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Administrative data

Description of key information

According to CLP 30% positive response in an adjuvant test warrants C&L. The test item was not considered to be a sensitiser under the conditions of the GMPT test.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 April 1999 - 11 June 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is performed according to OECD guidelines and GLP.
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
OECD 429 was not adopted until 2002
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River France, 76410 Saint-Aubin-les~Elbeuf, France.
- Age at study initiation: approximately 3 months old
- Weight at study initiation: 362 ± 16 g for the males and 364 ± 18 g for the females
- Housing:individually in polycarbonate cages (48 cm x 27 cm x 20 cm) equipped with a polypropylene bottle
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days before the beginning of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 30-70
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 29 April 1999 - 11 June 1999
Route:
intradermal and epicutaneous
Vehicle:
unchanged (no vehicle)
Concentration / amount:
Test substance concentrations were as follows:
Induction (treated group)
intradermal injections: at the concentration of 5% (w/w) in corn oil,
topical application: undiluted.
First challenge (all groups)
topical application: undiluted.
Second challenge (all groups): topical application: at the concentration of 50% (w/w) in com oil.
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
Test substance concentrations were as follows:
Induction (treated group)
intradermal injections: at the concentration of 5% (w/w) in corn oil,
topical application: undiluted.
First challenge (all groups)
topical application: undiluted.
Second challenge (all groups): topical application: at the concentration of 50% (w/w) in com oil.
No. of animals per dose:
Control: 10
Testgroup: 20
Details on study design:
RANGE FINDING TESTS:
A preliminary test was conducted in order to determine the concentrations to be tested in the main study.
By intradermal route:
- 24 hours before treatment. the dorsal region of the animals was clipped
- intradermal administrations of the test substance formulation (0.1 ml) at different concentrations were performed in the interscapular region.
- cutaneous reactions were evaluated approximately 24, 48 hours and 6 days after the injection

By cutaneous route:
- 24 hours before treatment, both flank regions of the animals were clipped
-a volume of 0.5 ml of the undiluted test substance or test substance formulation at the chosen concentration(s) was placed on a dry gauze pad (approximately 4 cm2 ) which was then applied to the skin and held in place by an occlusive dressing for 24 hours,
- cutaneous reactions were evaluated approximately 24 and 48 hours after removal of the dressings.

Criteria for selection of concentrations
The following criteria were used:
- the concentrations should be well-tolerated systemically and locally,
- intradermal injections should cause moderate irritant effects (no necrosis or ulceration of the skin),
- cutaneous application for the induction should cause at most weak or moderate skin reactions or be the maximal practicable concentration,
- cutaneous application for the challenge phase should be the highest concentration which does not cause irritant effects.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures:
Intradermal route
Three injections of 0.1 ml were made into each side of this interscapular region (i.e. three pairs of sites). as follows:
Injection sites:
Anterior, Middle, Posterior
Treated group:
1: FCA diluted at 50% (v/v) with 0.9% NaCl
2: test substance at 5% (w/w) in com oil
3: test substance at 5% (w/w) in a mixture FCA /0.9% NaCl 50/50 (v/v)
Control group:
1: FCA diluted at 50% (v/v) with 0.9% NaCI
2: vehicle
3: vehicle at 50% (w/w) in a mixture FCA 10.9% NaCl 50/50 (v/v)

Cutaneous route
On day 7, the interscapular area was clipped.
As the test substance was shown to be non-irritant during the preliminary test, the animals were treated with 0.5 ml of sodium lauryl sulfate at the concentration of 10% (w/w) in vaseline, in order to induce local irritation.
On day 8, a cutaneous application co the interscapular region was perfonned as follows:
Control group
- application of 0.5 ml of the vehicle.
Treated group
- application of 0.5 ml of the undiluted test substance.
The test substance or the vehicle was placed on a dry gauze pad, which was then applied to the interscapular region..
The pad was held in place for 48 hours by means of an adhesive hypoallergenic dressing and an adhesive analJergenic waterproof plaster. .
On removal of the dressing, no residual test substance was observed. The presence of cutaneous irritation was checked 1 hour after removal of the occlusive dressing.

- Exposure period: see above
- Test groups: 20 animals
- Control group: 10 animals
- Site: see above
- Frequency of applications: see above
- Duration:see above
- Concentrations:see above

B. CHALLENGE EXPOSURE
First challenge application
On day 22, the animals of both groups received an application of 0.5 ml of the undiluted test substance to the posterior right flank and 0.5 ml of the vehicle to the posterior left flank. This application was perfonned using a I ml plastic syringe (0.01 ml graduations). The test substance or the vehicle was placed on a dry gauze pad, which was then applied to a 4 cm2 (2 cm x 2 cm) clipped area of the skin.
The pads were held in contact with the skin for 24 hours by means of an occlusive, hypoallergenic dressing and an adhesive anallergenic waterproof plaster.
On removal of the dressing, no residual test substance was observed. As equivocal cutaneous reactions were noted, a second challenge application was perfonned after a rest period of 8 days.
Second challenge application
On day 33, the animals of both groups received an application of 0.5 m) of the test substance at the concentration of 50% (w/w) to the posterior left flank and 0.5 ml of the vehicle to the posterior right flank under the same experimental conditions as for the first challenge application.
On removal of the dressing, no residual test substance was observed.

Other
CLINICAL EXAMINATIONS
The animals were observed at least once a day during the study in order to check for clinical signs and mortality.
BODY WEIGHT
The animals were weighed individually on the day of allocation into the groups, on the first day of the study (day 1), on days 8, 15 and 25 and on the last day of the study.
Necropsy
At the end of the study, all the animals were killed by carbon dioxide asphyxiation. No necropsy was performed.
Skin samples
No skin samples were taken.
Positive control substance(s):
yes
Remarks:
2,4-Dinitro Chlorobenzene (DNCB)
Positive control results:
The species and strain which were used showed a satisfactory sensitization response in 90% animals trealed with DNCB.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
undiluted
No. with + reactions:
2
Total no. in group:
20
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: undiluted. No with. + reactions: 2.0. Total no. in groups: 20.0.

First challenge:

After 24 hours 4/20 animals in the treated group showed grade 1 erythema, no effects were observed in the control group.

A very slight or well-defined erythema (grade 1 or 2) was noted in 5/20 animals of the treated group and in 1/20 animals of the control group after 48 hours.

In the treated group 4/20 animals showed dryness of the skin and in 1/20 animal this prohibited scoring. In total 5/20 animals showed a very slight or well-defined erythema (grade 1 or 2).

In the control dryness of the skin was not observed.

Second challenge:

No cutaneous reactions were observed in the animals of the control group. In the treated group, at the 24-hour reading, a very slight or well-defined erythema (grade 1 or 2) was noted in 6/20 and 2/20 animals, respectively. At the 48-hour reading, a well-defined erythema (grade 2) persisted in one animal. Dryness of the skin was observed in 2/20 animals. The well-defined erythema recorded in 2/20 animals of the treated group was attributed to

delayed contact hypersensitivity.

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
4/20 (20%) guinea-pigs after 1nd challenge, 24-hours,
5/20 (25%) guinea-pigs after 1nd challenge, 48-hours.
2/20 (10%) guinea-pigs after 2nd challenge, 24-hours,
2/20 (10%) guinea-pigs after 2nd challenge, 48-hours.
The study appears to be well conducted only 2/20 animals giving a clear positive reaction. This is not sufficient for classification. There are a number of grade 1 reactions but these do not contribute to the positive reaction count.
Executive summary:

Thirty guinea-pigs were allocated to two groups: a control group 1 (five males and five females) and a treated group 2 (ten males and ten females).

On day 1, intradermal injections of Freund's complete adjuvant mixed with the test substance (treated group) or the vehicle (control group) were performed in the interscapular region.

On day 7, the same region received a topical application of sodium lauryl sulfate in vaseline (10%, w/w) in order to induce local irritation.

On day 8, the test substance (treated group) or the vehicle (control group) was applied to the same test site which was then covered by an occlusive dressing for 48 hours.

On day 22, after a rest period of 12 days, all animals of the treated and control groups were challenged by a cutaneous application of the test substance to the right nank. The left flank served as control and received the vehicle only. Test substance and vehicle were maintained under an occlusive dressing for 24 hours.

Skin reactions were evaluated approximately 24 and 48 hours after removal of the dressing. As equivocal cutaneous reactions were noted, a second challenge application was performed under the same experimental conditions after a rest period of 8 days, except that the test substance and the vehicle were applied to the left and right flanks, respectively.

Test substance concentrations were as follows:

Induction (treated group)

intradennal injections: at the concentration of 5% (w/w) in corn oil,

topical application: undiluted.

First challenge (all groups)

topical application: undiluted.

Second challenge (all groups): topical application: at the concentration of 50% (w/w) in com oil.

At the end of the study, animals were killed without examination of internal organs. No skin samples were taken from the challenge application sites.

The sensitivity of the guinea-pigs in CIT experimental conditions was checked with a positive sensitizer, 2,4-Dinitro Chlorobenzene (DNCB). During the induction period, the reference substance DNCB was applied at the concentrations of 0.1% (w/w) (day 1) and 1% (w/w) (day 8) in com oil. For the challenge application, the reference substance DNCB was applied at the concentration of 1% (w/w) in com oil.

Results

No clinical signs and no deaths related to treatment were noted during the study.

After 24 hours 4/20 animals in the treated group showed grade 1 erythema, no effects were observed in the control group.

A very slight or well-defined erythema (grade 1 or 2) was noted in 5/20 animals of the treated group and in 1/20 animals of the control group after 48 hours.

In the treated group 4/20 animals showed dryness of the skin and in 1/20 animal this prohibited scoring. In total 5/20 animals showed a very slight or well-defined erythema (grade 1 or 2).

In the control dryness of the skin was not observed.

After the second challenge application, no cutaneous reactions were observed in the animals of the control group.

In the treated group at the 24-hour reading, a very slight or well-defined erythema was noted in 6/20 and 2/20 animals, respectively. At the 48-hour reading, a well-defined erythema persisted in one animal. Dryness of the skin was observed in 2/20 animals. The well defined erythema recorded in 2/20 animals of the treated group was attributed to delayed contact hypersensitivity. The species and strain which were used showed a satisfactory sensitization response in 90% animals trealed with DNCB.

Conclusion

According to the authors under these experimental conditions the test substance induces delayed contact hypersensitivity in 2/20 (10%) guinea-pigs after 2nd challenge.

The study appears to be well conducted only 2/20 animals giving a clear positive reaction. This is not sufficient for classification. There are a number of grade 1 reactions but these do not contribute to the positive reaction count.

Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The study appears to be well conducted only 2/20 animals giving a clear positive reaction. This is not sufficient for classification. There are a number of grade 1 reactions but these do not contribute to the positive reaction count.

Migrated from Short description of key information:

4/20 (20%) guinea-pigs after 1nd challenge, 24-hours,

5/20 (25%) guinea-pigs after 1nd challenge, 48-hours.

2/20 (10%) guinea-pigs after 2nd challenge, 24-hours,

2/20 (10%) guinea-pigs after 2nd challenge, 48-hours.

Justification for selection of skin sensitisation endpoint:

K1: The study is performed according to OECD guidelines and GLP.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

According to CLP 30% positive response in an adjuvant test warrants C&L. The test item was not considered to be a sensitiser under the conditions of the GMPT test.