Registration Dossier

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: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no deviations from standard test guidelines and no methodological deficiences, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
Freund's complete adjuvant test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Description: colourless liquid
Container: one smoked glass flask
Storage conditions: at room tempertaure and protected from light
Purity: 99.5%

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male/female
Details on test animals and environmental conditions:
Number: 30 animals (15 males and 15 nulliparous and non-pregnant females)
Allocation of the animals to the groups: on day-1, the animals were weighted and randomly allocated to two groups. A control group 1 consisting of ten animals (five males and 5 females) and a treated group 2 consisting of 20 animals (ten males and ten females).
Weight: on day-1, the animals were approximatively three months old and had a mean body weight ± standard deviation of 306±19 g for the males and 309±22 g for the females.
Acclimatization: at least five days before the beginning of the study.
Identification of the animals: ear-tattoo.

During the acclimatization period and troughout the study, the conditions in the animal room were set as follows:
- temperature: 21±2°C
- relative humidity: 30 to 70%
- light/dark cycle: 12h/12h
- ventilation: about 12 cycles/hour of filtered, non-recycled air.
The temperature and relative humidity were recorded continuously and records retained.
The housing conditions were checked regularly.
During the acclimatization period and throughout the study, the animals were housed individually in polycarbonate cages (48x27x20 cm) equipped with a polypropylene bottle.
Dust-free sawdust was provided as litter.
Bacteriological analysis of the sawdust and detection of possible contaminants were performed periodically.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
paraffin oil
Concentration / amount:
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 were clipped,
- the test substance was prepared in an appropriate vehicule,
- intradermal administartions of the test substance (0.1 mL) at different concentrations were performed in the dorsal region between the shoulders,
- cutaneaous reactions were evaluated approximately 24, 48 hours and six days after injection.

By cutaneous route:
- 24 hours before treatment, both flank regions of the animals were clipped,
- if necessary, the test substance was prepared in an appropriate vehicule,
- the test substance (0.5 mL for each concentration) was applied to a dry gauze pad of approximately 4 cm2 which was held in place by an occlusive dressing for 24 hours,
- cutaneaous reactions were evaluated approximately 24, 48 hours after removal of the dressing.

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 effect (no necrosis or ulceration of the skin),
- topical application for the induction should cause at most weak or moderate skin reactions or be the maximal practicable concentration,
- topical application for the challenge should be the highest concentration which does not cause irritant effect.
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
paraffin oil
Concentration / amount:
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 were clipped,
- the test substance was prepared in an appropriate vehicule,
- intradermal administartions of the test substance (0.1 mL) at different concentrations were performed in the dorsal region between the shoulders,
- cutaneaous reactions were evaluated approximately 24, 48 hours and six days after injection.

By cutaneous route:
- 24 hours before treatment, both flank regions of the animals were clipped,
- if necessary, the test substance was prepared in an appropriate vehicule,
- the test substance (0.5 mL for each concentration) was applied to a dry gauze pad of approximately 4 cm2 which was held in place by an occlusive dressing for 24 hours,
- cutaneaous reactions were evaluated approximately 24, 48 hours after removal of the dressing.

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 effect (no necrosis or ulceration of the skin),
- topical application for the induction should cause at most weak or moderate skin reactions or be the maximal practicable concentration,
- topical application for the challenge should be the highest concentration which does not cause irritant effect.
No. of animals per dose:
A control group 1 consisting of ten animals (five males and 5 females) and a treated group 2 consisting of 20 animals (ten males and ten females).
Details on study design:
Preparation of the animals
For all animals and before each treatment, the applications sites were:
- clipped on days 1 and 7 (scapular area 4x2 cm),
- clipped and shaved on day 21 (each flank 2x2 cm),
- clipped again on day 25 (each flank 2x2 cm).

Intradermal route
On day 1, six injections wera made deep into the dermis of a clipped area (4x2 cm) in the dorsal region between the shoulders, using a needle mounted on a 1 mL glass syringe.

Three injections of 0.1 mL were made into each side of this shoulder region, as follows:

Injection sites* Treated group Control group
Anterior 1: FCA diluted at 50% (v/v) 1: FCA diluted at 50% (v/v)
with 0.9% NaCl with 0.9% NaCl

Middle 2: test substance at 0.5% (w/w) 2: vehicule
in paraffin oil

Posterior mixture of 50/50 (w/v) mixture of 505/50 (w/v)
of 1 and 2 of 1 and 2

*: three pairs of sites
FCA: Freund's complete adjuvant


Cutaneous route
As the test substance was shown to be irritant during the preliminary tests, a topical application with sodium laurylsulphate was not necessary on day 7.
On day 8, a topical application to the region of the intradermal injections (4x2 cm) was performed.

Control group : application of 0.5 mL of the vehicle.
Treated group: application of 0.5 mL of teh test substance at the chosen concentrations.

The test substance and the vehicle were prepared on a dry gauze pad which was then applied to the dorsal region between the shoulders and held in place for 48 hours by means of an adhesive hypoallergenic dressing and an adhesive anallergenic waterproof plaster.
On removal of the dressing, if present, any residual test substance was removed by means of a dry or a moistened gauze pad.
Cutaneous reactions were recorded one hour after removal of the occlusive dressing.

Challenge phase
On day 22, the animals from both groups received an application of 0.5 mL of the test substance at the chosen concentration to the posterior right flank, and 0.5 mL of the vehicle to the posterior left flank.
This application was performed using a 1 mL plastic syringe.
The test substance and the vehicle were prepared on a dry gauze pad, then applied to a 4 cm2 (2x2 cm) clipped area of the skin. The gauze pad was held in contact with the skin for 24 hous by means of an occlusive, hypoallergenic dressing and an adhesive anallergenic waterproof plaster.
On removal of the dressing, if present, any residual test substance was removed by means of a dry or a mointened gauze pad.
Positive control substance(s):
not required

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
0.5 mL at a concentration of 1% (w/w)
No. with + reactions:
2
Total no. in group:
20
Clinical observations:
Very slight erythema (barely perceptible)
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 0.5 mL at a concentration of 1% (w/w). No with. + reactions: 2.0. Total no. in groups: 20.0. Clinical observations: Very slight erythema (barely perceptible).
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
0.5 mL at a concentration of 1% (w/w)
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
Dryness of the skin for 7 animals (5 males and 2 females)
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 0.5 mL at a concentration of 1% (w/w). No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: Dryness of the skin for 7 animals (5 males and 2 females).

Any other information on results incl. tables

No clinical signs and no mortalities were observed during the study.

The body weight gain of the tretad animals was normal when compared to that of the control animals.

Challenge application : no residual test substance was observed after removal of the dressing.

Comparable very weak skin reactions were observed in both groups, they were attributed to a slight irritant effect of the test substance.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under experimental conditions and according to the maximization method of Magnusson and Kligman, no cutaneous reactions attributable to the sensitization potential of allyl bromide at a concentration of 1% (w/w) were observed un guinea-pigs.
Therefore, allyl bromide should not be considered as sensitizing.