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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:
Aug 05 - Nov 16, 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

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:
An in vivo skin sensitisation study according to OECD test guideline 406 was carried out in 1999, before the entry into force of the amendments to Annex VII.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

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 5 weeks
The mean initial body weight at the start of the study was 334 g (range from 301 to 376 g).

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 1 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 (10 females)
Group 2: test material group (20 females)

Housing:
--------
Five guinea pigs were housed in type 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, Altromin Standard TPF 3012, 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 20 to 22 °C and the relative atmospheric humidity 53 to 80%.

Study design: in vivo (non-LLNA)

Induction
Route:
intradermal and epicutaneous
Vehicle:
water
Concentration / amount:
Induction (intradermal): 25 g/L
Induction (topical); 100 g/L
Day(s)/duration:
intradermal induction: 1 week; topical induction: 1 week
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
Challenge: 1 g/L
Day(s)/duration:
1 day, 2 weeks after epicutaneouss induction; The challenge sites were investigated for reactions 48 hours after start of the challenge
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Total: 35 females
Pre-test: 5 females
Control group: 10 females
Test group 20 females
Details on study design:
RANGE FINDING TESTS:
- intradermal injection: 0, 0.1, 1, 10, 25, 50 g/L (aqueous preparation)
- topical applications, no pretreatment: 10, 50, 100, 200, 400 g/L (aqueous preparation)
- topical applications, with FCA: 1, 5, 10, 50 g/L (aqueous preparation)

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 group: 0.1 ml FCA + NaCl, 25g/L test item ad NaCl, 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 (100 g/L) ad NaCl
- Control group: NaCl aqueous solution
- Frequency of applications: single
- Duration: 48 h, occlusive
B. CHALLENGE EXPOSURE
- No. of exposures: single
- Day(s) of challenge: 1d
- Exposure period: 2 weeks after topical application
- Test groups: 1g/L test item ad NaCl
- Control group: NaCl
- 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

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
25 g/L (induction I), 100 g/L (induction II), 1 g/L (challenge)
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no signs of toxicity
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
test group
Dose level:
25 g/L (induction I), 100 g/L (induction II), 1 g/L (challenge)
No. with + reactions:
1
Total no. in group:
20
Clinical observations:
no signs of toxicity
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
1 g/L sodium chloride solution
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
1 g/L sodium chloride solution
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
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).

10 females were treated with the vehicle sodium chloride solution (group 1) and 20 females were treated with the test material (group 2).

Induction included intradermal injection of test material preparation (25 g/L with and without Freund's complete adjuvant) on experimental day 1, and topical application of test material preparation (100 g/L) for 48 hours on experimental day 8.

Challenge by topical application of the test material preparation (1 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 one positive reaction was observed in the second reading. This results in 5 % positive reactions.

Conclusion

According to EU Regulation No. 1272/2008 (CLP), the test material is not classified as a skin sensitizer.