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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:
Jun 27 - Sep 22, 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

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 data have been generated before the OECD 429 protocol was mandatory for REACH.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
Batch: E05095440

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 6 weeks

The mean initial body weight at the start of the study was 326 g (range from 301 to 354 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 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 (5 females)
Group 2: test material group (10 females)

Housing:
The guinea pigs were housed in a 10 m2 room of the Test Institute. Lighting was controlled by a timer to provide a 12-hour light - 12-hour dark regime.
Five guinea pigs were housed in type GM/5 (EBECO) Makrolon cages with a shelter and placed on mobile racks. The animals were kept on conventional softwood granulate as the bedding. The cages had been machine cleaned before the start of the study. The bedding was changed two times a week.
Temperature and atmospheric humidity were measured by a thermohygrograph. The room temperature within the study period was 22 to 24°C and the relative atmospheric humidity 45 to 75 %.
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.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
paraffin oil
Concentration / amount:
5 g/L
Day(s)/duration:
on Day 1
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
polyethylene glycol
Concentration / amount:
200 g/L
Day(s)/duration:
on Day 8/ 48 hours
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
polyethylene glycol
Concentration / amount:
100 g/L
Day(s)/duration:
24 hours
Adequacy of challenge:
highest non-irritant concentration
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:
- no pretreatment, intradermal injection: 0, 1, 5, 10, 25, 50 g/L given to one animal in paraffin oil
- topical applications (48 h), no pretreatment: 10, 50, 100, 200, and 400 g/L in polyethylene glycol 400
- topical applications (24 h), FCA pretreatment: 10, 50, 100, and 200 g/L in polyethylene glycol 400

MAIN STUDY
A1. INDUCTION EXPOSURE (intradermal injection)
After shaving of the shoulder region, six intradermal injections were given (three on each
side of the spinal column in a total area of 2 x 4 cm). The guinea pigs received the
following injections of 0.1 ml each:

Group 1 (vehicle: Liquid paraffin)
cranial: 0.1 ml Freund's complete adjuvant + sodium chloride solution
medial: 0.1 ml Liquid paraffin
caudal: 0.1 ml Freund's complete adjuvant + sodium chloride solution

Group 2 (test material)
cranial: 0.1 ml Freund's complete adjuvant + sodium chloride solution
medial: 0.1 ml (5 g/L ad liquid paraffin)
caudal: 0 .1 ml Freund's complete adjuvant + sodium chloride solution (5 g test material/L preparation)


A2. INDUCTION EXPOSURE (topical application)
One week after the intra.dermal injections, the shoulder area of the guinea pigs was shaven
again and on injection sites covered with a filter paper patch of about 8 cm2, fully soaked with 1 ml of the test material preparation or the vehicle. The patches were attached for 48
hours under occlusive conditions by a self-adhesive fabric (Fixomull® stretch, Beiersdorf).

Group 1: Polyethylene glycol 400
Group 2: Test material (200 g/L ad polyethylene glycol 400)


B. CHALLENGE EXPOSURE
Two weeks after topical induction the challenge was performed by fixing two filter papers of about 4 cm2, fully loaded with 0.5 ml of the test material preparation and soaked with 0.5 ml polyethylene glycol 400, to the shaven side ofthe animals.

Group 1:
Topical induction: Polyethylene glycol 400 (undiluted)
Challenge: Polyethylene glycol 400 (undiluted)
Primary irritation: (100 g/L ad polyethylene glycol 400)

Group 2:
Topical induction: test material (200 g/L ad polyethylene glycol 400)
Challenge: test material (100 g/L ad polyethylene glycol 400)
Challenge: Polyethylene glycol 400 (undiluted)

The patches were fixed for 24 hours under occlusive conditions by a self-adhesive fabric.

Observation schedule

Clinical investigations
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 challenge skin changes at the application sites were evaluated according to the following MAGNUSSON and KLIGMAN scheme.
Following the grading according to the EEC Directive 2001/59/EEC, a result in an adjuvant method is considered positive if 30% or more ofthe test animals showed positive
reactions.
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
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
100 g/L
No. with + reactions:
0
Total no. in group:
10
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:
100 g/L
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no signs of toxicity
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
0 g/L
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no clinical signs
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
0 g/L
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no clinical signs
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
positive control
Dose level:
10 g/L
No. with + reactions:
2
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
positive control
Dose level:
10 g/L
No. with + reactions:
5
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation

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.

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.