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Diss Factsheets

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:
2005-01-26 to 2005-05-09
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:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
adopted 17 July 1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
30 September 1996
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
OECD Guideline 406 "Skin Sensitisation" method (GPMT) was performed before the LLNA was set as preferred test method. Moreover, the chemical structure of the substance, a maleic acid derivative, may result in a false-positive result in the LLNA as described by Kreiling et al (2008) Food Chem Toxicol, 46, 1896-1904.

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: HsdPoc:DH
- Source: Harlan Winkelmann GmbH, Bochum, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approx. 7 weeks
- Weight at study initiation: 348g (mean)
- Housing: The guinea pigs were housed in a 10 m^2 room. 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.
- Diet: ad libitum (Provimi Kliba 3418.0)
- Water: ad libitum (Community tap water)
- Acclimation period: at least 7 days
- Indication of any skin lesions:

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 22°C
- Humidity: 35 - 64% (transiently outside the target range of 45 - 75%. This minor and short deviation did not influence the integrity of the study).
- Photoperiod: 12 hrs dark / 12 hrs light
- IN-LIFE DATES: From: 2005-02-15 To: 2005-03-11

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
paraffin oil
Remarks:
with and without Freund's complete adjuvant
Concentration / amount:
5g/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
Remarks:
PEG 400
Concentration / amount:
100g/L
Day(s)/duration:
48 hours (applied 1 week after intradermal injection)
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challenge
Route:
epicutaneous, occlusive
Vehicle:
polyethylene glycol
Remarks:
PEG 400
Concentration / amount:
50 g/L
Day(s)/duration:
24 hours (applied 2 weeks after topical induction)
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Total: 15 females
Control group: 5 females
Test group 10 females
Details on study design:
MAIN STUDY
A1. INDUCTION EXPOSURE (intradermal injection)
Group 1: Polyethylene glycol 400
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 (5 g/L in PEG 400)
cranial: 0.1 ml Freund's complete adjuvant + sodium chloride solution
medial: 0.1 ml test material (5 g/L liquid paraffin)
caudal: 0.1 ml Freund's complete adjuvant with test material + sodium chloride solution (5 g test material/L preparation)

A2. INDUCTION EXPOSURE (topical application)
Group 1: Polyethylene glycol 400
Group 2: test material (100 g/L polyethylene glycol 400)
- Frequency of applications: single
- Duration: 48 h, occlusive
- Concentrations: 100 g/L

B. CHALLENGE EXPOSURE
- No. of exposures: single topical application
- Day(s) of challenge: 1d
- Exposure period: 2 weeks after topical application

Group 1: Topical induction: PEG 400
Challenge: PEG 400
Primary irritation: test material (50 g/L in PEG 400)
Group 2: Topical induction: test material (100 g/L in PEG 400)
Challenge: test material (50 g/L in PEG 400)
Primary irritation: PEG 400


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.
-----------------------------------------------------------------------
Reaction type Scores Definition
-----------------------------------------------------------------------
Negative reaction 0 no reaction
Positive reaction 1 scattered mild redness
2 moderate and diffuse redness
3 intense redness and swelling
-----------------------------------------------------------------------


Following the grading according to the EEC Directive 2001 /59/EEC, a result in an adjuvant method is considered positive if 30% or more of the test animals showed positive reactions.

Body weight
The body weight of the guinea pigs was determined prior to the start of the experimental part (day 1), on experimental days 8, 15, 22, and at the end of the experimental part.

Positive control substance(s):
yes
Remarks:
The validity of the test is periodically tested using Hexalcinnamaldehyde as the positive control.

Results and discussion

Positive control results:
The GPMT test with alpha-Hexalcinnamaldehyde (Challenge: 25 g/L) resulted in 70% positive reactions (7/10 animals) and, thus, confirmed the validity of the test performed.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
50 g/L
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no signs of toxicity
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
50 g/L
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no signs of toxicity
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
50 g/L
No. with + reactions:
1
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 chemical
Dose level:
50 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:
positive control
Dose level:
25 g/L
No. with + reactions:
5
Total no. in group:
10
Clinical observations:
No clinical signs
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
positive control
Dose level:
25 g/L
No. with + reactions:
7
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:
After challenge, one animal showed positive reaction in the test material treated skin sites at the first reading (48 hours) but not in the second reading (72 hours). This results in 10 % positive reactions at challenge. Therefore, the test material is not considered as a sensitizer for the skin. According to the EEC Directive, the test material has not to be classified as a sensitizer.
Executive summary:

Purpose

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

Study Design

The test item was evaluated for skin sensitizing properties in the guinea pig maximization test according to MAGNUSSON and KLIGMAN (1969).
5 Female guinea pigs in the negative control group (group 1) treated with the vehicles (liquid paraffin and polyethylene glycol 400) and 10 females in the test material group (group 2) were investigated.

Induction included intradermal injection of test material preparation in liquid paraffin (5 g/L with and without Freund's complete adjuvant) on experimental day 1, and topical application for 48 hours of test material preparation in polyethylene glycol 400 (100 g/L) on experimental day 8.

Challenge was performed two weeks after topical induction by topical application for 24 hours of the test material preparation in polyethylene glycol 400 (50 g/L) and readings were taken at 48 hours and 72 hours.

Results

After intradermal injection, the common signs of irritation after injection of Freund's complete adjuvant were observed. The injection sites were swollen and red, later on, scabs developed.

After challenge with polyethylene glycol 400, no erythema or edema were observed at the readings.
A single treatment was performed with the test substance  (50 g/L polyethylene glycol 400) to exclude primary irritation of the test material concentration. No positive reactions were observed in the treated areas at any reading.

After challenge with the test substance (50 g/L preparation in polyethylene glycol 400) a single positive skin reaction was observed in one animal at the first reading. Thus resulting in 10 % positive reactions after challenge.
After challenge, no positive reactions were observed in the areas treated with polyethylene glycol 400 alone at any reading.

The clinical behavior of the guinea pigs was normal during the experimental part. All animals survived the experimental part.

The body weight development corresponded to that of the animals of the vehicle group.

Reactions after challange:

Induction
with		 Challenge
with		 Positive/animals
(48 h after challange)		 Positive/animals
(72 h after challange)		 Positive/animals
(overall)
test substance test substance 1/10 0/10 1/10


Conclusion

After challenge, one animal showed positive reaction in the test material treated skin sites at the first reading but not in the second reading. This results in 10 % positive reactions at challenge. Therefore, the test material is not considered as a sensitizer for the skin. According to the EEC Directive, the test material has not to be classified as a sensitizer.