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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:
2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD TG 406, EPA OPPTS 870.2600 and in accordance with the Principles of Good Laboratory Practice (GLP).
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Study conducted prior to LLNA requirements.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: dark green crystalline solid
Details on test material:
- Name of test material (as cited in study report): 1,4,5,8-Tetra (4'-n-butylphenylamino) Anthraquinone (TBPAAQ)
- Substance type: dark green crystalline solid
- Physical state: solid
- Analytical purity: (97% active ingredient
- Lot/batch No.: Lot #020801; TD No. 02-045
- Expiration date of the lot/batch: 31 March 2004
- Stability under test conditions: expected to be stable fro the duration of testing
- Storage condition of test material: room temperature

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Elm Hill Breeding Labs, Chelmsford, MA
- Age at study initiation: young adult
- Weight at study initiation: 328-375 grams at experimental start
- Housing: group housed in suspended stainless steel caging with mesh floors
- Diet (e.g. ad libitum): Pelleted Purina Guinea Pig Chow #5025 approximately 20 grams per guinea pig daily
- Water (e.g. ad libitum): Filtered tap water was supplied ad libitum by automatic water dispensing system
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-23°C
- Humidity (%): 44-68%
- Photoperiod (hrs dark / hrs light): 12 hour 1ighUda.t-k cycle

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
other: The test substance, as received, was a crystalline solid. To enhance skin contact, the test substance was moistened with mineral oil prior to application.
Concentration / amount:
Topical phase - 0.5 g of a 80% w/w mixture of the test substance in mineral oil
Challenge phase - 0.4 ml of a 80% (w/w) mixture of the test substance in mineral oil
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: The test substance, as received, was a crystalline solid. To enhance skin contact, the test substance was moistened with mineral oil prior to application.
Concentration / amount:
Topical phase - 0.5 g of a 80% w/w mixture of the test substance in mineral oil
Challenge phase - 0.4 ml of a 80% (w/w) mixture of the test substance in mineral oil
No. of animals per dose:
20 animals/dose
Details on study design:
RANGE FINDING TESTS: Preliminary irritation testing was performed on eight animals to determine appropriate concentrations of the test substance that could be used for both intradermal and topical induction as well as topical challenge.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: intradermal - 1 injection, topical application - 0.5 gram of a 80% (w/w) mixture of TBPAAQ in mineral oil
- Exposure period: single
- Test groups: one
- Control group: yes
- Site: Intradermal injection - The test animals received 6 intradermal injections (0.1 ml each) in the shaved suprascapular area. Topical application - Seven days after the intradermal injections, the topical induction phase was conducted. The suprascapular area over the injection sites was re-clipped free of fur. Twenty-four hours prior to the topical induction, the dose area of each test and sham control animal was pre-treated with 5% w/w sodium laurel sulfate (SLS) mixture in petrolatum in order to enhance sensitization by provoking a mild inflammatory reaction. The site remained uncovered until the topical induction patch was applied. Prior to the topical induction, the test sites were gently wiped with water, using a clean towel to remove any residual SLS. Approximately twenty-five hours after SLS application, readings were made of local reactions (erythema)
- Frequency of applications: single
- Duration: single
- Concentrations: intradernal injection - 0.5% w/w suspension of TBPAAQ in complete Freund's adjuvant (50% v/v in distilled water)

B. CHALLENGE EXPOSURE
- No. of exposures: 0.4 ml of an 80% w/w mixture of TBPAAQ in mineral oil
- Day(s) of challenge: 20 days after test initiation
- Exposure period: 24 hours
- Test groups: single
- Control group: yes
- Site: a naive site on the right side of each test and sham control animal was clipped free of fur. The following day, four-tenths of a milliliter of an 80% w /w mixture (HNIC) of the test substance in mineral oil was applied to a naive site on each test animal using an occlusive 25 mm Hill Top chamber. The chambers were secured in place and wrapped with non-allergenic Durapore adhesive tape to avoid dislocation of the chambers and to prevent evaporation. After the 24 hour exposure period, the chambers were removed and the sites were gently wiped with ethanol then water, using a clean towel to remove any residual substance
- Concentrations: 0.4 ml of an 80% w /w mixture (HNIC) of the test substance in mineral oil
- Evaluation (hr after challenge): 24 and 48 hours
Challenge controls:
alpha-hexylcinnamaldehyde, technical grade (85% active ingredient) as a positive control substance)
Positive control substance(s):
yes
Remarks:
alpha-hexylcinnamaldehyde

Results and discussion

Positive control results:
Nine of the ten test animals exhibited signs of a sensitization response (faint to moderate erythema 24 hours after patch removal. Faint erythema (1) persisted at seven sites through 48 hours. Desquamation was evident at several sites following challenge.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
The test substance was applied as an 80% w/w mixture in mineral oil
No. with + reactions:
2
Total no. in group:
20
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: The test substance was applied as an 80% w/w mixture in mineral oil. No with. + reactions: 2.0. Total no. in groups: 20.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
The test substance was applied as an 80% w/w mixture in mineral oil
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: The test substance was applied as an 80% w/w mixture in mineral oil. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
sham control
No. with + reactions:
1
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: sham control. No with. + reactions: 1.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
sham control
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: sham control. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
HCA Technical (85% A.I.) applied as a 50% w/w solution in mineral oil
No. with + reactions:
9
Total no. in group:
10
Clinical observations:
Nine of the ten test animals exhibited signs of a sensitization response (faint to moderate erythema [l-2]) 24 hours after patch removal.
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
HCA Technical (85% A.I.) applied as a 50% w/w solution in mineral oil
No. with + reactions:
7
Total no. in group:
10
Clinical observations:
Faint erythema (1) persisted at seven sites through 48 hours. Desquamation was evident at several sites following challenge.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of the study, TBPAAQ is not considered to be a contact sensitiser
Executive summary:

A Magnusson-Kligman sensitization test was conducted with guinea pigs to determine the potential for TBPAAQ to evaluate dermal skin sensitization reactions.

The study was conducted using three stages; preliminary irritation screens, a two-stage induction phase and a challenge phase as described below. Preliminary irritation testing was performed on eight animals to determine appropriate concentrations of the test substance that could be used for both intradermal and topical induction as well as topical challenge.

The first induction phase involved six intradermal injections into the suprascapular area of each of 20 guinea pigs. These doses were comprised of pairs of injections of the test substance, the test substance combined with Complete Freund's Adjuvant as well as Adjuvant alone. A sham control group (ten animals) was maintained under the same environmental conditions and received injections of mineral oil, mineral oil combined with Complete Freund's Adjuvant as well as Adjuvant alone.

Approximately one week later, the second phase of induction was conducted. The test substance (test group) or mineral oil (sham control group) was applied topically for a period of 48 hours to the area encompassing the intradermal injection sites. Approximately one hour after the topical induction patches were removed, all animals were scored for erythema.

Approximately two weeks later, a challenge dose of the test substance mixed with mineral oil was applied for 24 hours to a naive site on each test and sham control animal. Approximately 24 and 48 hours after challenge patch removal the animals were scored for a sensitization response (erythema).

Based on the results of this study, TBPAAQ is not considered to be a contact skin sensitizer. The positive response observed in the historical positive control validation study with a-hexylcinnamaldehyde, technical grade (85% active ingredient) validated the test system used in this study.