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Administrative data

Description of key information

A Magnusson-Kligman Maximisation test in male guinea pigs subjected to intradermal (day1; 10%) and topical induction (day 7; 50%) and topical challenge (day 22; 50%) under occlusive dressing failed to elicit a delayed hypersensitivity response.

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
Study period:
1984-08-29 to 1984-10-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Principles of method if other than guideline:
Method referenced to Magnusson B and Kligman A M, "The identification of contact allergens by animal assay, the guinea pig maximisation test",
Journal of Investigative Dermatology, Vol. 52, No. 3, pp. 268-276, 1969.
GLP compliance:
not specified
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The study was conduced before REACH Guidance on in vivo LLNA testing was generated.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Olac 1976 Ltd., Bicester, Oxfordshire, England
- Age at study initiation: no data
- Weight at study initiation: 315-408 g
- Housing: ten guinea-pigs of the same sex housed in open-top, anodised aluminium cages (90 x 60 x 27 cm) with solid floors
- Diet: ad libitum (Guinea Pig F.D.I. from Special Diet Services), plus a daily supplement of autoclaved hay
- Water: ad libitum tap water
- Acclimation period: 6 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): target 18 (acceptable range: 15-23)
- Humidity (%): target 55 (acceptable range: 40-70)
- Air changes (per hr): 17 without recirculation
- Photoperiod (hrs dark / hrs light): 12/12
Route:
intradermal and epicutaneous
Vehicle:
olive oil
Concentration / amount:
Primary Induction:
0.1 ml of 10% w/v AN-701 in olive oil and 10% w/v AN-701 in Freunds Complete Adjuvant

Secondary induction: after sodium lauryl sulphate 10% in petrolatum was applied the day before, 0.4ml of 50% w/v AN-701 in olive oil

Challenge:
0.2 ml of 50% AN-701 in olive oil
Route:
epicutaneous, occlusive
Vehicle:
olive oil
Concentration / amount:
Primary Induction:
0.1 ml of 10% w/v AN-701 in olive oil and 10% w/v AN-701 in Freunds Complete Adjuvant

Secondary induction: after sodium lauryl sulphate 10% in petrolatum was applied the day before, 0.4ml of 50% w/v AN-701 in olive oil

Challenge:
0.2 ml of 50% AN-701 in olive oil
No. of animals per dose:
20 test animals and 20 control animals, evenly divided as to sex
Details on study design:
RANGE FINDING TESTS: for skin irritation

Intradermal injections: an area of 4 x 6cm over scapula on 4 guinea pigs was shaved. An intradermal injections of 0.1ml of 0.5%, 2%, 5%, 10%, 25% and 50% w/v AN-701 in olive oil was used for two of the test subjects or in Freunds complete adjuvant for the remaining two test subjects. Reactions to the treatment were assessed at 24 and 48 hours post injection.

Topical application: The hair was shaved from both flanks of five guinea pigs on the day before dosing. The test sites were treated with 0.2ml of 1%, 4%, 10%, 20% or 50% w/v AN-701 in olive oil, absorbed onto a two-ply gauze patch (10x10mm) and covered by an air tight occlusive dressing for 24 hours. Reactions to the test material were assessed at 24 and 48 hours after the removal of the dressing.

Criteria for selection of treatment regimes for main study: The concentration selected must be well tolerated locally and systemically. Intradermal injections must not cause necrosis or ulceration of skin and concentration used at challenge must be at sub-irritant levels.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 exposures (intradermal and epicutaneous)

- Exposure period: Day 1, intradermal induction (primary induction). On day 7 the shaven dorsum of all animals were subjected to inunction with 10% w/v sodium lauryl sulphate in petrolatum to enhance dermal absorption the next day. On Day 8 the dermal areas were treated with test material for a 48 hour time period (secondary induction).
- Test groups: 20 animals (10 males, 10 females)

- Control group: 20 animals (10 males, 10 females)

- Site: Primary induction; 3 pair of injections were made deep into the dermis, such that on either side of the dorsal median line there were three injection sites in a row parallel to the spinal column. All injection sites lay near the periphery of a dermal test site 40mm x 20mm long, overlaying the scapula. The anterior and middle sites were positioned close together and distant from the posterior sites. Secondary induction: A shaven area on the dorsum of all animals. The doses were absorbed onto 2-ply gauze (25x25 mm) which were applied to the skin and covered by an occlusive dressing.

For the primary induction (test group):
Anterior sites: 0.1 of ml Freunds complete adjuvant
Middle sites: 0.1 ml of 10% w/v AN-701 in olive oil
Posterior sites: 0.1 ml of 10% w/v AN-701 in Freunds complete adjuvant
For the primary induction (control group):
Anterior sites: 0.1 ml of Freunds complete adjuvant
Middle sites: 0.1 ml of olive oil
Posterior sites: 0.1 ml of Freunds complete adjuvant

For secondary induction (test group):
0.4ml of 50% w/v AN-701 in olive oil
For secondary induction (control group):
0.4ml of olive oil

- Duration: Secondary induction: 48 hours

B. CHALLENGE EXPOSURE
-Methodology: On day 21 both flanks on the trunk of all animals (test and control group) were shaven. The following day the left side was treated with by topical application of 0.2ml of olive oil while the opposite side was treated with 0.2ml of 50% w/v AN-701, absorbed onto 2 ply gauze (10x10 mm). Both sides were then covered by an occlusive dressing for a period of 24 hours. Three hours after the removal of the challenge treatment and dressing the treatment sites were gently depilated with a cream of calcium thioglycolate and then washed.

- Evaluation (hr after challenge):
The challenge sites were examined at 24 and 48 hours after the removal of treatment.


OTHER:
The presence or absence of erythema and swelling after the challenge treatment was recorded and scored in blind.
Challenge controls:
olive oil and Freunds complete adjuvant.
Positive control substance(s):
no
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0.2 ml of olive oil
No. with + reactions:
1
Total no. in group:
20
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 0.2 ml of olive oil. No with. + reactions: 1.0. Total no. in groups: 20.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
0.2 ml of olive oil
No. with + reactions:
1
Total no. in group:
20
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 0.2 ml of olive oil. No with. + reactions: 1.0. Total no. in groups: 20.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0.2 ml of olive oil
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 0.2 ml of olive oil. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
0.2 ml of olive oil
No. with + reactions:
1
Total no. in group:
20
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 0.2 ml of olive oil. No with. + reactions: 1.0. Total no. in groups: 20.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0.2ml of 50% w/v AN-701
No. with + reactions:
3
Total no. in group:
20
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 0.2ml of 50% w/v AN-701. No with. + reactions: 3.0. Total no. in groups: 20.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
0.2ml of 50% w/v AN-701
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 0.2ml of 50% w/v AN-701. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0.2ml of 50% w/v AN-701
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 0.2ml of 50% w/v AN-701. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
0.2ml of 50% w/v AN-701
No. with + reactions:
1
Total no. in group:
20
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 0.2ml of 50% w/v AN-701. No with. + reactions: 1.0. Total no. in groups: 20.0.
Interpretation of results:
GHS criteria not met
Conclusions:
AN-701 failed to elicit a delayed hypersensitivity response in guinea pigs.
Executive summary:

The potential for 2,6-di-tert-butylphenol, AN-701, to cause delayed hypersensitivity in guinea-pigs was assessed by the Magnusson-Kligman Maximisation test. Ten female and ten male guinea pigs were subjected to intradermal injections of Freunds complete adjuvant, 10% w/v AN-701 in olive oil, and 10% w/v AN-701 in Freunds complete adjuvant on day 1. Seven days later the same area of skin was treated by a topical treatment of 50% w/v of AN-701 in olive oil and the test site was covered by an occlusive dressing. A control group was treated in the same method with the test material omitted. On day 22 all animals were challenged by the application of olive oil on one flank and 50% w/v AN-701 in olive oil on the opposite flank and challenge sites were covered by an occlusive dressing. The occlusive dressing was removed after 24 hours and the test material was removed. The degree of a hypersensitivity reaction was assessed 24 and 48 hours after the removal of test material. The incidence and severity of the reaction to AN-701 in suspension was no greater than the vehicle control group. The conclusion reached therefore is that AN-701 had failed to elicit a delayed hypersensitivity response in guinea pigs.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The test material was evaluated for its sensitisation potential to guinea pig skin. The test material failed to elicit a delayed hypersensitivity response and therefore 2,6-di-tert-butylphenol should be classified as non-sensitising according to the EEC labelling regulations. No symbol and risk phrase are required.

Test method:

The method used followed that described in the OECD Guidelines for Guideline 406 (Skin Sensitisation). Ten female and ten male guinea pigs were subjected to intradermal injections of Freunds complete adjuvant, 10% w/v AN-701 in olive oil, and 10% w/v AN-701 in Freunds complete adjuvant on day 1. Seven days later the same area of skin was treated by a topical treatment of 50% w/v of AN-701 in olive oil and the test site was covered by an occlusive dressing. A control group was treated in the same method with the test material omitted. On day 22 all animals were challenged by the application of olive oil on one flank and 50% w/v AN-701 in olive oil on the opposite flank and challenge sites were covered by an occlusive dressing. The occlusive dressing was removed after 24 hours and the test material was removed. The degree of a hypersensitivity reaction was assessed 24 and 48 hours after the removal of test material.

Justification for classification or non-classification

The test material failed to elicit a delayed hypersensitivity response and therefore in accordance with EU CLP (Regulation (EC) No. 1272/2008) 2,6-di-tert-butylphenol should be classified as non-sensitising. No symbol and risk phrase are required.