Registration Dossier

Administrative data

Description of key information

Key, GPMT, OECD 406, GLP: < 30 %, mild sensitiser ( Magnusson and Kligman)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 MAY 2000 - 25 SEP 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
July 1992
Deviations:
yes
Remarks:
The temperature and relative atmospheric humidity in the animal room was transiently outside the target range of 17 to 23 °C and 30 to 70 % (acc to guideline). This minor and short deviation did not influence the integrity of the study.
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Test was conducted prior to the adaption of the OECD guideline for the LLNA.
Species:
guinea pig
Strain:
other: HsdPoc:DH
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen
- Age at study initiation: about 6 weeks
- Weight at study initiation: 346 g (range 307 - 407 g).
- Housing: The guinea pigs were housed in a 10 m² room of the Institute of Toxicology. Two guinea pigs were housed in a Makrolon cage type with a shelter placed on mobile racks. The animals were kept on conventional softwood granulate as bedding. The cages had been machine cleaned before the start of the study. The bedding was changed two times a week.
- Diet (e.g. ad libitum): ad libitum, the diet is checked periodically by an independent and German Government approved laboratory according to the specifications of the manufacturer. Analysis includes both qualitative and quantitative evaluation for heavy metals, aflatoxins, pesticides and antibiotics.
- Water (e.g. ad libitum): ad libitum, the drinking water was periodically analyzed according to the German regulations for human drinking water.
- Acclimation period: at least 7 d
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 27
- Humidity (%): 46 - 84
- Photoperiod (hrs dark / hrs light): 12/12
Route:
intradermal and epicutaneous
Vehicle:
paraffin oil
Concentration / amount:
Concentration of test material and vehicle used at induction:
a) intradermal: 2.5 g/l (0.25%)
b) topical: 200 g/l (20%)
Day(s)/duration:
Day 1 (intradermal), Day 8-10 (epicutaneous)
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
paraffin oil
Concentration / amount:
Concentration of test material and vehicle used for each challenge:
1. challenge: 50 g/l (5%)
2. challenge: 25 g/l (2.5%)
Day(s)/duration:
Day 22-23
Adequacy of challenge:
highest non-irritant concentration
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
paraffin oil
Concentration / amount:
Concentration of test material and vehicle used for each challenge:
1. challenge: 50 g/l (5%)
2. challenge: 25 g/l (2.5%)
Day(s)/duration:
Day 29-30
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Number of animals in test group: 20
Number of animals in negative control group: 10
Details on study design:
RANGE FINDING TESTS: To determine the concentrations suitable for the main study, a pretest with single intradermal or topical administrations of the vehicle and of the test item was performed. Intradermal (i.d.) injections were given to one animal. Four further animals were used to determine the topical concentrations for the main study. Two of the guinea pigs were exposed for 48 hours without pretreatment to determine the concentration for the dermal induction. The other two animals were treated according to the control group including intradermal injections with FCA. One week after topical induction the two animals of the pretest group were exposed for 24 hours to different test material concentrations to find out the challenge concentration. In the pretest the following concentrations were used:
1. Liquid paraffin: intradermal
2. test item with liquid paraffin as vehicle:
intradermal: 50, 25, 10, 5, and 1 g/L
topical: 400, 200, 100, and 10 g/L
topical with FCA: 100, 50, 10, and 1 g/L

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2
- Exposure period: epicutaneous induction for 24 h
- Test groups:
1. induction (intradermal)
cranial: 0.10 ml Freund's complete adjuvant + Sodium cloride solution
medial: 0.10 ml test item (2.5 g/L liquid paraffin)
caudal: 0.10 ml Freund's complete adjuvant with test item + Sodium cloride solution (2.5 g test material/L preparation)
2. induction (epicutaneous)
One week after the intradermal injections, the shoulder area of the guinea pigs was shaven again and covered with a filter paper patch of about 8 cm² fully soaked with 1 ml of the test material preparation (200 g/L in liquid paraffin). The patches were attached for 48 hours under occlusive conditions.
- Control group:
1. induction (intradermal)
cranial: 0.10 ml Freund's complete adjuvant + Sodium cloride solution
medial: 0.10 ml Liquid paraffin
caudal: 0.10 ml Freund's complete adjuvant + Sodium cloride solution
2. induction (epicutaneous)
One week after the intradermal injections, the shoulder area of the guinea pigs was shaven again and covered with a filter paper patch of about 8 cm² fully soaked with 1 ml of the vehicle. The patches were attached for 48 hours under occlusive conditions.
- Site: shoulder region (cranial, medial, caudal)
- Frequency of applications: day 1 and day 8
- Duration: day 1 (intradermal), day 8 (epicutaneous for 24 h)
- Concentrations:
1. induction (intradermal): 2.5 g/L (0.25%)
2. induction (epicutaneous): 200 g/L (20%)

B. CHALLENGE EXPOSURE
- No. of exposures: 2
- Day(s) of challenge: 22-23; 29-30
- Exposure period: 24 h
- Test groups: Two and three weeks after the topical induction the challenge was performed by fixing filter papers of about 4 cm² fully loaded with 0.5 ml of the test item preparation to the shaven flanks of the animals.
- Control group: yes
- Site: flanks
- Concentrations:
1. challenge 50 g/L
2. challenge 25 g/L
- Evaluation (hr after challenge): 48 and 72 h after start of challenge

Challenge controls:
10 females were treated with the vehicle liquid paraffin during induction and challenged with the test item at concentrations of 50 g/L (challenge 1) and 25 g/L (challenge 2)
Positive control substance(s):
yes
Remarks:
a-Hexylcinnamaldehyde, 10 g/L (data from the GLP study T14666. Experimental part: April 11 until May 05, 2000.)
Positive control results:
80 % positive reactions (8 of 10 animals) were observed after treatment with a-Hexylcinnamaldehyde (10 g/L)
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
5 %
No. with + reactions:
4
Total no. in group:
20
Reading:
2nd reading
Hours after challenge:
72
Group:
test group
Dose level:
5 %
No. with + reactions:
2
Total no. in group:
20
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
2.5 %
No. with + reactions:
4
Total no. in group:
20
Reading:
rechallenge
Hours after challenge:
72
Group:
test group
Dose level:
2.5 %
No. with + reactions:
4
Total no. in group:
20
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
5 %
No. with + reactions:
0
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
5 %
No. with + reactions:
0
Total no. in group:
10
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
2.5 %
No. with + reactions:
0
Total no. in group:
10
Reading:
rechallenge
Hours after challenge:
72
Group:
negative control
Dose level:
2.5 %
No. with + reactions:
0
Total no. in group:
10
Reading:
1st reading
Hours after challenge:
48
Group:
positive control
Dose level:
1 %
No. with + reactions:
7
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
72
Group:
positive control
Dose level:
1 %
No. with + reactions:
4
Total no. in group:
10

After the first challenge 6/20 animals of the test group showed effects. 4 animals showed findings only at the first reading and 2 other animals showed findings only at the second reading. This inconsistent result was interpreted to be more the result of an irritation than sensitisation. Therefore, a rechallenge was performed with 2.5 % test item treatment. After challenge II with 25.0 g/L test material, 4 of 20 animals (20%) showed positive reactions. Now the reactions were seen in the same animals at both readings.

for details on results, please refer to tables under 'Attached background material'

Interpretation of results:
other: EU GHS criteria not met
Conclusions:
Challenge I with a test item concentration of 50.0 g/L, led to positive reactions of the skin in 6 of 20 animals (30%). Four animals showed a clear positive reaction at the first reading (after 48 h) but two others reacted at the second reading (after 72 h). So the result was regarded as inconsistent with signs of irritation and a second challenge prepared. After challenge II with 25.0 g/L test material, 4 of 20 animals (20%) showed positive reactions. Now the reactions were seen in the same animals at both readings.
Executive summary:

The test item was evaluated for skin sensitising properties in a study according to OECD Guideline 406 and following GLP. 10 female guinea pigs in the negative control group (group 1) treated with the vehicle liquid paraffin and 20 females in the test material group (group 2) were investigated. Induction included intradermal injection of test material preparation (2.5 g/L with and without Freund's complete adjuvant) on day 1, and topical application of test material preparation (200.0 g/L) on day 8. Challenge by topical application of the test material preparation was performed two weeks (challenge I) and three weeks (challenge II) after topical induction. Test material concentrations were 50.0 g/L (Challenge I) and 25.0 g/L (Challenge II).

Challenge I with a test item concentration of 50.0 g/L, led to positive reactions of the skin in 6 of 20 animals (30%). Four animals showed a clear positive reaction at the first reading (after 48 h) but two others reacted at the second reading (after 72 h). So the result was regarded as inconsistent with signs of irritation and a second challenge prepared. After challenge II with 25.0 g/L test material, 4 of 20 animals (20%) showed positive reactions. Now the reactions were seen in the same animals at both readings. Challenge in group 1 with the vehicle to exclude sensitisation and the single application of the concerned test material concentration to find out signs of lowering the irritation level, did not lead to any skin reaction.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Skin sensitisation

The test item was evaluated for skin sensitising properties in a study according to OECD Guideline 406 and following GLP. 10 female guinea pigs in the negative control group (group 1) treated with the vehicle liquid paraffin and 20 females in the test material group (group 2) were investigated. Induction included intradermal injection of test material preparation (2.5 g/L with and without Freund's complete adjuvant) on day 1, and topical application of test material preparation (200.0 g/L) on day 8. Challenge by topical application of the test material preparation was performed two weeks (challenge I) and three weeks (challenge II) after topical induction. Test material concentrations were 50.0 g/L (Challenge I) and 25.0 g/L (Challenge II).

Challenge I with a test item concentration of 50.0 g/L, led to positive reactions of the skin in 6 of 20 animals (30%). Four animals showed a clear positive reaction at the first reading (after 48 h) but two others reacted at the second reading (after 72 h). So the result was regarded as inconsistent with signs of irritation and a second challenge prepared. After challenge II with 25.0 g/L test material, 4 of 20 animals (20%) showed positive reactions. Now the reactions were seen in the same animals at both readings. Challenge in group 1 with the vehicle to exclude sensitisation and the single application of the concerned test material concentration to find out signs of lowering the irritation level, did not lead to any skin reaction.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the sensitisation rate of < 30 % in the GPMT (with adjuvants) the test item is not subject to classification in accordance with Regulation (EC) No 1272/2008.