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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:
March and April 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
according to the method described by Buehler

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: method described by Buehler
Version / remarks:
E.V. Buehler, delayed contact hypersensitivity in the guinea pig, Arch. Dermatol. 91, 1965, S. 171 - 177
GLP compliance:
yes
Type of study:
Buehler test
Justification for non-LLNA method:
The substance has been tested comparable to the OECD Guideline 406, Bühler method before the LLNA test model was selected to be the preferred test system under the REACH Regulation.

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: Hsd/Poc:DH
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Porcellus, Firgrove Farm, Heathfield, Sussex, UK
- Age at study initiation: "young adults"
- Weight at study initiation: 403-537g
- Housing: individually ind stainless steel cages
- Diet: RGP Diet, ad libitum
- Water: ad libitum
- Acclimation period: at least six days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 +/- 2
- Humidity (%): 55 +/- 15
- Air changes (per hr): 25-30
- Photoperiod (hrs dark / hrs light): 12 hrs/12 hrs

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
Induction: 60% (w/v)
Challenge: 3%, 10%, 30%, 60% (w/v)
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
Induction: 60% (w/v)
Challenge: 3%, 10%, 30%, 60% (w/v)
No. of animals per dose:
10 animals in the control group
20 animals in the test group
Details on study design:
RANGE FINDING TESTS:
- Induction stage: Concentrations of 30% and 60% (highest achievable concentration) (w/v) were applied to the flanks of two females (one concentration per animal) in a similar fashion as described in the main study. Yellow staining of the application site prevented assessment of erythema. In the absence of any other signs of irritation, the 60% (w/v) preparation was used in the main study.
- Challenge stage: Concentrations of 3%, 10%, 30% and 60% (highest achievable concentration) (w/v) in corn oil were applied to each of two females as described in the main study. Application sites were stained yellow by the test material, preventing assessment of erythema.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: two weeks
- Test groups: 400 mg of 60% (w/v) preparation in corn oil
- Control group: 0.4 ml of vehicle
- Site: scapular region
- Frequency of applications: 7-day intervals
- Duration: 6 hrs


B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: two weeks after the final induction exposure
- Exposure period: one day
- Duration: at least 6 hrs
- Site: both flanks
- Concentrations: 3%, 10%, 30%, 60% (w/v) in corn oil, appr. 200 mg, animals received four patches with different concentrations.
- Evaluation: 24 and 48 hrs after removal of the dressings


OTHER: Yellow discoloration of the application sites prevented assessment of erythema. Therefore, skin samples were submitted for histopathological examination.
- skin samples from the challenge sites of test and control animals and untreated skin were examined
- fixation in Bouin's solution and wax,
- staining with haematoxylin and eosin
- light microscope
Challenge controls:
- none
Positive control substance(s):
yes
Remarks:
hexylcinnamaldehyde

Results and discussion

Positive control results:
The positive control substance stained the skin of all test animals yellow during the induction phase, but this did not prevent the assessment of irritation. Signs of slight irritation were seen in the test animals during the induction phase. There were no signs of irritation in any of the control animals.

After challenge with an undiluted sample of the positive control substance scattered mild redness was seen in seven out of twenty animals (scattered mild redness in 7/20 animals at first reading after 24 hours; in 5/20 animals at second reading after 48 hours). No reactions were observed in the control animals. The net percentage response was calculated to be 35%.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
3%, 10%, 30%, 60% preparation in corn oil
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Staining by the test material prevented a clinical assessment of erythema. Histopathological investigation revealed a low grade skin irritation reaction.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 3%, 10%, 30%, 60% preparation in corn oil. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: Staining by the test material prevented a clinical assessment of erythema. Histopathological investigation revealed a low grade skin irritation reaction..
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
3%, 10%, 30%, 60% preparation in corn oil
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Staining by the test material prevented a clinical assessment of erythema. Histopathological investigation revealed a low grade skin irritation reaction.
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 3%, 10%, 30%, 60% preparation in corn oil. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: Staining by the test material prevented a clinical assessment of erythema. Histopathological investigation revealed a low grade skin irritation reaction..
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
3%, 10%, 30%, 60% preparation in corn oil
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
Staining by the test material prevented a clinical assessment of erythema. Histopathological investigation revealed a low grade skin irritation reaction.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 3%, 10%, 30%, 60% preparation in corn oil. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: Staining by the test material prevented a clinical assessment of erythema. Histopathological investigation revealed a low grade skin irritation reaction..
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
3%, 10%, 30%, 60%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
Staining by the test material prevented a clinical assessment of erythema. Histopathological investigation revealed a low grade skin irritation reaction.
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 3%, 10%, 30%, 60%. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: Staining by the test material prevented a clinical assessment of erythema. Histopathological investigation revealed a low grade skin irritation reaction..
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
100%
No. with + reactions:
7
Total no. in group:
20
Clinical observations:
no effects
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
100%
No. with + reactions:
5
Total no. in group:
20
Clinical observations:
no effects

Any other information on results incl. tables

Staining by the test material prevented a clinical assessment of erythema. Histopathological investigation revealed a low grade skin irritation reaction: A predominantly minimal to slight inflammatory reaction with acanthosis and inflammatory cell infiltration was recorded in approximately equal proportions in skin samples from control and test animals treated with 3%, 10% or 30% (w/v) preparations of the test material in corn oil. There was considered to be no significant difference in the frequency of this effect between test and control samples. Following application of the 60% (w/v) preparation in corn oil only a very minor effects was observed in the test group.

Additional investigations were performed on skin irritation potential of corn oil, the vehicle used in this study, under Buehler challenge conditions. There were no skin reactions visible on clinical examination. Histopathological findings were similar to those reported in this test with the test item.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
regulation (EC) no 1272/2008
Conclusions:
The test item is non-sensitising under the conditions of the test.
Executive summary:
The sensitisation potential of the test item was assessed in female guinea pig (20 animals in the test and 10 animals in the control group) using a method based on that described by Buehler (1965). Following challenge with 60%, 30%, 10% and 3% w/v preparations of the test material in corn oil, all application sites were stained yellow preventing assessment of erythema. On this basis skin samples from all application sites and untreated sites were excised and fixed in Bouin's fixative. Skin samples from all the challenge sites, together with samples of untreated skin were processed and examined using light microscopy.

Histopathological examination revealed a background level of minimal irritation to the skin for all the concentrations used at challenge. There was considered to be no significant difference in the skin response between test and control animals. In a positive control study, challenge of previously induced guinea pigs with an undiluted preparation of hexylcinnamaldehyde elicited a moderate skin sensitisation response, confirming the validity of the assay.

Therefore, the test item has not to be classified as skin sensitiser according to Regulation (EC) No 1272/2008.