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Description of key information

The Guinea Pig Maximization Test (GPMT) was performed to identify the sensitizing effect of the test chemical on the skin of the guinea pigs following the procedures mentioned in OECD 406 Guidelines.

The intradermal induction with 5% test substance formulations caused moderate and confluent erythema and swelling in all test group animals . After the percutaneous induction with a 10% test substance formulation incrustation, partially open (caused by the intradermal induction) could be observed in addition to moderate and confluent erythema and swelling in all test group animals .Two challenges were performed 14 and 21 days after the percutaneous induction . After the challenge(s) with a 5% test substance formulation, moderate and confluent or intense erythema and swelling could be observed in test group animals .Based on the results of this study and applying the evaluation criteria, it was concluded that the test chemical has a sensitizing effect on the skin of the guinea pig in the Maximization Test under the test conditions chosen .

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
data is from experimental reports
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Principles of method if other than guideline:
The Guinea Pig Maximization Test (GPMT) was performed to identify the sensitizing effect of the test chemical on the skin of the guinea pigs
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The Guinea Pig Maximization Test (GPMT) has been carried out as an animal test to predict human sensitization for over a decade and is recommended by international test guidelines such as OECD .
Species:
guinea pig
Strain:
other: Pirbright White, Dunkin Hartley Crl : (HA) BR [SPF ]
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River GmbH - Wiga,Kisslegg, FRG
- Age at study initiation: Young adult animals
- Weight at study initiation: 331 - 378 g
- Housing: Stainless steel wire mesh cages with plastic-coated grating, floor area 40 cm x 51 cm
- Bedding : bedding in the cages ; sawdust in the waste trays .
- Diet (e.g. ad libitum): Kliba Labordiät (Kaninchen-Meerschweinchen-Haltungsdiät), ad libitum
- Water (e.g. ad libitum): Water ad libitum (tap water ; about 2 g of ascorbic acid per10 l water was added to the drinking water twice a week)
- Identification of the animals : Ear tattoo
- Acclimation period: 7 days before the beginning of the study in the laboratory for dermal toxicity


ENVIRONMENTAL CONDITIONS
- Temperature (°C): temperature in the range of 21 - 25°C
- Humidity (%): relative humidity in the range of 30 -70 % .
- Air changes (per hr): The animals were housed in fully air-conditioned rooms in which a central air-conditioning system
- Photoperiod: 12 h light ( 6 .00 am - 6 .00 pm); 12 h darkness ( 6 .00 pm - 6 .00 am)
No. of animals per dose:
The study was performed on 10 guinea pigs in the test group and 5 animals in each of control groups 1 and 2 .
Additionally all control group animals have been treated with the vehicle .
Details on study design:
RANGE FINDING TESTS: The pretest was performed with 4 female animals for percutaneous and with 4 female animals for intradermal application.
For the dermal induction treatment the highest concentration of the test substance that causes slight to moderate irritation and for the challenge the maximum non-irritant concentration was determined with the pretest . For detecting a possible influence on irritating effects of previous intradermal treatment with Freund's adjuvant, animals pretreated with Freund's
adjuvant / 0 .9% aqueous NaCl-solution (1 : 1) each, in the same manner as intradermal pretest referring front row (A) and back row (C) without test substance about 3 or 4 weeks prior to the
application of the test substance were used . The test substance or the vehicle (empty formulation) was applied to the skin of the flanks for 2 x 24 hours under occlusive dressing .The minimum irritant concentration was found to be a 10% test substance formulation in a oil/water emulsion (mixture of the test substance in aqua bidest . (neutralized with 10 M NaOH), paraffin oil DAB 10 and Cremophor A 25) . The maximum non-irritant concentration was found to be a 5% test substance formulation in a oil/water emulsion (mixture of the test substance in distilled water (neutralized with 10 M NaOH), paraffin oil DAB 10 and Cremophor A 25) .



MAIN STUDY
A. INDUCTION EXPOSURE
Intradermal exposure
- No. of exposures: 6 intradermal injections in groups of two per animal were given .
- Exposure period:
- Test groups: 10
- Control group: 10
- Site: shoulder
- Frequency of applications: 2 injections per concentration
- Duration: single
- Concentrations: test substance formulation 5% in a oil/water emulsion or in Freund' s adjuvant / 0 .9% aqueous NaCl-solution (1 : 1) (test group) and empty formulation (oil/water emulsion without test substance) or 50 % preparation of the empty formulation with Freund's adjuvant / 0 .9% aqueous NaCl-solution (1 : 1) (control groups) with test substance at the selected concentration .

Epicutaneous Exposure
- No. of exposures: 6 intradermal injections in groups of two per animal were given .
- Exposure period: 48 hours
- Test groups:test substance formulation 10% in a oil/water emulsion (test group)
- Control group: empty formulation (oil/water emulsion without test substance) (control groups )
- Site:shoulder, same area as in the case of the previous intradermal application
- Frequency of applications: single
- Duration: 48 hours
- Concentrations: test substance formulation 10% in a oil/water emulsion (test group)

B. CHALLENGE EXPOSURE
- No. of exposures: single
- Day(s) of challenge: The first challenge was performed 14 days after the percutaneous induction . A second challenge was carried out one week after the first one .
- Exposure period: 24 hours
- Test groups: 10
- Control group: 10
- Site: flank
- Concentrations: First challenge : The test group and control group 1 were treated with the test substance formulation 5% in an oil/water emulsion or empty formulation (oil/water emulsio n
without test substance). Control group'2 only received the empty formulation. 2nd challenge : test substance formulation 5% in a oil/water emulsion or empty formulation (oil/water emulsio n without test substance)
- Evaluation (hr after challenge): 24 hours and 48 hours after the removal of the patch

OTHER: Assessment of the skin reactions
The evaluation of the skin reactions was performed according to the following grading scale of Magnusson and Kligman :
0 = no visible change
1= discrete or patchy erythema
2 = moderate and confluent erythema
3 = intense erythema and swelling
Descriptions of any dermal findings not covered by this scale were recorded .
Positive control substance(s):
yes
Remarks:
Alpha-Hexylcinnamaldehyde techn . was used as positiv control
Positive control results:
The positive control with Alpha-Hexylcinnamaldehyde techn . 85 % showed that the chosen guinea pig strain was able to detect sensitizing compounds under the laboratory conditions chosen .
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
5% test substance formulation
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
5% test substance formulation, moderate and confluent or intense erythema and swelling could be observed in test group animals .
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
5% test substance formulation
No. with + reactions:
9
Total no. in group:
10
Clinical observations:
5% test substance formulation, moderate and confluent or intense erythema and swelling could be observed in test group animals .
Remarks on result:
positive indication of skin sensitisation

The number of animals with skin findings after the lst challenge and after the 2nd challenge is summarized in the following table :

 

1stchallenge

2ndchallenge

5% test formulation in a oil/water emulsion*

Vehicle control- empty formulation oil/water emulsion

5% test formulation in a oil/water emulsion*

Vehicle control- empty formulation oil/water emulsion

Control group 1

0/5

0/5

0/5

0/5

Control group 2

No application of test chemical

0/5

0/5

0/5

Test group

10/10

0/10

9/10

0/10

x/y : number of positive reactions/number of animals tested ( reading at 24 h and/or 48 h after the removal of the patch )

mixture of 5 g test substance + 83 g aqua bidest . (including the necessary amount of 10 M NaOH for neutralization of the acid solution) + 12 g mixture of paraffin oil/Cremophor A 25 (10 paraffin oil viscous DAB 10 + 2 g Cremophor A 25) .

** : mixture of 88 g aqua bidest . + 12 g mixture of paraffin oil/Cremophor A 25 (10 g paraffin oil viscous DAB 10 + 2 g Cremophor A 25) .

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
The intradermal induction with 5% test substance formulations caused moderate and confluent erythema and swelling in all test group animals . After the percutaneous induction with a 10% test
substance formulation incrustation, partially open (caused by the intradermal induction) could be observed in addition to moderate and confluent erythema and swelling in all test group animals .
Two challenges were performed 14 and 21 days after the percutaneous induction . After the challenge(s) with a 5% test substance formulation, moderate and confluent or intense
erythema and swelling could be observed in test group animals .Based on the results of this study and applying the evaluation criteria, it was concluded that the test chemical has a sensitizing effect on the skin of the guinea pig in the Maximization Test under the test conditions chosen .
Executive summary:

The Guinea Pig Maximization Test (GPMT) was performed to identify the sensitizing effect of the test chemical on the skin of the guinea pigs following the procedures mentioned in OECD 406 Guidelines. Young adult FemalePirbright White, Dunkin Hartley Crl : (HA) BR [SPF] guinea pigs were chosen for the study.

The pretest was performed with 4 female animals for percutaneous and with 4 female animals for intradermal application.

For the dermal induction treatment the highest concentration of the test substance that causes slight to moderate irritation and for the challenge the maximum non-irritant concentration was determined with the pretest . For detecting a possible influence on irritating effects of previous intradermal treatment with Freund's adjuvant, animals pretreated with Freund's adjuvant / 0 .9% aqueous NaCl-solution (1 : 1) each, in the same manner as intradermal pretest referring front row (A) and back row (C) without test substance about 3 or 4 weeks prior to the application of the test substance were used . The test substance or the vehicle (empty formulation) was applied to the skin of the flanks for 2 x 24 hours under occlusive dressing .The minimum irritant concentration was found to be a 10% test substance formulation in a oil/water emulsion (mixture of the test substance in aqua bidest . (neutralized with 10 M NaOH), paraffin oil DAB 10 and Cremophor A 25) . The maximum non-irritant concentration was found to be a 5% test substance formulation in a oil/water emulsion (mixture of the test substance in distilled water (neutralized with 10 M NaOH), paraffin oil DAB 10 and Cremophor A 25) .

The main study was performed on 10 guinea pigs in the test group and 5 animals in each of control groups 1 and 2 .

The intradermal induction with 5% test substance  formulations caused moderate and confluent erythema and swelling in all test group animals . After the percutaneous induction with a 10% test substance formulation incrustation, partially open (caused by the intradermal induction) could be observed in addition to moderate and confluent erythema and swelling in all test group animals .Two challenges were performed 14 and 21 days after the percutaneous induction . After the challenge(s) with a 5% test substance formulation, moderate and confluent or intense erythema and swelling could be observed in test group animals .Based on the results of this study and applying the evaluation criteria, it was concluded that the test chemical has a sensitizing effect on the skin of the guinea pig in the Maximization Test under the test conditions chosen .

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Various studies have been summarized to ascertain the level of dermal sensitization caused by test chemical in living organisms. These results include in vivo experimental studies performed on humans, guinea pigs for the test chemical.

The Guinea Pig Maximization Test (GPMT) was performed to identify the sensitizing effect of the test chemical on the skin of the guinea pigs following the procedures mentioned in OECD 406 Guidelines. Young adult Female Pirbright White, Dunkin Hartley Crl : (HA) BR [SPF] guinea pigs were chosen for the study.

The pretest was performed with 4 female animals for percutaneous and with 4 female animals for intradermal application.

For the dermal induction treatment the highest concentration of the test substance that causes slight to moderate irritation and for the challenge the maximum non-irritant concentration was determined with the pretest . For detecting a possible influence on irritating effects of previous intradermal treatment with Freund's adjuvant, animals pretreated with Freund's adjuvant / 0 .9% aqueous NaCl-solution (1 : 1) each, in the same manner as intradermal pretest referring front row (A) and back row (C) without test substance about 3 or 4 weeks prior to the application of the test substance were used . The test substance or the vehicle (empty formulation) was applied to the skin of the flanks for 2 x 24 hours under occlusive dressing .The minimum irritant concentration was found to be a 10% test substance formulation in a oil/water emulsion (mixture of the test substance in aqua bidest . (neutralized with 10 M NaOH), paraffin oil DAB 10 and Cremophor A 25) . The maximum non-irritant concentration was found to be a 5% test substance formulation in a oil/water emulsion (mixture of the test substance in distilled water (neutralized with 10 M NaOH), paraffin oil DAB 10 and Cremophor A 25) .

The main study was performed on 10 guinea pigs in the test group and 5 animals in each of control groups 1 and 2 .

 

The intradermal induction with 5% test substance formulations caused moderate and confluent erythema and swelling in all test group animals . After the percutaneous induction with a 10% test substance formulation incrustation, partially open (caused by the intradermal induction) could be observed in addition to moderate and confluent erythema and swelling in all test group animals .Two challenges were performed 14 and 21 days after the percutaneous induction . After the challenge(s) with a 5% test substance formulation, moderate and confluent or intense erythema and swelling could be observed in test group animals .Based on the results of this study and applying the evaluation criteria, it was concluded that the test chemical has a sensitizing effect on the skin of the guinea pig in the Maximization Test under the test conditions chosen .

 

BUEHLER Test was also conducted to determine sensitizing effect of the test chemical on the skin of the guinea pig. The study was performed according to OECD Guideline 406 (Skin Sensitization), EU Method B.6 (Skin Sensitization), EPA OPP 81-6 (Skin Sensitization) Guidelines. Young adult Female Pirbright White, Dunkin Hartley Crl : (HA) BR [SPF] guinea pigs were chosen for the study.

 

A pretest was performed with 8 female animals. The test substance was applied to the skin of the flanks for 2 x 6 hours under occlusive dressing . The reactions were read and scored at 24 and 48 h after the removal of the patch. Assessment of skin findings was performed according to Draize, J .H . (1959). In accordance with the Guidelines EEC, OECD and EPA a slightly irritating concentration should be used in the main test for induction whereas the maximum non-irritant concentration should be applied for challenge .

The following concentrations for induction and the challenge were selected on the basis of the pretests :

1st, 2nd and 3rd induction =test substance 50 % in distilled water

Challenge = test substance 50% or 5% in distilled water.

3 inductions were conducted. 0.5 g of the test substance formulation was applied for 6 hours to each animal; one application per week ; days 0, 7 and 14 on the same application area. Readings were taken 24 hours after removal of the patch.

A challenge was carried out 14 days after the third induction. 0.5 g or 0 .5 ml of the 50% test substance formulation : anterior right flank; 5% test substance formulation : posterior right flank were applied for 6 hours to each animal .The test group and control group 1 were treated with the test substance formulations (control group 2 remained untreated) . Reading was taken 24, 48 hours after patch removal. Alpha-Hexylcinnamaldehyde techn was used as the positive control.

The first and second induction with 50 % test substance preparations did not cause any sign of skin irritation. After the third induction with a 50 % test substance preparation only 1 test group animal showed well-defined erythema. The 50% test substance preparation caused very slight to well-defined skin reaction in 1 test group animal on the anterior right flank . The same animal exhibited very slight skin reaction on the posterior right flank which was treated with the 5% test substance preparation .

Based on the results of this study and applying the evaluation criteria, it was concluded that the test chemical does not have a sensitizing effect on the skin of the guinea pig in the BUEHLER Test under the test conditions chosen.

Over the 3-year period, between January 2003 and December 2005, 1693 patients underwent patch testing to extended British Contact Dermatitis Society (BCDS) standard series to identify the contact allergens in various cosmetics. Of these, 13 patients[5 male, 8 female] were additionally patch tested with 10% of the test chemical. Patch testing followed the standard protocol using Finn Chambers on Scanpor tape (Bio-Dignostics Ltd., UK) with results being recorded at 48 hr (D2) and 96 hr (D4). Positive reactions were graded from + to +++ according to international recommendations.

The test chemical produced a cosmetic dermatitis of the face and neck in 13/13 of the patients tested, but it also needs to be considered as a cause of sensitization at other sites.

Based on these observations, the test chemical can be considered to be sensitizing to skin.

The above results are supported by a scratch test and patch test performed to evaluate the dermal sensitization potential of the test chemical.

A black man with a broad-spectrum light sensitivity simultaneously showed an urticarial and contact sensitivity to a preparation containing the test chemical. To identify the intenstity of the dermal reaction elicited by the test chemical, a scratch test as well as a patch test was performed.

   

A scratch test of a 1% test chemical solution resulted in a 2+ response; a 1% test chemical solution also elicited a 2+ reaction. A single 24 hour patch test of a 5% test chemical solution (aqueous) revealed a 2+ papular reaction at 24 and 48 hours.

Hence, the test chemical can be considered to be sensitizing to skin.

Even though the results of the Buehler test indicates that the test chemical lacks the potential to cause sensitization to guinea pig skin, but it is clearly contradicted by the results observed on guinea pigs and humans. Taking into considerations, all these factors, the test chemical was considered to be sensitizing to skin. Comparing the above annotations with the criteria of CLP Regulation, the test chemical can be classified under the category “Category 1”

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Even though the results of the Buehler test indicates that the test chemical lacks the potential to cause sensitization to guinea pig skin, but it is clearly contradicted by the results observed on guinea pigs and humans. Taking into considerations, all these factors, the test chemical was considered to be sensitizing to skin. Comparing the above annotations with the criteria of CLP Regulation, the test chemical can be classified under the category “Category 1”