Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04/05/10 to 04/06/18
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report date:
2004

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test

Test material

Constituent 1
Reference substance name:
LCE04026
IUPAC Name:
LCE04026
Details on test material:
- Storage: ambient temperature, out of the light, in a room specially fitted out to that effect
- Batch: 41645

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
male
Details on test animals and environmental conditions:
- Species: Hartley albino guinea-pigs
- Origin: SCEA (24610 Villefranche-de-Lonchat, France)
- Acclimatization: for at least 5 days before the beginning of the test
- Identification: each animal was identified individually by auricular ring and the corresponding number was written on a label put on its cage
- Housing: the animal were housed one by cage
- Temperature: 21 +/-3 °C
- Relative humidity: 50 +/- 20%
- Renewal of air: 10 cycles per hour
- Feeding: the complete diet was supplied under pelleted from 106, which guaranteed an appropriate ascorbic acid content, delivered by UAR (91360 Epinay sur Orge, France)
- Drinking: the tap water was distributed in polypropylene bottle with stainless steel teat.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
water
Remarks:
(distilled)
Concentration / amount:
- Preliminary tests:
determination of the highest concentration to cause mild to moderate skin irritation by intradermal injection(MIC ID): 10, 5, 2.5, and 1.25% in distilled water
determination of the highest concentration to cause mild to moderate skin irritation by topical application (MIC T): 50, 25, 12.5 and 6.25% in distilled water.
determination of the maximal non irritating concentration (MNIC) by topical application: 50, 25, 12.5 and 6.25% in distilled water
- Main study:
intradermal induction: 1.25% in distilled water
topical induction: 50 % in distilled water
challenge application: MNIC (50%) and MNIC/2 (25%)
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
water
Remarks:
(distilled)
Concentration / amount:
- Preliminary tests:
determination of the highest concentration to cause mild to moderate skin irritation by intradermal injection(MIC ID): 10, 5, 2.5, and 1.25% in distilled water
determination of the highest concentration to cause mild to moderate skin irritation by topical application (MIC T): 50, 25, 12.5 and 6.25% in distilled water.
determination of the maximal non irritating concentration (MNIC) by topical application: 50, 25, 12.5 and 6.25% in distilled water
- Main study:
intradermal induction: 1.25% in distilled water
topical induction: 50 % in distilled water
challenge application: MNIC (50%) and MNIC/2 (25%)
No. of animals per dose:
11 animal for the treated group
5 animals for the control group
Details on study design:
- Preliminary test
Determination of the MIC by intradermal injection - (MICID):
few days before the main test, 2 guinea-pigs were closely-clipped at dorsal lumbar region over an area of approximately 30 cm². They received, on each side of the spinal column, the test item at 4 different concentrations in intradermal injection, of 0.1 ml volume.
The concentrations were chosen according to the type of the test item and the vehicle.
The choice of the highest dose was limited by the diameter of the injection needle (≥ 21 G).
24 hours ± 1 hour after the injection, the skin reactions were assessed according to the grading scale used in the main test

Determination of the MIC by topical application (MICT):
Few days before the main test, 2 guinea-pigs were closely-clipped and depilated at dorsal lumbar region over an area of approximately 30 cm².
The guinea-pigs received the test item and its dilutions in the chosen vehicle.
For each animal, the 4 different concentrations (50 %, 25 %, 12.5 % and 6.25 %) of the test item, in the chosen vehicle (distilled water) were deposited on patches composed of a 2 cm x 2 cm square of filter paper (i.e. 4 cm²), put on a 4 cm² square of gauze, itself put on a square of Blenderm occlusive film of appropriate size. The occlusive dressings were held in contact with a Micropore hypoallergenic adhesive film which surrounded the trunk of the animal without hindering from breathing.
The quantities put on each patch, were of 0.25 ml.
The patches were maintained for 48 hours ± 1 hour in contact with the skin and then removed.
The skin reactions were assessed after patch removal, according to the grading scale used in the main test

Determination of the MNIC by topical application
One week before the main test, on D-7, 2 guinea-pigs were closely-clipped at shoulder region over an area of approximately 60 cm². They received, on each side of the spinal column, 3 pairs of intradermal injections of 0.1 ml volume, of the 3 following solutions :
(1) Freund’s Complete Adjuvant (FCA) diluted at 50 % with distilled water (v/v)
(2) Distilled water (vehicle)
(3) Mixture 50/50 of the solutions (2) and (1) (v/v)
Following a rest period of 17 days, animals were closely-clipped and depilated at dorsal lumbar region.
3 days later, 4 different concentrations (50 %, 25 %, 12.5 % and 6.25 %) of the test item in the chosen vehicle (distilled water) (defined in v/v from the results obtained during the determination of the MICT) were put on occlusive patches. They were maintained on the body of the animal with a Micropore hypoallergenic adhesive film.
The quantities put on each patch were of : 0.20 ml.
The patches were maintained for 24 hours ± 1 hour in contact with the skin and then removed.
The skin reactions were assessed, according to the grading scale used in the main test, 24, 48 and 72 hours (± 1 hour) after patch removal.

- Main test
Experimental schedule and distribution of the animals per group
Animals were closely-clipped, at shoulder area, on D-1 (during acclimatization period) avoiding abrading the skin.
On D1, the 16 animals were distributed between the treated group (11 animals) and the control group (5 animals) at random, according to the Moses and Oakford's random table and then weighed and put in individual cages.
The treated group was submitted to all the stages of the experimentation: induction and challenge with the test item.
The control group only received the test item in primary application, concurrently to the challenge test performed on the treated group.

Induction exposures
On D1: intradermal injections
- Control group = group 1
Three pairs of intradermal injections of 0.1 ml volume were given in the shoulder region cleared of hair on each side of the midline.
(1) FCA diluted at 50 % with distilled water (v/v)
(2) Distilled water (vehicle)
(3) mixture 50/50 of the solutions
- Treated group = group 2
Three pairs of intradermal injections of 0.1 ml volume were given in the same sites and order as in the control animals.
(1) FCA diluted at 50 % with distilled water (v/v)
(2) test item at the dose previously defined (MICID)
(3) test item at the same dose as in (2) in a 50/50 mixture (v/v) of FCA and distilled water (v/v) (the mixture was homogenized under magnetic stirring).
In injection 3, the water soluble test item was dissolved in the aqueous phase prior to mixing with FCA .
Injections 1 and 2 were given close to each other and nearest the head, while the injection 3 was given towards the caudal part of the test area.
On D8 : following a rest period of 7 days, animals were closely-clipped again in the shoulder region.
As the test item did not induce sufficient skin reactions (erythema < 2) during the determination of the MCIT, the test area was painted with 0.5 ml of a 10 % sodium lauryl sulfate preparation in petrolatum, in order to create a local irritation. This treatment was performed in the 2 groups of animals (treated and control).
On D9 : topical application
- Control group = group 1
0.5 ml of vehicle was deposited on the patch composed of a 8 cm² rectangle of filter paper (2 cm x 4 cm), put on a 8 cm² rectangle of gauze (2 cm x 4 cm) itself put on a 25 cm² square of Blenderm occlusive film. The occlusive dressing was held in contact for 48 hours ± 1 hour with a Micropore hypoallergenic adhesive film which surrounded the trunk of the animal,.
- Treated group = group 2
0.5 ml of the test item at the MICT was applied according to the method previously defined and let in contact with the skin for 48 hours ± 1 hour.
On D11 : patches were removed and skin reactions were observed and reported in the work document reserved for that effect.

Challenge exposure
Following a rest period of 5 days, the guinea-pigs from the 2 groups were closely-clipped (D17) and depilated (D18) on one side at dorsal lumbar level, in an area which has never been in contact with the test item before.
On D21 : topical application
0.20 ml of the test item at its MNIC and 0.20 ml of the test item at its MNIC/2 were respectively applied to 2 patches each one composed of a square of 4 cm² filter paper, put on a 4 cm² square of gauze, itself put on a Blenderm occlusive film of appropriate size. The patches were held on the depilated flank of the animal by a Micropore hypoallergenic adhesive film.
The occlusive dressings were let in contact for 24 hours ± 1 hour.

Observations and grading of skin reactions
On D22 : patches were removed in the animals from 2 groups.
On D23 : 24 hours after patch removal and therefore 48 hours from the start of the challenge application, the skin reactions were observed and graded, for each animal, according to the Magnusson and Kligman grading scale shown below :
— no visible change....................................................................................................................0
— discrete or patchy erythema....................................................................................................1
— moderate and confluent erythema............................................................................................2
— intense erythema and swelling.................................................................................................3
On D24 and D25 : 24 and 48 hours later, a second and a third observations (i.e. 48 and 72 hours after patch removal and therefore 72 and 96 hours after application) were made.
All animals were weighed after the third reading.

All skin reactions and any unusual findings (oedemae...), including systemic reactions, resulting from induction and challenge procedures were observed and recorded in the work document reserved for that effect.
Challenge controls:
Challenge exposure
Following a rest period of 5 days, the guinea-pigs from the 2 groups were closely-clipped (D17) and depilated (D18) on one side at dorsal lumbar level, in an area which has never been in contact with the test item before.
On D21 : topical application
0.20 ml of the test item at its MNIC and 0.20 ml of the test item at its MNIC/2 were respectively applied to 2 patches each one composed of a square of 4 cm² filter paper, put on a 4 cm² square of gauze, itself put on a Blenderm occlusive film of appropriate size. The patches were held on the depilated flank of the animal by a Micropore hypoallergenic adhesive film.
The occlusive dressings were let in contact for 24 hours ± 1 hour.

On D22 : patches were removed in the animals from 2 groups.
On D23 : 24 hours after patch removal and therefore 48 hours from the start of the challenge application, the skin reactions were observed and graded, for each animal.
Positive control substance(s):
yes
Remarks:
2-mercaptobenzothiazole (CAS# 149-30-4)

Results and discussion

Positive control results:
The percentage of reactive animals during the challenge exposure was equal to 40 %.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
50%
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
50%
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
25%
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
25%
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
50%
No. with + reactions:
0
Total no. in group:
11
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 11.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
50%
No. with + reactions:
0
Total no. in group:
11
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 11.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
25%
No. with + reactions:
0
Total no. in group:
11
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 11.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25%
No. with + reactions:
0
Total no. in group:
11
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 11.0.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
Taking into account the criteria defined by the Directive 67/548/EEC and its successive amendments, the test item LCE04026 applied at the challenge dose of 50 % with distilled water was not classified among the chemicals which may cause a sensitisation by skin contact.
Executive summary:

The aim o the study was to assess, in 15 guinea pigs, the skin sensitisation potential of the test item. The effect was potentiated by the injection of Freund’s Complete Adjuvant. The 10 treated animals were exposed to the test item by intradermal injection at the concentration of 1.25 % with distilled water and epidermal application at the concentration of 50 % with distilled water. Concurrently, the 5 control animals received the vehicle only by intradermal and epidermal application (distilled water). Following a rest period of 10 to 14 days ending the induction period, all the animals were exposed to the non irritant challenge dose of 50 % with distilled water. The extent and degree of skin reaction to the challenge exposure in the test animals were compared with those demonstrated by control animals.

The test item was classified in accordance with the percentage of reactive animals according to the criteria defined in the decree of 20/04/1994 published in the Official Journal of the French Republic of May 08,1994 taken in enforcement from the basic Directive 67/548/EEC published in th the Official Journal of the European Communities of June 27, 1967 and its successive amendments.

Experimental dates : from 04/05/13 to 04/06/18

Results : Percentage of reactive treated animals : 0 % Percentage of reactive control animals : 0 %

Conclusion : The test item was not classified among the chemicals which may cause sensitisation by skin contact.