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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:
22 July 1993 to 22 December 1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
followed the maximisation method of Magnusson and Kligman (1969). However, 1% was chosen for intradermal induction in the main test while it was a necrotizing dose, different vehicle was used for intradermal injection of control group, and no positive control or reliability check data included in the study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1993

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
1% was chosen for intradermal induction in the main test while it produced necrosis 48h post dosing. Different vehicle was used for intradermal injection of control group. No positive control or reliability check data included in the study
Principles of method if other than guideline:
The test was performed according to the maximisation method of Magnusson and Kligman: Magnusson, B. ; Kligman, A.M. : The identification of contact allergens by animal assay. The guinea pig maximization test. J. Invest. Derm. 52: 268-276 (1969).
GLP compliance:
no
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Study performed before establishment of current requirements

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid powder
Details on test material:
Other: dark brown powder
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Centre d'élevage Lebeau, 78950 Gambais, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: no data
- Age at study initiation: no data
- Weight at study initiation: 403 ± 27 g for males and 387 ± 24 g for females
- Housing: Animals were housed individually in sterilizable polycarbonate cages (48 x 27 x 20 cm) equiped with a water bottle made of polypropylene. Animals are placed on litter made of sawdust sieved and dedusted (SISCA, 94142 Alforville, France). The litter is periodically checked for the presence of possible residues or contaminants (Laboratoire Wolff, 92110 Clichy, France).
- Diet (e.g. ad libitum): free access to a standard guinea pig diet "Cobayes entretien référence 106" (U.A.R., 91360 Villemoisson-sur Orge, France). Composition of the diet is detailed in the study report.
- Water (e.g. ad libitum): 0.22 µm filtered drinking water (Millipore, 78140 Vélizy, France) was supplied ad libitum via water bottles. Bacteriological and chemical analysis are periodically performed on the drinking water. The presence of main contaminats is also checked (pesticides, heavy metals, nitrosamines) (Laboratoire Municipal et Régional de Rouen, 76000 Rouen, France - Centre de Nutrition Humaine, 54000 Nancy, France - Laboratoire Départmental d'Analyses, 27000 Evreux, France).
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): no data (air is filtered by absolute type filters)
- Photoperiod (hrs dark / hrs light): 12/12

- IN-LIFE DATES: From: 22 July 1993 To: 23 August 1993

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
other: a mix of dimethylacetamide/acetone/ethanol (40/30/30)
Remarks:
the choice of the vehicle was not justified in the study report and the control group received paraffin oil
Concentration / amount:
1%
Adequacy of induction:
other: necrotizing dose
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100%
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100 %
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Control group: 10 animals (5 males and 5 females)
Test group: 20 animals (10 males and 10 females)
Details on study design:
PREPARATION OF ANIMALS FOR BOTH THE RANGE FINDING AND THE MAIN TEST:
The application sites of all animals were:
- clipped before intradermal injections on Day -1 (scapular region 4 cm x 2 cm),
- clipped before topical applications of the induction phase on Day 7 (same region),
- clipped and shaved before topical applications of the challenge phase on Day 21 (each flank 2 cm x 2 cm)


RANGE FINDING TEST: a preliminary test was peformed in order to determine the concentrations to be used in the main test.

A. INTRADERMAL INJECTION
- Test group: no data on number of animals used
- Concentrations: the maximum concentration tested was 1% in a mix of dimethylacetamide/acetone/ethanol (40/30/30)
- Observations: approximately 24 and 48 hours after dosing

B. TOPICAL APPLICATION
- Test group: no data on number of animals used
- After removal of the dressing, the treated sites were gently cleaned by washing with water for injections.
- Concentrations: the maximum concentration of the test item in vehicle tested was 10% in a mix of dimethylacetamide/acetone/ethanol (40/30/30) (several concentrations tested as reported in the study report but those concentrations were not reported). Test item was also tested at 100% (undiluted).
- Observations: approximately 24 hours after removal of the dressing.


MAIN STUDY:

A. INDUCTION EXPOSURE
- Test group: 10 males and 10 females
- Control group: 5 males and 5 females

A1. INTRADERMAL INDUCTION (Day 1)
- Dosing: at Day 1, 3 pairs of intradermal injections were made at the prepared skin site of each animal. All injections were made at the edge of the prepared site and the anterior and median injections were positioned close together and distant from the posterior injections.
- Treatment of the Test group:
* Anterior site: 0.1mL of Freund's adjuvant at 50% (v/v) in 0.9% NaCl
* Median site: 0.1mL of 1% test item in the vehicle (dimethylacetamide/acetone/ethanol (40/30/30))
* Posterior site: 0.1mL of 1% test item suspended in dimethylacetamide/acetone/ethanol (40/30/30) in Freund's adjuvant at 50% (v/v) in 0.9% NaCl
- Treatment of the Control group:
* Anterior site: 0.1mL of Freund's adjuvant at 50% (v/v) in 0.9% NaCl
* Median site: 0.1mL of paraffin oil
* Posterior site: 0.1mL of paraffin oil mixed at 50/50 (v/v) with Freund's adjuvant at 50% (v/v) in 0.9% NaCl

A2. TOPICAL INDUCTION (Day 8)
- Preparation of the site: on Day 7, 0.5 mL of Sodium Lauryl Sulphate at 10% in petrolatum were smeared over the skin of the scapular region in order to create a local irritation.
- Dosing: on Day 8, a topical application in the scapular region of 0.5 mL of paraffin oil was performed for animals of the control group and 0.5 g of the undiluted test material for animals of the treated group, and maintained for 48h under occlusive dressing.
After 48 hours contact, the dressings were removed and the treated sites gently cleaned by a compress soaked in water for injections.
- Observations: approximately 1 hour after removal of the dressings, any cutaneous reactions were recorded.

B. CHALLENGE EXPOSURE (Day 22)
- Dosing: on Day 22, animals of the treated group received a topical application of 0.5 g of the undiluted test material over a 4cm2 posterior region of the the right flank and 0.5 mL of the vehicle in the posterior region of the the left flank, while animals of the control group received a topical application of 0.5 g of the undiluted test material over the anterior region of the the right flank. The test material was prepared in an humidified compress (water for injections) while the vehicle was prepared in a dry compress that were applied on the skin. Then, they were covered with a strip of synthetic anallergic film and the trunk wrapped with an elastic hypoallergenic adhesive bandage to maintain the test item and vehicle in contcat with the skin (occlusive barrier) for 24 hours. The adhesive dressings and gauze patches were removed after 24 hours contact and the treated sites gently cleaned by a compress soaked in water for injections.
- Observations: Observations of the treated sites was carried out approximately 24 hours and 48 hours after the removal of the dressing (48 and 72 hours after the topical challenge respectively).

SCORING OF THE CUTANEOUS REACTIONS
24 and 48 h 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:

Erythema and Eschar Formation
No erythema ...................................................................................................................................0
Very slight erythema (barely perceptible) ......................................................................................1
Well defined erythema ....................................................................................................................2
Moderate to severe erythema ........................................................................................................3
Severe erythema (beet redness) with or without eschar formation (deep lesions)......................4

Oedema Formation
No oedema .........................................................................................................................0
Very slight oedema (barely perceptible)..............................................................................1
Slight oedema (edges of area well defined by definite raising) ........................................2
Moderate oedema (raised approximately 1 mm) ................................................................3
Severe oedema (raised more than 1 mm and extending beyond area of exposure) .........4

Any other skin reactions were recorded.

OTHER 1: IN VIVO CLINICAL OBSERVATIONS
- Animals were daily observed for clinical signs and mortality.
- Body weight: animals of the main study were weighed at the start of treatment (Day1), on Day 8 and 15 and on completion of the challenge (Day 25).

OTHER 2: TERMINAL STUDIES
- Termination and euthanasia method: all the surviving animals were killed by carbon dioxide narcosis following the end of the experimental procedure.
- Necropsy: Animals that died or were sacrified during the study were necopsied and subject to macroscopic examination.
- After ethanasia, skin removal at the site of application was performed at each right and left posterior flank of all animals of the treated group (as skin reactions were recorded). Skin samples were preserved in 10% buffered formol.
- Miscroscopic examination: no histologic examination was performed

OTHER 3 : RELIABILITY CHECK
No concomittent positive control was performed and no historical positive control data were presented in the study report.
Challenge controls:
no
Positive control substance(s):
no

Results and discussion

Positive control results:
Not applicable.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
0% Test item
No. with + reactions:
0
Total no. in group:
9
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
0% Test item
No. with + reactions:
0
Total no. in group:
9
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
1% intradermal injection and 100% topical induction and challenge
No. with + reactions:
0
Total no. in group:
18
Clinical observations:
a discrete erythema (grade 1) was noted in 10/18 animals
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
test group
Dose level:
1% intradermal injection and 100% topical induction and challenge
No. with + reactions:
0
Total no. in group:
18
Clinical observations:
the discrete erythema (grade 1) noted in 10/18 animals at 48h reversed in 7/10 of the concerned animals
Reading:
1st reading
Hours after challenge:
48
Group:
positive control
Remarks on result:
not measured/tested
Reading:
2nd reading
Hours after challenge:
72
Group:
positive control
Remarks on result:
not measured/tested

Any other information on results incl. tables

RANGE FINDING STUDY

- Administration by intradermal route: the maximal tested concentration was 1% in dimethylacetamide/acetone/ethanol (40/30/30). This concentration caused irritation 24 h post dosing and then necrosis 48 h post dosing. This concentration was chosen for the main test.

- Administration by topical route: 24 h after the removal of dressing, both concentrations (10% in dimethylacetamide/acetone/ethanol (40/30/30) and undiluted) caused no skin irritation, with a brown coloration of the skin by the test material. Based on these results, the concentration chosen for both the induction and challenge phases was 100% (undiluted test material).

MAIN STUDY

- Clinical examinations: One female of the treated group had a slight decreased spontaneous activity and a decreased body weight during the second week of observation. One male of the control group had a slight decreased spontaneous activity, tremors and piloerection at Day 22 that preceded the death of the animal by Day 23. One male of the treated group was sacrified at Day 8 because of severe pre-death clinical signs (hypokinesia, thinness, cold to the touch and dehydration). One female of the treated group was found dead on Day 22 without apparent clinical sign. As such spontaneous mortality is sometimes observed in this species, it was not attributed to treatment with the test material.

- Body weight: body weight gain of 95% of the treated animals was similar to that of controls (detailed results are presented in the study report). An important weight loss was observed at Day 8 for the male that was sacrified.

- Induction phase reactions: signs of skin irritation were recorded on Day 10 after patch removal at the intradermal injection sites of animals of the control group. Evaluation of the skin reactions of animals of the treated group was not possible due to the black coloration of the skin by the test material.

- Challenge phase reactions: No cutaneous reactions were observed for the animals of the control group 48 and 72 h after dosing. In the treated group, a discrete erythema (grade 1) was noted in 10/18 animals the 48 h post challenge. 72 h post dosing, the discrete erythema (grade 1) only persisted in 3 animals. No edema or other cutaneous reactions were recorded.

- Macroscopic findings: no findings after macroscopic examination of the main organs of animals found dead or sacrified during the study.

Applicant's summary and conclusion

Interpretation of results:
other: not sensitising under the conditions of this test
Conclusions:
Under the experimental conditions of this study, the test material did not elicit a skin sensitisation response in the guinea pig.
Executive summary:

This non-GLP study was performed to assess the potential of the test material to induce and elicit delayed dermal sensitization according to the guinea pig maximization test of Magnusson and Kligman described in Magnusson B and Kligman AM., 1969.

Material and methods

Concentrations of the test material used in the main study were determined by performing a range finding study. The selected concentrations were: 1% in dimethylacetamide/acetone/ethanol (40/30/30) for the intradermal injection and undiluted test material for the induction and the challenge topical applications (necrotizing dose and highest non irritant dose respectively).

The main study was undertaken using 30 male and female guinea pigs (20 in the test group and 10 in the control group). Test animals were intradermally injected with FCA at 50% (v/v) in 0.9% NaCl (anterior sites), the test material at 1% in

dimethylacetamide/acetone/ethanol (40/30/30) (middle sites), and the test material suspended in the vehicle at 1% in FCA at 50% (v/v) in 0.9% NaCl (posterior sites). One week later, animals were treated by topical application of Sodium Lauryl Sulfate at 10% (w/w) in petrolatum in order to induce a local irritation, and then boosted by topical application of the undiluted test material under an occlusive dressing for 48h. Control animals were treated in the same manner but using the vehicle praffin oil instead of the test material. Two weeks after the second induction stage, animals of the treated group were challenged by topical application-under occlusive dressing for 24h- of both the vehicle and the undiluted test material, while animals of the control group were challenged by

topical application-under occlusive dressing for 24h- of the undiluted test material only. Skin reactions were evaluated 24 and 48 h after removal of the dressing.

Results

No systemic clinical signs and no deaths related to treatment were noted during the study. The body weight gain of 95% of the animals of the treated group was comprabale to that of the control group.

No well-defined macroscopic cutaneous reactions were observed after the challenge application. The discrete erythema observed in 10/18 animals of the treated group 48 h after dosing (reversing at 72h post dosing in 7of the 10 reacting animals) was considered as a slight primary skin irritation reaction.

Conclusion

Under the experimental conditions of this study, the test material did not elicit a skin sensitisation response in the guinea pig.