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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:
03-02-2000 to 21-07-2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
Method employed in this study for the detection of delayed contact hypersensitivity was the guinea-pig maximization test described by B. Magnusson and A.M. Kligman - "The identification of contact allergens by animal assay, the guinea pig maximisation method"; Invest. Dermatol. 1969. 52, 268-276
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: September 1999 ; signature: December 1999
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Scientific justification and information as to the availability of the study is attached by the applicant in 'attached background material'.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: Liquid
- Storage condition of test material: At room temperature, protected from light and under nitrogen gas
- Other: colourless

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Remarks:
Cri: (HA) BR
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Recognised supplier
- Age at study initiation: Young adult (approximately 3 months old).
- Weight at study initiation: 362 ±16 g males and 343 ±15 g females
- Housing: Individually in polycarbonate cages (48 cm x 27 cm x 20 cm) with dust free sawdust and polypropylene water bottle.
- Diet (e.g. ad libitum): Complete maintenance diet for guinea pigs (details in the full study report).
- Water (e.g. ad libitum): filtered (0.22 micron) water ad libitum
- Acclimation period: The acclimatization period was at least 5 days before the start of treatment under laboratory conditions; identical to the test

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ±2 °C
- Humidity (%): 30 – 70%
- Air changes (per hr): at least 12 per hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark

IN-LIFE DATES: From: To: 08-02-2000 to 13-03-2000

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
olive oil
Concentration / amount:
- Intradermal: 25% test material in Corn Oil
Day(s)/duration:
Day 1
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
Route:
epicutaneous, occlusive
Concentration / amount:
- Topical: 100% test material (undiluted)
Day(s)/duration:
Day 8
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
Challengeopen allclose all
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Remarks:
Corn oil for the intradermal injections for the topical applications
Concentration / amount:
- Challenge: 100% (or in corn oil as applicable)
Day(s)/duration:
Day 22
Adequacy of challenge:
highest non-irritant concentration
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Remarks:
Corn oil for the intradermal injections for the topical applications
Concentration / amount:
- Challenge: 30% in corn oil
Day(s)/duration:
Day 32
No. of animals per dose:
Test group: 20; Control group: 10
Details on study design:
RANGE FINDING TESTS:
A preliminary irritation study was conducted in order to select test substance concentrations to be used in the main study. By intradermal route: tested concentrations: 75%, 50% and 25% (w/w). Cutaneous reactions were evaluated approximately 24, 48 hours and 6 days after the injections. By cutaneous route (occlusive dressing, 24 hours) tested concentrations: 100% and 75% (w/w). Reactions were evaluated approximately 24 and 48 hours after removal. At 24 and 48 hours: intradermal route 25%w/w was selected due to slight irritation to irritation, and which remained at 6 days. In higher concentrations crust formation was observed at 6 days. At 24 and 48 hours: cutaneous route: no reactions were observed at 75% (w/w) or 100% concentration. Final concentrations for definitive testing based on preliminary irritation study:
- Intradermal: 25% test material in Corn Oil
- Topical: 100% test material (undiluted)
- Challenge: 1st challenge: 100% (undiluted) ; 2nd challenge: 30%w/w in Corn Oil
Vehicle: Corn oil (intradermal induction and topical challenge), was chosen on the basis of the most suitable formulation at the required concentrations and maximising the solubility of the test substance. The preparation of the test item at 0.9% NaCl solution (v/v) was non-homogenous with two phases, within in pre-testing solubility testing.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 1 intradermal induction; 1 epidermal induction (topical booster)
- Exposure period: Day 1 intradermal induction and Day 8 topical induction (topical booster)
- Test groups: duplicate injections as follows: 2 ID: Freund's Complete Adjuvant diluted at 50% in 0.9% NaCl; 2 ID: test item at 25% in corn oil ; 2 ID: a test mixture of the test item at 25% in FCA at 50% and in 0.9% NaCl equal volumes v/v.
- Control group: Vehicle and 50:50 FCA in 0.9% NaCl, and vehicle at 50% in 50:50 FCA/0.9% NaCl, only.
- Site: intradermal induction – three pairs of injections in clipped interscapular region;
- Frequency of applications:
- Duration: 0-7 days. On day 7, clipped and SLS at 10% application. On day 8 - 48 hours for epidermal induction. The dressing was removed after 48 hours exposure
- Concentrations: Intradermal induction: A) 1:1 w/w mixture of Freunds' Complete Adjuvant with 0.9% NaCl for injection; B) Test item at a 25% concentration ; C) A 1:1 w/w mixture of the test item, at twice the concentration used in (B) and Freunds' Complete Adjuvant/0.9% NaCl. Topical induction: The scapular area between the injection sites was clipped and brushed with a solution of SLS at 10%. Then the following day subsequently treated with 0.5 mL of a 100% test item concentration using occlusive dressing.
The control group were treated as described for the experimental group except that, instead of the test item, the vehicle was administered along with injections of (A) and (C) in three sites.

B. CHALLENGE EXPOSURE
- No. of exposures: 1 initial challenge ; 1 further challenge
- Day(s) of challenge: 24 hours (topical challenge)
- Exposure period: Day 22 the dressing was removed after 24 hours exposure. The treated sites were assessed for challenge reactions 24 and 48 hours after removal of the dressing. The second challenge was performed after a 9 day rest period.
- Test groups: 1 challenge; test item 100% and 2 challenge: 30%w/w in corn oil vehicle.
- Control group: 1; vehicle only
- Site: One flank (clipped)
- Concentrations: 100 and 30% using occlusive dressing.
- Evaluation (hr after challenge): 24 and 48 hours after dressing removal (at Day 23 and 24 and/or day 32).
The control group were treated as described for the experimental group except that, instead of the test item, the vehicle was administered.

OTHER: Mortality, toxicity and body weights along with irritation were examined as part of the study
Challenge controls:
(Naive) negative control groups consisting of 5 females and 5 males were exposed to the vehicle in the induction and challenge, consistent the main study with the difference that instead of test item only the vehicle was administered during induction.
Positive control substance(s):
yes
Remarks:
Mercaptobenzothiazole (1%w/w intradermal and 20%w/w topical)

Results and discussion

Positive control results:
A reliability check was performed (presented in the full study report) to check the sensitivity of the test system and the reliability of the experimental techniques used. The study used the same conditions as the main study using Mercaptobenzothiazole (at 1%w/w intradermal induction and and 20%w/w topical concentrations) as positive control.
The skin reactions observed in seven experimental animals in response to the > 20 % PC item concentration in the challenge phase were considered indicative of sensitisation, based on the absence of any response in the control animals. These results lead to a sensitisation rate of 80% to the 1%w/w induction and 20%w/w challenge concentrations.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
100%
No. with + reactions:
0
Total no. in group:
19
Clinical observations:
Total no. in groups: 19.0. (one mortality at day 12) ; Clinical observations: None reported; maximum score = 1 (n=10) to 2 (n=3) (discrete or moderate erythema).
Remarks on result:
no indication of skin sensitisation
Remarks:
Were within the range of control group
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
100%
No. with + reactions:
0
Total no. in group:
19
Clinical observations:
Total no. in groups: 19.0. (one mortality at day 12) ; Clinical observations: None reported; maximum score = 1 (n=9) to 2 (n=1) (discrete or moderate erythema).
Remarks on result:
no indication of skin sensitisation
Remarks:
Were within the range of control group
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Total no. in groups: 10.0. ; Clinical observations: None reported; maximum score = 1 (n=7) to 2 (n=1) (discrete or moderate erythema).
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Total no. in groups: 10.0. ; Clinical observations: None reported; maximum score = 1 (n=7) (discrete erythema).
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
1% intradermal induction and 20% cutaneous
No. with + reactions:
7
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Remarks:
non-concurrent PC conducted within 6 months of the definitive test; % sensitised was based on comparison of challenge sites with test and controls.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study, the test item is not considered to be a contact sensitizer.
Executive summary:

The study was performed according to guideline OECD TG 406 and EU Method B.6 and consistent with the Magnusson-Kligman Guinea Pig Maximisation test under GLP to assess the skin sensitisation potential of the test item. The concentrations selected for the main study were based on the results of a preliminary study. In the main study, after induction (intradermic injection at 25%w/w in corn oil vehicle and topical application at 100%, on days 1 and 8 respectively) with 10% SLS application on day 7. A treatment group of ten males/females respectively and a control group of five males/females were on day 22, challenged with 100% (undiluted) test item along with parallel control challenged at 100% (undiluted) test item. The left flank was treated with vehicle only. Skin reactions were evaluated 24 to 48 hours after removal of the occlusive dressing. Due to equivocal reactions a second challenge was conducted on day 32 with 30%w/w test item in corn oil. One mortality (male) was found on day 12 in the treatment group. No clinical signs were observed prior to mortality death. This was not attributed to. treatment with the test item. There were no clinical signs during the study. Bodyweight gain in the treated group was comparable to controls. In the first challenge, a discrete or moderate erythema (grade 1 or 2) was noted in 8/10 of the control group and in 13/19 of the treated group. Dryness of the skin was observed in one member of each group. In the second challenge, a discrete or moderate erythema (grade 1 or 2), sometimes together with dryness of the skin, was noted in 10/10 of the control group and in 13/19 of the treated group. As the cutaneous reactions observed after both challenge applications of the treated group were of similar incidence and severity when compared to those recorded in the control group, they were attributed to the irritant properties of the test item. Under the conditions of this study, the test item is not considered to be a contact skin sensitizer.