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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:
January 11, 1994 - February 11, 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report date:
1995

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
adopted July, 1997
Qualifier:
according to guideline
Guideline:
other: EEC directive 92/69, Methods for the determination of toxicity, Publication No. L383A, B.6.: Acute Toxicity-Skin Sensitization
Version / remarks:
December 29, 1992
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The Guinea Pig Maximazitation Test met the previous requirements before the entry into force of REACH. The GPMT is suitable and reliable to cover this endpoint. For this reason and for animal welfare reasons, no further in vivo study (LLNA test) needs to be performed.

Test material

Constituent 1
Chemical structure
Reference substance name:
2-Ethylhexyl (R)-2-(2-methyl-4chlorophenoxy)propionate
EC Number:
630-324-3
Cas Number:
861229-15-4
Molecular formula:
C18H27ClO3
IUPAC Name:
2-Ethylhexyl (R)-2-(2-methyl-4chlorophenoxy)propionate
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- batch No.of test material: 5007
- sample No.:1505E
- Expiration date of the lot/batch: March 1993

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 4°C in the dark

In vivo test system

Test animals

Species:
guinea pig
Strain:
Pirbright-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River GmbH - Wiga, Sulzfeld, FRG
- Age at study initiation: young adult animals
- Weight at study initiation: 296 -355 g
- Housing: Makrolon cage, type IV; 5 animals per cage
- Bedding: Granulat Typ 3/4, SSNIFF
- Diet: ad libitum, Kliba Labordiät 341, Klingentalmühle AG, Kaiseraugst, Switzerland
- Water: ad libitum, tap water; about 2 g of ascorbic acid per 10 L water was added to the drinking water twice a week
- Acclimation period: 7 days
- Indication of any skin lesions:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light):12h light/12h dark

Clipping of the test animals: about 3 h prior application of test substance at the appropriate application sites

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
other: olive oil DAB 10 or Freund´s adjuvant/0.9% aqueous NaCl-solution (1:1) or olive oil
Concentration / amount:
5%
Day(s)/duration:
48 hours
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
0.3 g (unchanged test substance)
Day(s)/duration:
48 hours
Challengeopen allclose all
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
unchanged test substance
Day(s)/duration:
24 hours
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
unchanged test substance
Day(s)/duration:
24 hours
No. of animals per dose:
number of animals per control group 10
number of animals of the test group 20
Details on study design:
PRETEST (Dose finding)
- Amount applied: filter paper soaked in test substance or test substance formulations; animal was exposed to about 0.15 g of the test substance formulations
- Application: 2 x 2 cm filter paper strips were applied to the skin of the flanks under occlusive dressing (bandage consists of rubberized linen patches 4 x 4 cm from Russka, test patch 5 x 5 cm of Idealbinde from Pfälzische Verbandstoff-Fabrik,and Fixomull Strech adhesive fleece)
- Exposure period: test substance was applied 2 times for 24 h within a period of 96 h in order to detect non-specific phenomena that are not caused by a sensitization reaction but could possibly be attributed to a shift in the irritation threshold
- Site of application: flank, respective on the same area
- Number of test animals: 4 per test concentration (unchanged, 75%, 50% and 25%)
- Readings: about 24 and 48 h after the beginning of application
- Assessment of skin findings: scores according to Draize et al. 1959:

Erythema and eschar formation:

No erythema 0
Very slight erythrema (barely perceptible) 1
well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beet redness) to slight eschar formation (injuries in depth) 4

Oedema formation:

No oedema 0
Very slight oedema (barely perceptible) 1
Slight oedema (edges of area well-defined by definite raising) 2
Moderate oedema (raised approximately 1 millimetre) 3
Severe oedema (raised more than 1 millimetre and extending beyond the area of exposure) 4

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 6 intradermal injections in groups of two per animal
- Test groups: 20 animals
- Control group: 10 animals
- Site: shoulder
A.front row with 2 injections each of 0.1 mL Freund´s adjuvant without test substance emulsified with 0.9% aqueous NaCl-Solution in a ratio of 1:1
B.middle row with 2 injections each of 0.1 mL of the test substance formulation
C.back row with 2 injections each of 0.1 mL Freund´s adjuvant /0.9% aqueous NaCl-solution (1:1) with test substance
- Injections for control groups 1 and 2:
The animals were given the same injections (A,B,C) but without test substance, only with the formulating agent
- Readings: 24 h after application
- Assessment of skin findings: analogous to the pretest

Percutaneous induction: one week after intradermal induction
- Amount applied: 2 x 4 cm filter paper strips to the skin of the shoulder under occlusive dressing; the filter paper strip was soaked in the test substance and exposed to about 0.3 g of the test substance
-control groups not treated, since the test substance was applied unchanged and thus no solvent was used
- Duration of exposure; 48 h
- Site: shoulder, same area as in the case of the previous intradermal application
- Readings: 48 h after the beginning of application
- Assessment of skin findings: analogous to the pretest

B. CHALLENGE EXPOSURE
First challenge 21 days after intradermal induction. To clarify the results obtained in the first challende, a second challenge was carried out after the first one.
- amount applied: 2 x 2 cm filter paper strips to the skin of the flank under occlusive dressing; the filter paper strip was soaked in the test substance
- 1st challenge: treatment of the test group and of the control group 1 with the test substance (control group 2 remained untreated)
- 2nd challenge: treatment of the test group and of the control groups 1 and 2 with the test substance
- Duration of exposure: 24 h
- Site: intact flank
- Readings: 24 h and 48 h after the removal of the patch
- Assessment of skin findings: analogous to the pretest


Challenge controls:
yes
Positive control substance(s):
yes
Remarks:
1-chloro-2,4-dinitrobenzene (not included in this study, but seperate study was performed and results presented; Project no. 30H0383/912273)

Results and discussion

Positive control results:
The positive control with 1-chloro-2,4-dinitrobenzene showed that the test system was able to detect sensitizing compounds under the laboratory conditions chosen.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
unchanged substance
No. with + reactions:
8
Total no. in group:
20
Clinical observations:
5 animals with very slight erythema and 3 animals with well-defined erythema
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
vehicle
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
0.5% in ethanol
No. with + reactions:
15
Total no. in group:
20
Clinical observations:
well-defined erythema in 8 animals and very slight erythema in 7 animals observed
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
unchanged substance
No. with + reactions:
3
Total no. in group:
20
Clinical observations:
2 animals with very slight erythema and 1 animal with well.defined erythema
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
vehicle
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
positive control
Dose level:
0.5% in ethanol
No. with + reactions:
19
Total no. in group:
20
Clinical observations:
17 animals with very slight erythema and 2 animals with well-defined erythema
Remarks on result:
positive indication of skin sensitisation

Any other information on results incl. tables

Table 1: Number of animals with skin findings after the 1st challenge and after the 2nd challenge.

 

1st challenge

2nd challenge

unchanged test substance

unchanged test substance

Control group 1

0 / 10

0 / 10

Control group 2

No application of test substance

 

Test group

8 / 20

3 / 20

x/y: number of positive reactions/number of animals tested; readings 24 h after the removal of the patch

Table 2: Number of animals with visual skin findings after the 1st challenge and after the 2nd challenge

 

1st challenge

2nd challenge

Positive ctr 0.5% in ethanol conc.

Ethanol conc.

Positive ctr 0.5% in ethanol conc.

Ethanol conc.

Control group 1

0 / 10

0 / 10

5 / 10

0 / 10

Control group 2

No application of test substance

0 / 10

0 / 10

0 / 10

Test group

15 / 20

0 / 20

19 / 20

0 / 20

x/y: number of positive reactions/number of animals tested; readings 24 h after the removal of the patch

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Based on the results of the study under the test conditions chosen MCPP-P 2-EHE has a sensitizing effect on the skin of the guinea pigs in the Maximization Test.
Executive summary:

A GLP study according OECD 406 was performed based on the method of Magnusson and Kligman to test for its skin sensitization potential of MCPP-P 2-EHE. A pretest was carried out to detect non-specific phenomena that are not caused by a sensitization reaction but could possibly be attributed to a shift in the irritation threshold. Based on this test, concentrations for intradermal (5%) and percutaneous (unchanged) induction and challenge (unchanged) were selected.

In the main test, after intradermal induction with 5% test substance, slight to well-defined signs of irritation could be observed in the treated animals. The percutaneous induction with the unchanged test substance led to increstation, partially open (caused by the intradermal induction), in addition to well-defined erythema and slight edema. The challenges with the unchanged test substance caused very slight to well-defined erythema. After the 1st challenge (21 days after intradermal induction) with the unchanged test substance, 8 out of 20 animals of the test group were observed with skin findings (40%). Most of the skin findings were mild (erythema grad 1), only three cases grad 2 erythema were noted 24h after removal of the patch. The control group did not show any skin reactions. After the 2nd challenge (28 days after intradermal induction), three animals out of 20 (15%) showed skin reactions in form of very slight erythema (2) and well defined erythema (1). Again, the control groups did not show any skin reactions. A significant lowering of the incidence of skin findings after a second challenge is a rare event with chemicals causing only mild sensitization. The positive control with 1 -chloro-2, 4 -dinitrobenzene showed that the test system was able to detect sensitizing compounds under the laboratory conditions chosen. Based on the results of the study under the test conditions chosen MCPP-P 2-EHE has a sensitizing effect on the skin of the guinea pigs in the Guinea Pig Maximization Test. The evaluation "sensitizing" results because more than 30% of the experiment animals exhibit skin reactions.