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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Principles of method if other than guideline:
The potential of the test item SR94653 to induce delayed contact hypersensitivity was evaluated in guinea pigs according to the maximization method of Magnusson and Kligman and to OECD (No. 406, 17th July 1992) and EC (96/54/EEC, B.6, 30 July 1996) guidelines. The study was conducted in compliance with the principles of Good Laboratory Practice Regulations.
GLP compliance:
yes
Type of study:
guinea pig maximisation test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): (R)-2-chloromandelic acid
- Physical state: white powder
- Analytical purity: 99.7 %
- Lot/batch No.: 1500175 and CR-UY-002
- Stability under test conditions: guaranteed by the sponsor
- Storage condition of test material: at room temperature
- Other: The correspondence between both batch numbers (1500175 at receipt and CR-UY-002 on the analytical certificate) was confirmed by the Study Monitor in a statement dated 11 June 2001

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Saint-Aubin-les-Elbeuf, France.
- Age at study initiation: 1-3 months
- Weight at study initiation: mean body weight ± standard deviation of 387± 25 g for the males and 356 ± 13 g for the females
- Housing: individually
- Diet:106 pelleted diet (UAR, Villemoisson, Epinay-sur-Orge, France).
- Water: Drinking water filtered by a FG Millipore membrane (0.22 micron) was provided ad libitum
- Acclimation period: at least 5 days before the beginning of the study


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 30-70
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

The temperature and relative humidity were under continuous control and recording. The records were checked daily and filed. In addition to these daily checks, the housing conditions and corresponding instrumentation and equipment are verified and calibrated at regular intervals. During the acclimation period and throughout the study, the animals were housed individually in polycarbonate cages with stainless steel lid (48 cm x 27 cm x 20 cm) equipped with a polypropylene bottle. Each cage contained autoclaved sawdust (SICSA, Alfortville, France). Sawdust is analysed by the supplier for composition and contaminant levels.

Food is analysed regularly by the supplier for composition and contaminant levels. Bacteriological and chemical analyses of water are performed regularly by external laboratories. These analyses include the detection of possible contaminants (pesticides, heavy metals and nitrosamines).
No contaminants were known to have been present in the diet, drinking water or bedding material at levels which may be expected to have interfered with or prejudiced the outcome of the study.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
other: see below
Concentration / amount:
induction phase intradermal: 1%
induction phase epicutaneous: 50%
challenge phase: 50 %
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: see below
Concentration / amount:
induction phase intradermal: 1%
induction phase epicutaneous: 50%
challenge phase: 50 %
No. of animals per dose:
30
Details on study design:
RANGE FINDING TESTS:
Preliminary test
A preliminary test was conducted in order to determine the concentrations to be tested in the
main study.
By intradermal route (tested concentrations: 10%, 5% and 1% (w/w)):
- 24 hours before treatment, the dorsal region of the animals was clipped,
intradermal injections of the dosage form preparations (0.1 mL) were performed in die
interscapular region,
- cutaneous reactions were evaluated approximately 24, 48 hours and 6 days after the
injections.

By cutaneous route
- Under the conditions of the induction phase (tested concentration: 50% (w/w)):
- 24 hours before treatment, the interscapular region of the animals was clipped,
- a filter paper (approximately 8 cm ) was fully-loaded with a dosage form preparation and was then applied to the clipped area of the skin. The filter paper was held in place by means of an occlusive dressing for 48 hours,
- cutaneous reactions were evaluated 24 and 48 hours after removal of the dressing.

Under the conditions of the challenge phase (tested concentration: 50% (w/w)):
- 24 hours before treatment, both flanks of the animals were clipped and shaved,
- the filter paper of a chamber (Finn Chamber®) was fully-loaded with a dosage form preparation. The chamber was then applied to the clipped area of the skin (one concentration per flank). The chamber was held in place by means of an occlusive dressing for 24 hours,
- cutaneous reactions were evaluated 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
Main study
Preparation of the animals
For all animals, the application sites were:
- clipped on days -1 and 7 (interscapular region 4 cm x 2 cm),
- clipped and shaved on day 21 (each flank 2 cm x 2 cm),
- shaved on day 24 (each flank 2 cm x 2 cm),
- clipped on day 25, before skin sampling (each flank 2 cm x 2 cm).

Induction phase by intradermal and cutaneous routes
Intradermal route
On day 1, six injections were made deep into the dermis of a 4 cm x 2 cm clipped interscapular area, using a needle (diameter: 0.50 x 16 mm) mounted on a 1 mL plastic syringe (mL graduations).

Cutaneous route
As the test item was shown to be non-irritant during the preliminary test, all the animals except 2/20 animals of the treated group (male Nos. 10 and 15), which already have showed marked irritation, were treated on day 7 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 pad of filter paper (approximately 8 cm2) was fully-loaded with the test item at the concentration of 50% (w/w) and was then applied to the interscapular region of the animals of die treated group. The animals of the control group received an application of the vehicle alone under the same experimental conditions. The pad was held in place for 48 hours by means of an adhesive hypoallergenic dressing and an adhesive antiallergenic waterproof plaster.

Challenge phase
On day 22, the animals of treated and control groups received an application of the test item and vehicle. The filter paper of a chamber (Finn Chamber®) was fully-loaded with the test item at the concentration of 50% (w/w) and was then applied to a clipped area of the skin of the posterior right flank of all animals. The vehicle was applied under the same experimental conditions to the skin of the posterior left flank. The chambers were held in contact with the skin for 24 hours by means of an adhesive antiallergenic waterproof plaster.

SCORING OF CUTANEOUS REACTIONS
Twenty-four and 48 hours after removal of the dressing of the challenge application, both flanks
of the treated and control animals were observed in order to evaluate cutaneous reactions,
according to the following scale:
0 no visible change
1 discrete or patchy erythema
2 moderate and confluent erythema
3 intense erythema

Any observed oedema was recorded. Any other lesions were noted.

CLINCAL 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) and on the last day of the study (day 25).

PATHOLOGY
Necropsy
At the end of the study, all the animals were killed by carbon dioxide asphyxiation. No necropsy was performed.

Skin samples
At the end of the study, skin samples were taken from the posterior left and right flanks of all the animals. The samples were preserved in 10% buffered formalin.

Microscopic examination
No histological examination was performed.

DETERMINATION OF THE ALLERGENICITY LEVEL
The animals of the treated group show a positive reaction if macroscopic cutaneous reactions are clearly visible (score ≥ 1) and are of greater intensity and/or duration of response than the maximum reaction seen in control animals, or if macroscopic reactions are confirmed at microscopic examination as being due to the sensitization process.

Determination of the allergenic level
The allergenic level of the test item is calculated by comparing the number of animals showing positive reactions with the number of surviving treated animals at the end of the study.

% of animals showing Allergenicity level Classification
a reaction
0-8 I weak
9-28 II mild
29 - 64 III moderate
65 - 80 IV strong
81 - 100 V extreme

According to the Commission Directive 93/21/EEC, when the reactions are positive in at least 30% of the treated animals, the test item has sensitization properties and the symbol Xi, the indication of danger “Irritant" and the sentence “R 43: May cause sensitization by skin contact" must be applied.

The sensitivity of the experimental technique is regularly assessed using a known moderate sensitizer, MERCAPTOBENZOTHIAZOLE. In a recent study performed under CIT experimental conditions, the strain of guinea pigs used showed a satisfactory sensitization response in 100% animals.


OTHER:
In order to respect the criteria for the selection of concentrations (the concentration should be well-tolerated systemically and locally, intradermal injections should cause moderate irritant effect but no necrosis or ulceration of the skin), concentration chosen for the main study was 1%(w/w).

In order to respect the criteria for the selection of concentrations (the concentrations should be well-tolerated systemically and locally, 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 effect), concentration chosen for the topical application of the induction phase (day 8) and for the challenge application (day 22) was 50% (w/w).
Positive control substance(s):
yes
Remarks:
Mercaptobenzothiazole (historical control)

Results and discussion

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:
10
Clinical observations:
nothing abnormal detected
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: 10.0. Clinical observations: nothing abnormal detected.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
50 %
No. with + reactions:
3
Total no. in group:
10
Clinical observations:
dryness of skin
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 50 %. No with. + reactions: 3.0. Total no. in groups: 10.0. Clinical observations: dryness of skin.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
50 %
No. with + reactions:
6
Total no. in group:
20
Clinical observations:
discrete erythema
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50 %. No with. + reactions: 6.0. Total no. in groups: 20.0. Clinical observations: discrete erythema.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
50 %
No. with + reactions:
4
Total no. in group:
20
Clinical observations:
moderate erythema
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50 %. No with. + reactions: 4.0. Total no. in groups: 20.0. Clinical observations: moderate erythema.

Any other information on results incl. tables

Results

Clinical examinations

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

 

Bodyweight

The overall body weight gain of the treated animals was similar to that of controls.

 

Challenge phase - Scoring of cutaneous reactions

On removal of the dressing, any residual test item was removed by means of a moistened cotton pad.

 

No cutaneous reactions were observed in the animals of the control group at the 24-hour reading; a dryness of the skin was noted in 3/10 animals at the 48-hour reading.

 

In the treated group, at the 24-hour reading, a discrete or moderate erythema (grade 1 or 2) in addition to a dryness of the skin, was observed in 3/20 and 1/20 animals, respectively.

 

At the 48-hour reading, a discrete or moderate erythema (grade 1 or 2) was recorded in 6/20 and 4/20 animals, respectively. A dryness of the skin was also recorded in 11/20 animals. The cutaneous reactions observed in the animals of the treated group, which were of higher severity than those recorded in the animals of the control group, were attributed to delayed contact hypersensitivity.

 

 

Conclusion

Under our experimental conditions and according to the maximization method of Magnusson and Kligman, the test item SR94653 induces delayed contact hypersensitivity in 10/20(50 %) guinea pigs.

 

According to the classification criteria laid down in Commission Directive 93/21/EEC, the test item should be classified as sensitizing to the skin

Applicant's summary and conclusion

Conclusions:
Classification: sensitizing