dressing for six hours after application of test article. Plastic Elizabethan collars were used during the exposure period.
Vehicle: Pyrogen-free sterile water for Group 1 (controls), Group 2 (5 mg/kg/day), and Group 3 (50 mg/kg/day). Group 4 (250 mg/kg/day) was treated with undiluted test article.
- Concentration in vehicle:
5 and 50 mg/kg/day group: 2.5mg/mL and 25 mg/mL, respectively.
250 mg/kg/day group: as received, 78.5%.
- Total volume applied:
0, 5, and 50 mg/kg/day group: 2 mL/kg bodyweight.
250 mg/kg/day group: 0.216 mL/kg/day.
Individual volumes were adjusted according to the most recently recorded bodyweight.
- Duration of exposure: Semi-occlusion for 6 hours. Application site was not washed between doses.
- Removal of test substance: None.
- Controls: Pyrogen-free sterile water.

Treatment sites were subdivided into quadrants, and application was rotated among quadrants daily.
Duration of treatment / exposure:
90 days
Frequency of treatment:
6 hours/day, 5 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
0 (water), 5, 50, 250 mg/kg bw (corresp. to concentrations of ca. 2.5%, 25% and the undiluted test substance)
Basis:
nominal per unit body weight
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
Observations:
Animals were observed and the appearance of the application sites was assessed daily.
- Clinical signs: Yes, animals were observed daily for changes in condition or behavior.
- Mortality: Yes. Mortality checks were performed twice daily.
- Body weight: Yes. Body weights were recorded weekly.
- Food consumption: Yes. Food consumption was recorded weekly
- Water consumption: Not measured.
- Ophthalmoscopic examination: Yes. Ophthalmoscopic examinations were performed before the start of treatment. All high dose and control animals were reexamined during week 13 prior to blood sampling.
- Haematology: Yes. Haematology was evaluated in all animals during the final week of treatment. Haematology Parameters: packed cell volume, haemoglobin concentration, erythrocyte count, total and differential leukocyte count, coagulation test (prothrombin time, partial thromboplastin time).
- Clinical Chemisty: Yes. Blood chemistry was evaluated in all animals during the final week of treatment. Blood Chemistry Parameters evaluated were: blood urea nitrogen, glucose, alanine aminotransferase, aspartate aminotransferase, total protein, albumin, A/G ratio, sodium, potassium, calcium, chloride, inorganic phosphorus, bilirubin, and creatinine.
- Urinalysis: Yes. Urinalysis was performed on all animals during the final week of treatment. Parameters evaluated were: volume, specific gravity, pH, protein, glucose, ketones, bilirubin, urobilinogen, blood pigments, erythrocytes, leucocytes, crystals, and casts.
Sacrifice and pathology:
- Organ Weights: Yes. Thymus, heart, liver, spleen, kidneys, adrenals, testes or ovaries, and brain were weighed.
- Gross and histopathology: Yes. All animals were examined at the end of treatment for gross and histopathology. Tissues of organs were preserved for microscopic analysis. All tissues from all control and high dose animals, as well as lungs, liver, kidneys, skin, and all gross lesions from all animals were examined microscopically.
Statistics:
Statistical analysis of body weights, organ weights, & haematological data was by analysis of variance, and where differences were significant at the 5% level, by pairwise t-tests between control and treated groups. Blood chemistry data were examined for significant differences by Kruskal-Wallis test for between group differences, and where differences were significant at the 5% level, by with Wilcox rank sum test.

Results and discussion

Results of examinations

Details on results:
Observations:
- Clinical signs: Other than yellow staining at the application site in the 50 and 250 mg/kg/day groups, there were no treatment-related clinical signs. Animals in all groups exhibited periorbital, perinasal, and cranial fur staining, which was attributed to effects of the Elizabethan collar.
- Mortality: No animals died during treatment
- Body weight gain: Body weight gain was not adversel affected by treatment
- Food consumption and compound intake: Food consumption was not affected by treatment.
- Ophthalmoscopic examination: No treatment-related ocular changes occurred.

- Haematology: All haematological parameters, i.e., packed cell volume, haemoglobin concentration, erythrocyte count, total leucocyte count, and leukocyte differential count, as well as prothrombin time and partial thromboplastin time, were within the normal range for the performing laboratory.

- Clinical chemistry: Statistical differences from controls occurred as follows: 250 mg/kg/day: sodium (male) and glucose (female); 50 mg/kg/day: Sodium (female) and chloride (male); 5 mg/kg/day alanine aminotransferase (male), inorganic phosphorus (female), and chloride (female).
However, all the listed parameters were within the normal range for rats of this age and sex. None of the changes were dose-related or associated with other pathological findings. Therefore, the differences in blood chemistry from controls were not considered to be biologically significant or related to administration of the test material.

- Urinalysis
No changes in urine composition or cellularity occurred.

Sacrifice and pathology:
- Organ weights: Statistically significant increases in absolute and brain weight-related spleen weights in females from all treated groups were attributed to a slightly increased bodyweight in the affected animals. Body weight related organ weights were unaffected.
- Gross and histopathology:
Macroscopic pathology: One male from the 250 mg/kg/day group had a small area of scab formation at the application site, and two females from the same group had abnormal raised brown areas at the application site. Areas of hairloss and scab formation also occurred on untreated areas in one male and one female from the 250 mg/kg/day group, and hair loss at an untreated area occurred in one female from the 50 mg/kg/day group.
Microscopic pathology: No systemic changes showing a treatment-related distribution were observed. Animals from the 5 mg/kg/day group did not show any significant pathological changes.
Abnormal histopathological findings occurred at the application sites of all treated groups. Incidences of epidermal hyperplasia, scab formation, and ulceration of application sites were slightly higher at 250 mg/kg/day than at 50 mg/kg/day. Animals in the 5 mg/kg/day group did not show any significant pathological changes.

Effect levels

open allclose all
Key result
Dose descriptor:
NOEL
Remarks:
local effects (skin)
Effect level:
5 mg/kg bw/day
Sex:
male/female
Basis for effect level:
dermal irritation
Key result
Dose descriptor:
NOAEL
Remarks:
systemic effects
Effect level:
> 250 mg/kg bw/day (nominal)
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
LOAEL
Remarks:
systemic effects
Effect level:
> 250 mg/kg bw/day
Sex:
male/female

Any other information on results incl. tables

Table 1: Mortality:

Dose

[mg/kg/day]

Number dead /
number investigated

0

0/10

5

0/10

50

0/10

250

0/10

 

Table 2: Results of blood chemistry investigations – group mean values:

Parameter

Unit

Control

Low Dose

Medium Dose

High Dose

Dose

mg/kg/day

0

5

50

250

Blood Chemistry – Males

Alanine minotransferaseA

CalciumA

 

U/L

mmol/L

 

35

99.7

 

44*

99.8

 

30

101.2*

 

37

99.8

Blood Chemistry – Females

GlucoseA

Inorganic PhosphorusA

ChlorineA

PotassiumA

 

mg%

mg%

mmol/L

mmol/L

 

1.9

4.9

106.5

3.5

 

113

4.3*

104.0**

3.5

 

115

4.4

106.1

3.7

 

128*

4.5

108.3

3.8*

A All values were within expected laboratory ranges.

* = significantly different from controls, p<0.05.

** = significantly different from controls, p<0.01.

 

Table 3: Absolute and Bodyweight-related Spleen Weights, and Bodyweight Gains - Group Mean Values:

Parameter

Unit

Control

Low Dose

Mediu Dose

High Dose

Dose

mg/kg/day

0

5

50

500

Absolute spleen weight

Males

Females

grams

grams

0.68

0.51

0.73

0.59**

0.67

0.59*

0.78

0.59*

Brain-related spleen weight

Males

Females

grams

grams

34.08

26.35

35.76

30.62*

34.03

31.81*

38.83

30.74*

Total bodyweight gain

Males

Females

grams

grams

180

39

178

48

175

40

177

48

* = significantly different from controls, p<0.05.

** = significantly different from controls, p<0.01.

 

Applicant's summary and conclusion