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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:
Between 30 March and 4 May 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report date:
1994

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
An LLNA is not performed because other reliable information is available.

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
416-210-4
EC Name:
-
Cas Number:
128119-70-0
Molecular formula:
C14H26O2
IUPAC Name:
2-methyl-3-({1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl}oxy)propan-1-ol
Test material form:
liquid

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
Fifteen healthy female nulliparous and non-pregnant albino guinea-pigs of the Dunkin/Hartley strain were obtained from D. Hall, Newchurch, Staffordshire, England. The animals were in the weight range of 313 to 375 g on arrival and approximately six to seven weeks of age. All the guinea-pigs were acclimatised to the experimental environment for 13 days prior to allocation to the main study. An additional six animals, from the same supplier, were used for the preliminary investigations.

The animals on the main study were allocated without conscious bias to two groups as follows:
Group Number of animals Animal Numbers
Control animals 5 1020 to 1024
Test animals 10 1030 to 1039

- The guinea-pigs were housed in groups of ten in suspended metal cages with wire mesh floors in Building R 17 Room 13.
- A vitamin C enriched guinea-pig diet FD 1 and drinking water were provided ad libitum. Hay was given weekly. The batch of diet used for the study was not analysed for nutrients, possible contaminants or microorganisms. However, other batches of diet are periodically tested, by the Supplier, and were found to be within the set limits. Results of routine physical and chemical examination of drinking water at source, as conducted usually weekly by the supplier, are made available to Huntingdon Research Centre Ltd. as quarterly summaries.
- Animal room temperature was maintained at approximately 21°C and relative humidity at 30 - 70%. These environmental parameters were recorded daily. Air exchange was maintained at approximately 15 air changes per hour and lighting was controlled by means of a time switch to give 12 hours of artificial light (0700 - 1900 hours) in each 24 hours period.

Study design: in vivo (non-LLNA)

Induction
Route:
intradermal and epicutaneous
Vehicle:
other: Alembicol D
Concentration / amount:
Induction intradermal injection: 5% v/v and Induction topical application: 100%.
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challenge
Route:
epicutaneous, occlusive
Vehicle:
other: Alembicol D
Concentration / amount:
50% and 100%
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
10 test
5 control
Details on study design:
Preliminary study
The intradermal and topical irritancy of a range of dilutions of the test substance was investigated to identify where possible (a) concentrations of the test substance that would produce irritation suitable for the induction phase of the main study and (b) a maximum non-irritant concentration by the topical route of administration for the challenge phase.

Main study
Induction intradermal injections - test animals
- A 40 x 60 mm area of dorsal skin on the scapular region of the guinea-pig was clipped free of hair with electric clippers. Three pairs of intradermal injections were made into a 2 x 4 cm area within the clipped area.
Injectables for the test animals were prepared as follows:
1. Freund's complete adjuvant was diluted with an equal volume of water for irrigation (ph.Eur.).
2. Bornafix, 5% v/v in Alembicol D.
3. Bornafix, 5% v/v in a 50 : 50 mixture of Freund's complete adjuvant and Alembicol D.

Induction topical application - test animals
- The preliminary investigations indicated that the maximum practical concentration of the test substance for topical application (as supplied) did not produce skin irritation. Therefore, six days after the injections, the same 40 x 60 mm interscapular area was clipped and shaved free of hair and the site was pre-treated by gentle rubbing with 0.2 ml per site of 10% w/w sodium lauryl sulphate in petrolatum. Twenty-four hours later a 20 x 40 mm patch of Whatman No. 3 paper was saturated with approximately 0.4 ml of Bornafix, as supplied. The patch was placed on the skin of the test animals and covered by a length of impermeable plastic adhesive tape (50 mm width "Blenderm"). This in turn was firmly secured by elastic adhesive bandage (50 mm width "Elastoplast") wound round the torso of the animal and fixed with "Sleek" impervious plastic adhesive tape. The dressing was left in place for 48 hours.

Induction - control animals
- During the induction phase, the control animals were treated similarly to the test animals with the exception that the test substance was omitted from the intradermal injections and topical application.
The dermal reactions observed after each induction phase in both control and test animals by group are shown in Table 1 - attachment 4.

Challenge
- The control and test animals were challenged topically two weeks after the topical induction application using Bornafix, as supplied and 50% v/v in Alembicol D. Hair was removed by clipping and then shaving from an area on the left flank of each guinea-pig. A 20 x 20 mm patch of Whatman No. 3 paper was saturated with approximately 0.2 ml of Bornafix, as supplied and applied to an anterior site on the flank. Bornafix, 50% v/v in Alembicol D was applied in a similar manner to a posterior site. The patches were sealed to the flank for 24 hours under strips of "Blenderm" covered by "Elastoplast" wound round the trunk and secured with "Sleek" .

Observations
- Clinical signs: All animals were observed daily for signs of ill health or toxicity.
- Bodyweight: The bodyweight of each guinea-pig on the main study was recorded on Day 1 (day of intradermal injections) and on the last day observations were made of dermal responses to the challenge applications.
- Dermal responses: The diameter (mm) of the dermal response at the intradermal injection sites was recorded in the preliminary study only to assist in the choice of concentrations for the main study. Any other lesion not covered by this scoring system was described. The challenge sites were evaluated 24, 48 and 72 hours after removal of the patches.The dermal reactions resulting from intradermal injection and topical application on the preliminary study, and topical application at the challenge were assessed using the following numerical system:
-- Erythema and eschar formation: No erythema: 0, Slight erythema: 1, Well-defined erythema: 2, Moderate erythema: 3, Severe erythema (beet redness) to slight eschar formation (injuries in depth): 4
-- Oedema formation: No oedema: 0, Slight oedema: 1, Well-defined 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

Interpretation of results
- Dermal reactions in the test animals elicited by the challenge application were compared with the findings simultaneously obtained in the control animals.
- A test animal was considered to show positive evidence of delayed contact hypersensitivity if the observed dermal reaction at challenge was definitely more marked and/or persistent than the maximum reaction seen in animals of the control group.
- If the dermal reaction seen in a test animal at challenge was slightly more marked and/or persistent than (but not clearly distinguishable from) the maximum reaction seen in control animals, the result for that test animal was classified as inconclusive
- A test animal was considered to show no evidence of delayed contact hypersensitivity if the dermal reaction resulting from the challenge application was the same as, or less marked and/or persistent than the maximum reaction seen in animals of the control group.
Challenge controls:
During the induction phase, the control animals were treated similarly to the test animals with the exception that the test substance was omitted from the intradermal injections and topical application.
Positive control substance(s):
yes
Remarks:
The sensitivity of the guinea-pig strain used is checked periodically at HRC with hexyl cinnamic aldehyde, a known sensitiser.

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
100%
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
100%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Localised dermal reaction (restricted to a small area of the challenge site) in one animal. Dryness and sloughing of the epidermis in 10 animals
Key result
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
100%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Dryness and sloughing of the epidermis in 4 animals
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
50%
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
50%
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
50%
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
100%
No. with + reactions:
0
Total no. in group:
5
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
100%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
Localised dermal reaction (restricted to a small area of the challenge site) in one animal. Dryness and sloughing of the epidermis in 5 animals
Key result
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
negative control
Dose level:
100%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
Dryness and sloughing of the epidermis in 2 animals
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
50%
No. with + reactions:
0
Total no. in group:
5
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
50%
No. with + reactions:
0
Total no. in group:
5
Key result
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
negative control
Dose level:
50%
No. with + reactions:
0
Total no. in group:
5
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
100 and 50%
No. with + reactions:
29
Total no. in group:
30

Any other information on results incl. tables



CLINICAL SIGNS


- No signs of ill health or toxicity were recorded.


 


BODYWEIGHT


- Anticipated bodyweight increases were recorded for all guinea-pigs over the period of the study.


 


INDUCTION


- Intradermal injections: Necrosis was recorded at sites receiving Freund's Complete Adjuvant in test and control animals. Slight irritation was seen in test animals at sites receiving Bornafix, 5% v/v in Alembicol D and also in control animals receiving Alembicol D.


- Topical application: Slight erythema was observed in test animals following topical application with Bornafix, as supplied. No erythema was seen in the control guinea-pigs.


 


CHALLENGE


- The dermal reactions seen in the test animals were the same as or less marked than those seen in the controls.

Applicant's summary and conclusion

Interpretation of results:
other: Substance is not a skin sensitiser in accordance with EU CLP (1272/2008 and its amendments)
Conclusions:
In this test, Bornafix did not produce evidence of skin sensitisation (delayed contact hypersensitivity) in any of the animals.
Executive summary:

A GPMT study according to OECD 406 was performed to assess the skin sensitisation potential of Bornafix using the guinea-pig. Based on the results of a preliminary study and in compliance with the guideline, the following dose levels were selected based on the skin irritation in the preliminary study: Intradermal injection: 5% v/v in Alembicol D and topical application: 100%. Challenge application: 100 and 50% v/v in Alembicol D. Ten test and five control guinea-pigs were used in this study. In this study, Bornafix did not produce evidence of skin sensitisation (delayed contact hypersensitivity) in any of the test animals.