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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report date:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
Buehler test
Justification for non-LLNA method:
Study conducted prior to adoption of LLNA guideline by the OECD.

Test material

Constituent 1
Chemical structure
Reference substance name:
4,4'-cyclohexylidenedi-o-cresol
EC Number:
219-110-7
EC Name:
4,4'-cyclohexylidenedi-o-cresol
Cas Number:
2362-14-3
Molecular formula:
C20-H24-O2
IUPAC Name:
4-[1-(4-hydroxy-3-methylphenyl)cyclohexyl]-2-methylphenol
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): Dimethyl cyclohexyl bisphenol (DMBPC; CAS No. 2362-14-3)
- Synonyms: Dimethyl bisphenolcyclohexane; 4,4’-cyclohexylidene di-o-cresol; 1,1-Bis(4-hydroxy-3-ethyl)cyclohexane; Bis-OC-Z
- Appearance: fine white powder

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
Nineteen male and 19 female (nulliparous and non-pregnant) Hartley guinea pigs were received from the supplier. They weighed 380.9 to 427.2 g and were at least 21 days old. They were group-housed upon arrival in stainless steel suspended cages. The animals were acclimated for at least 5 days prior to dosing. Water and feed were provided ad libitum. The temperature and humidity were maintained at 68 ± 5 °F and 30 - 70 %, respectively. Room lights were on a 12-hour light/dark cycle.

Study design: in vivo (non-LLNA)

Induction
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100 % for induction doses
Challenge
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100 % for challenge doses
No. of animals per dose:
10 males/10 females Test group (100 % DMBPC topical)
5 males/5 females Negative control group
3 males/2 females Positive control group
1 male/2 females for the preliminary irritation test
Details on study design:
A preliminary irritation test was conducted, in which DMBPC, at concentrations of 100 % (neat) and 50, 25 and 10 % in 80 % ethanol was applied to the skin of 3 guinea pigs for 6 hours. The response was scored 24 hours after the test substance application and the test substance was determined not to be an irritant. Therefore, DMBPC at a concentration of 100 % (neat) was chosen for the topical induction and challenge doses.

For the main test, the application sites were prepared by clipping and shaving or depilating the skin of the scapular region in an area of about 3 x 4 cm. Animals were weighed on Day 0 (before induction application) and again at the end of the test (Day 30) and any clinical observations were recorded.

For the induction, on Days 0, 7 and 14, closed patches were prepared with the test substance and applied directly to the skin on one side of the animal and covered with a gauze pad of approximately 4-6 cm². The test substance was used at 100 % since the preliminary irritation screen indicated that it was not an irritant. The patch was kept in place with occlusive bandaging. After 6 hours the patch was removed and any residual test substance was wiped off with a gauze pad.

During induction, the positive control substance (0.4 mL of 0.1 % DNCB in acetone) was applied in the same manner. Naïve animals (i.e. untreated during the induction phase) served as a negative control group.

For the challenge, a 4 x 3 cm virgin skin site was shaved on the flanks of the experimental and control animals. The challenge test was performed on the freshly clipped skin sites of the test and naïve control animals in the same manner as the 6-hour closed patch test of the induction phase. The skin was exposed to the test substance at 100 % (the highest non-irritating concentration determined in the preliminary irritation study) for 6 hours.

The experimental and negative control animals were exposed to one dose of test substance on week 05; 0.1 % Dinitrochlorobenzene (DNCB) in acetone was used to challenge the positive control animals.

At approximately 21 hours after removal of the challenge dose, the area of the challenge was marked and the entire back was shaved. Approximately three hours after shaving, the test site was examined for erythema and oedema. The reaction was graded according to the following Magnusson and Kligman Grading Scale for the Evaluation of Challenge Patch Test reactions.

0 = No visible change
1 = Discrete or patchy erythema
2 = Moderate and confluent erythema
3 = Intense erythema and swelling

Evaluation of the challenge area was repeated at approximately 48 hours after removal of the challenge dose patch and the skin reactions were graded.

The results are summarised and expressed in the following terms:
- Incidence: The number of animals showing a response of 1 or more at 24 and 48 hours divided by the number of animals tested; expressed as a percentage.
- Severity: The sum of the test grades divided by the number of animals tested.

At the end of the study, all animals were weighed, then sacrificed by CO2 inhalation.
Challenge controls:
5 males/5 females Negative control group
Positive control substance(s):
yes
Remarks:
0.1% DNCB in acetone

Results and discussion

Positive control results:
A sensitization response was observed in all positive control animals confirming the validity of the test method.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
100 %
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 100 %. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
100 %
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 100 %. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
100 %
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 100 %. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: none.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
100 %
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 100 %. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: none.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
0.1 %
No. with + reactions:
5
Total no. in group:
5
Clinical observations:
none
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 0.1 %. No with. + reactions: 5.0. Total no. in groups: 5.0. Clinical observations: none.
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
0.1 %
No. with + reactions:
5
Total no. in group:
5
Clinical observations:
none
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: 0.1 %. No with. + reactions: 5.0. Total no. in groups: 5.0. Clinical observations: none.

Any other information on results incl. tables

- Preliminary Irritation Trial: Irritation (erythema or oedema) was not observed on any test site at test article concentrations as high as 100 % at the 24 hour observation period.


 


- Induction and Challenge Phases: All animals gained weight during the course of the study and no abnormal clinical observations were noted. No test animals showed signs of erythema or oedema at the 24 and 48 hour observations points for the challenge phase. No reactions were observed in the negative control group and 100 % reactivity was observed in the positive control group at challenge. The incidence and severity of the responses post-challenge were calculated as shown below.


 


 


Results







































 



Negative Control



Test



Positive Control



 



24 hours



48 hours



24 hours



48 hours



24 hours



48 hours



Incidence*



0/10 = 0%



0/10 = 0%



0/20 = 0%



0/20 = 0%



5/5 = 100%



5/5 = 100%



Severity**



0/10 = 0



0/10 = 0



0/20 = 0



0/20 = 0



13/5 = 2.6



11/5 = 2.2



*Incidence – The number of animals showing a response of 1 or more divided by the number of animals tested; expressed as a percentage.


**Severity – The sum of the test reaction grades divided by the number of animals tested.


 


Remarks on results: All animals gained weight over the course of the study. No systemic signs of toxicity were observed in any animal over the course of the study. None of the test animals exhibited any signs of erythema at any of the observation points. None of the negative control animals exhibited erythema at either the 24- or 48-hour observation periods following challenge application. 

Applicant's summary and conclusion

Interpretation of results:
other: Not classified according to EU criteria
Conclusions:
Under the conditions of this study, DMBPC was not considered to be a skin sensitiser.
Executive summary:

The potential of the test material to cause skin sensitisation was investigated in a Buehler test conducted in accordance with the standardised guidelines OECD 406 and EPA OPPTS 870.2600 under GLP conditions.

Male and female Hartley guinea pigs were exposed to the neat test material during both the challenge and induction phases following a preliminary test.

For the induction, on Days 0, 7 and 14, closed patches were prepared with the test substance and applied directly to the skin .The patch was kept in place with occlusive bandaging; after 6 hours the patch was removed and any residual test substance was wiped off with a gauze pad. The positive control substance (0.4 mL of 0.1 % DNCB in acetone) was applied in the same manner. Naïve animals (i.e. untreated during the induction phase) served as a negative control group.

The challenge test was performed on the freshly clipped skin sites of the test and naïve control animals in the same manner as the 6-hour closed patch test of the induction phase. The skin was exposed to the test substance at 100 % for 6 hours. The experimental and negative control animals were exposed to one dose of test substance on week 05; 0.1 % Dinitrochlorobenzene (DNCB) in acetone was used to challenge the positive control animals. 

At approximately 24 hours after removal of the challenge dose, the test site was examined for erythema and oedema. Evaluation of the challenge area was repeated at approximately 48 hours after removal of the challenge dose patch and the skin reactions were graded.

No test animals showed signs of erythema or oedema at the 24 and 48 hour observations points for the challenge phase. No reactions were observed in the negative control group and 100 % reactivity was observed in the positive control group at challenge. All animals gained weight during the course of the study and no abnormal clinical observations were noted. 

Under the conditions of this study, DMBPC was not considered to be a skin sensitiser.