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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jan 29 - Sep 24, 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Principles of method if other than guideline:
None
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The present study was available before the implementation of REACH.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: waxy mass

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: HsdPoc:DH
Sex:
female
Details on test animals and environmental conditions:
** Test System
Guinea pig, HsdPoc:DH, females
Breeder: Harlan Winkelman GmbH, Borchen Age: about 3 - 4 weeks
The mean initial body weight at the start of the study was 318 g (range from 300 to 338 g).
In a large number of tests, with a variety of test materials, the guinea pig and the specific strain used has proven to be a species well suited for skin sensitization studies.

** Identification and adaption
Healthy young animals were allocated to the study groups at least 7 days before dosing to allow them to acclimatize. The guinea pigs were identified by colour mark, whereas the pretest animals were marked with a different colour than the animals of group I and 2 (details are documented in the raw data).


Assignment
20 guinea pigs were used in this study.

Pretest: 5 females
Group 1: negative control group (5 females) Group 2: test material group (10 females)


- Housing:
Five guinea pigs were housed in type GMI5 (EBECO) Makrolon cages with a shelter and placed on mobile racks. The animals were kept on conventional softwood granulate as the bedding. The bedding was changed two times a week.

- Diet (e.g. ad libitum):
Diet and community tap water from Makrolon drinking bottles were available to the guinea pigs ad libitum.

The diet, Provimi Kliba 3418.0, was checked according to the specifications of the manufacturer by independent laboratories. Analysis included both qualitative and quantitative evaluation for heavy metals, aflatoxins, pesticides and antibiotics. The tap water was analyzed periodically according to the German regulations for human drinking water. The softwood granulate was analytically checked by independent laboratories.



ENVIRONMENTAL CONDITIONS
Temperature and atmospheric humidity were measured by a thermohygrograph. The room temperature within the study period was 22 to 23 °C and the relative atmospheric humidity 52 to 61 %.

Study design: in vivo (non-LLNA)

Induction
Route:
other: intradermal and topical
Vehicle:
paraffin oil
Challenge
Route:
other: topical
Vehicle:
paraffin oil
No. of animals per dose:
Total: 20 females
Pre-test: 5 females
Control group: 5 females
Test group 10 females
Details on study design:
RANGE FINDING TESTS:
- intradermal injection: 0, 1, 5, 10, 25, 50 g/L given to one animal in paraffin oil
- topical applications, no pretreatment: 10, 100, 200, and 400 g/L in polyethylene glycol 400
- topical applications, FCA pretreatment: 10, 50, 100, and 200 g/L in polyethylene glycol 400

MAIN STUDY
A1. INDUCTION EXPOSURE (intradermal injection)
- No. of exposures: 6 injections of 0.1 mL each, three on each side of the spinal column)
- Test groups: 0.1 mL FCA + NaCl, 0.1 ml 25g/L test item ad liquid paraffin, 0.1 mL FCA + 25 g/L test item + NaCl
- Control group: 0.1 mL FCA + NaCl, 0.1 mL liquid paraffin, 0.1 mL FCA + NaCl
- Site: cranial, medial, caudal
- Frequency of applications: single


A2. INDUCTION EXPOSURE (topical application)
- No. of exposures: one week after injection (A1) single topical application of a filter plate soaked with either test solution, or vehicle
- Test groups: Test item (400 g/L) ad PEG 400
- Control group: PEG 400
- Site: cranial, medial, caudal
- Frequency of applications: single
- Duration: 48 h, occlusive


B. CHALLENGE EXPOSURE
- No. of exposures: single two weeks after topical induction (A2)
- Day(s) of challenge: 1d
- Exposure period: 2 weeks after topical application
- Test groups: 100 g/L test item ad PEG 400
- Control group: PEG 400
- Site: cranial, medial, caudal
- Evaluation (hr after challenge): 48 and 72 h

-- Observations
During the induction phase, the guinea pigs were examined on days 2, 3, 7, 11, 12, 15, and 22 of the experimental part for local skin reactions and the results were documented.
The behavior and general condition of all animals were monitored daily. The challenge sites were investigated for reactions 48 hours after start of the challenge. Further inspections followed 72 hours after start of the challenge, to detect weak or slowly developing reactions.
After the challenge, skin changes at the application sites were evaluated according to the following MAGNUSSON and KLIGMAN scheme.



Positive control substance(s):
yes
Remarks:
Hexylcinnamaldehyde

Results and discussion

Positive control results:
50% positive reactions with control substance

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
25 g/L (intradermal induction I), 400 g/L (topical induction II), 100 g/L (topical challenge)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no signs of toxicity
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25 g/L (intradermal induction I), 400 g/L (topical induction II), 100 g/L (topical challenge). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no signs of toxicity.
Reading:
2nd reading
Hours after challenge:
72
Group:
test group
Dose level:
25 g/L (intradermal induction I), 400 g/L (topical induction II), 100 g/L (topical challenge)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no signs of toxicity
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 25 g/L (intradermal induction I), 400 g/L (topical induction II), 100 g/L (topical challenge). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no signs of toxicity .
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
0 g/L
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no signs of toxicity
Reading:
1st reading
Hours after challenge:
72
Group:
positive control
Dose level:
10 g/L
No. with + reactions:
5
Total no. in group:
10
Clinical observations:
no signs of toxicity

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the given experimental conditions, the test material induced no reactions. According to EU Regulation No. 1272/2008 (CLP), the test material is not classified as a skin sensitizer.
Executive summary:

Purpose

The purpose of this GPMT assay was to identify the contact allergenic potential of teh test material. This study should provide a rational basis for risk assessment to the sensitising potential of the test item in man.

Study Design

The test material was investigated for skin sensitizing properties in the guinea pig maximization test according to MAGNUSSON and KLIGMAN (1969).

5 females were treated with the vehicles liquid paraffin and polyethylene glycol 400 (group 1) and 10 females were treated with the test material (group 2).

Induction included intradermal injection of the test material preparation in liquid paraffin (25 g/L with and without Freund's complete adjuvant) on experimental day 1, and topical application of test material preparation in polyethylene glycol 400 (400 g/L) for 48 hours on experimental day 8.

Challenge by topical application of the test material preparation in polyethylene glycol 400 (100 g/L) for 24 hours was performed two weeks after topical induction and readings were taken at 48 hours and 72 hours after start of treatment.

Results

The animals did not show any clinical signs during the course of the study and no cases of mortality were observed. After challange no positive reactions were seen 48 or 72 hours after treatment with the test material.

Conclusion

Under the given experimental conditions, the test material induced no reactions. According to EU Regulation No. 1272/2008 (CLP), the test material is not classified as a skin sensitizer.