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: Study conducted in compliance with GLP and appropriate OECD Guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
denomination:
- protocol: Iso-PROPYL BROMIDE
- labelling: Iso-PROPYL BROMIDE
batch number:
- protocol: 0-302-1
- labelling: 0-302-1 CAL 4319/94
description: colourless liquid
· container: 1 glass flask
· date of receipt: 4.10.94
storage conditions: at room temperature, protected from light
· purity: 99.9%

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
Animals

Species and strain: Dunkin-Hartley guinea-pigs.
Reason for this choice: species recommended by the international regulations for sensitization studies. The strain used has been shown to produce a satisfactory sensitization response using known positive sensitizers.
Breeder: Centre d'Elevage Lebeau, 78950 Gambais, France.
Number: 15 nulliparous and non-pregnant females.
Allocation of the animals to the groups: on day -1, the animals were weighed and randomly allocated to 2 groups: a control group 1 consisting of 5 females and a treated group 2 consisting of 10 females.
Weight: on day 1, the animals had a mean body weight of 351 ± 13 g.
Acclimatization: at least 5 days before the beginning of the study.
Identification of the animals: the animals were identified individually by an ear-tattoo.

Environmental Conditions

During the acclimatization period and throughout the study, the conditions in the animal room
were as follows:
temperature: 21 ± 2°C
relative humidity: 30 to 70%
light/dark cycle: 12 h / 12 h
ventilation: about 12 cycles/hour of filtered, non-recycled air.
During the acclimatization period and throughout the study, the animals were housed individually in polycarbonate cages (48 x 27 x 20 cm) equipped with a polypropylene bottle. Calibrated and dust-free sawdust was provided as litter (SICS A, 92142 Alfortville, France). An
analysis of potential residues and major contaminants is performed periodically (LaboratoireWolff, 92110 Clichy, France).

Food and Water

During the study, the animals had free access to 106 diet" (U.A.R., 91360 Villemoisson-sur-Orge,France).
Food was periodically analysed (composition and contaminants) by the supplier.
Animals had free access to drinking water, filtered by a F.G. Millipore membrane (0.22 micron).

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
paraffin oil
Concentration / amount:
25% w/w in vehicle for induction; 100% for challenge
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
paraffin oil
Concentration / amount:
25% w/w in vehicle for induction; 100% for challenge
No. of animals per dose:
10 females for treated group; 5 females for control group
Details on study design:
Preliminary Test

A preliminary test was performed to define the concentration to be tested in the main study.

By intradermal route

Determination of the Minimum Irritant Concentration (M.I.C.):
24 hours before treatment, the dorsal region of the animals was clipped, the test substance was prepared in an appropriate vehicle, intradermal administration of the test substance (volume 0.1 ml) at increasing concentrations was performed in order to determine the minimum concentration which does causes an irritation, evaluation of the potential cutaneous reactions, 24 and 48 hours after injection.

By cutaneous route

Determination of the Minimum Irritant Concentration (M.I.C.) and Maximum Non-Irritant Concentration (M.N.I.C.):
24 hours before treatment, the dorsal region of the animals was clipped, 0.5 ml of the test substance undiluted was applied to a gauze pad of approximately 4 cm2 and then held in place by an occlusive dressing for 24 hours, potential cutaneous reactions were evaluated 24 and 48 hours after removal of the gauze pads.

Main Study

Preparation of the animals

For all animals and before each treatment, the application sites were:
clipped on days -1 and 7 (scapular area 4 cm x 2 cm), clipped again on days 21 and 25 (each flank 2 cm x 2 cm) and shaved on day 21 (each flank
2 cmx 2 cm).

Induction phase by intradermal and cutaneous routes

Intradermal route

On day 1, 6 intradermal injections were made into a clipped area (4 em x 2 em) in the scapular region, using a needle (diameter: 0.50 x 16 mm, Terumo: e.M.L., 77140 Nemours, France) mounted on a 1 ml glass syringe (0.01 ml graduations, Record: Carrieri, 75005 Paris, France).

Three injections of 0.1 rnl were injected into each side of the animal, as follows:

Control group
Freund's complete adjuvant diluted to 50% (v/v) with a sterile isotonic saline solution (0.9% NaCl), vehicle,a mixture of 50/50 (w/v) Freund's complete adjuvant diluted to 50% (v/v) with a sterile isotonic aqueous NaCl solution and the vehicle.

Treated group
Freund's complete adjuvant diluted to 50% (v/v) with a sterile isotonic saline solution (0.9% NaCl), test substance at a concentration of 25% (w/w) in the vehicle, a mixture 50/50 (w/v) of Freund's complete adjuvant diluted to 50% (v/v) with a sterile isotonic saline solution (0.9% NaCl), and, the test substance at a concentration of 25% (w/w) in the vehicle.

Cutaneous route

On day 7, the scapular area was clipped. As the test substance is shown to be non-irritant after occlusive cutaneous treatment during preliminary test, the animals were treated with 0.5 ml of sodium laurylsulphate (10%) in vaseline to provoke local irritation. On day 8, a cutaneous application on the 6 injection areas (4 em x 2 em) of the scapular region was performed.

Control group -- application of 0.5 ml of the vehicle.
Treated group -- application of 0.5 ml of a non-irritant concentration of the test substance i.e. in its original form.
The test substance and the vehicle were prepared on a dry gauze pad (Semes France, 54183 Heillecourt, France), which was then applied to the scapular region and held in place for 48 hours by means of an adhesive hypoallergenic dressing (Laboratoires de Pansements et d'Hygiene, 21300 Chenove, France) and an adhesive anallergenic waterproof plaster (Laboratoire des Professions Medicales, 92240 Malakoff, France). No residual test substance was observed at removal of the dressing.
One hour after removal of the occlusive dressing, cutaneous reactions were recorded.

Challenge phase

At the end of the rest period on day 22, the test substance was applied at the Maximum NonIrritant Concentration (M.N.I.C.) i.e. in its original form.
On day 22, the animals from both groups received an application of 0.5 ml of the M.N.I.C. of the test substance on the posterior right flank, and 0.5 ml of the vehicle on the posterior left flank. This application was performed using a 1 ml plastic syringe (0.01 ml graduations, Terumo: e.M.L., 77140 Nemours, France). The test substance and vehicle were prepared on a dry gauze pad (Semes France, 54183 Heillecourt, France), then applied to a 4 cm2 (2 cm x 2 cm) clipped area of the skin. The gauze pad was held in contact with the skin for 24 hours by means of an occlusive, hypoallergenic dressing (Laboratoires de Pansements et d'Hygiene, 21300 Chenove, France) and an adhesive anallergenic waterproof plaster (Laboratoire des Professions
Medicales, 92240 Malakoff, France). No residual test substance was observed at removal of the dressing.


SCORING OF CUTANEOUS REACTIONS

Twenty-four and 48 hours after removal of the dressing from the challenge application site, the 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) to slight eschar formation (injuries in depth) 4

Oedema formation
· No oedema 0
· Very slight oedema (barely perceptible) 1
· Slight oedema (visible swelling with well-defined edges) 2
· Moderate oedema (visible swelling raised more than 1 millimetre) 3
· Severe oedema (visible swelling raised more than 1 millimetre and extending beyond the area of exposure) 4

Any other lesions were noted.

CLINICAL EXAMINATIONS

The animals were observed twice a day during the study in order to record clinical signs and to check for 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), then on days 8, 15 and 25.

PATHOLOGY

Necropsy
On day 25, after the 48-hour observation period, the animals were killed by CO2 inhalation in excess.

Cutaneous samples
On day 25, a skin sample was taken from the treatment sites of the posterior left and right flanks of all animals. The samples were preserved in 10% buffered formalin.

Microscopic examination
Tissues taken for microscopic examination were embedded in paraplast, sectioned at approximately 4 microns in thickness and stained with hemalum-eosin. At the request of the study monitor, a histological examination was performed on the right flank of treated female Nos. 112, 113, 114, 117 and 119. No histological examinations were performed in all other animals.

DETERMINATION OF THE ALLERGENICITY LEVEL
The treated animals show a positive reaction if macroscopic cutaneous reactions are clearly visible (erythema and/or oedema >= 2) and more marked than the most severe reactions of the control animals, or, if "doubtful" macroscopic reactions are confirmed at microscopic examination as being due to the sensitization process. Sensitization reactions are characterized at microscopic examination by basal spongiosis, reactional acanthosis of the epidermis and infiltration of mononucleated cells into the dermis.

Determination of the allergenicity level
The allergenicity level of the test substance 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 Allergenicity Classification showing a reaction level
o - 8 I very weak
9 - 28 II weak
29 - 64 III moderate
65 - 80 IV strong
81 - 100 V very strong
According to the E.E.C. directive 931211E.E.C. published in the Journal Officiel des Communautes Europeennes, when the reactions are positive in at least 30% of the treated animals, the test substance has sensitization properties and the sentence "R 43: May cause sensitization by skin contact" must be applied.


Positive control substance(s):
no

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0%
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: 0%. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0
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: 0. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
100%
No. with + reactions:
7
Total no. in group:
10
Clinical observations:
Very slight erythema (grade 1) was observed in 7/10 animals 24 hr after removal of the dressing. hese very slight reactions observed 24 hr after removal of the dressing were due to a slight irritant effect of the undiluted test substance. No oedema.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 100%. No with. + reactions: 7.0. Total no. in groups: 10.0. Clinical observations: Very slight erythema (grade 1) was observed in 7/10 animals 24 hr after removal of the dressing. hese very slight reactions observed 24 hr after removal of the dressing were due to a slight irritant effect of the undiluted test substance. No oedema..
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
100%
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
Very slight erythema (grade 1) was observed in 1/10 animals 48 hr after removal of the dressing. Dryness of the skin was observed in 5.10 animals at the 48 hr reading. No oedema were observed 48 hr after removal of the dressing.
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 100%. No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: Very slight erythema (grade 1) was observed in 1/10 animals 48 hr after removal of the dressing. Dryness of the skin was observed in 5.10 animals at the 48 hr reading. No oedema were observed 48 hr after removal of the dressing..

Any other information on results incl. tables

Skin of the treated right flank (test substance at 100% concentration) was examined in 5/10 females showing cutaneous reactions. Slight to moderate acanthosis was noted in all 5 females, together with minimal hyperkeratosis in 3 females. Minimal mononuclear cell infiltration of the upper dermis was noted in 5/10 examined females, together with minimal granulocyte infiltration in 2 females. No treated skin from the 5/10 examined females showed spongiosis and in absence of the classical association of spongosis, acanthosis and mononuclear infiltration of dermis, the above-mentioned lesions reported in 5/10 examined females were equivocal.

Acanthosis can be seen in cases of irritative inflammatory reaction and the minimal microscopic changes noted in the 5/10 examined females might have been related to application of the test substance at 100% concentration and/or shaving.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
Under the experimental conditions used in this study and according to the maximization method established by Magnusson and Kligman, no cutaneous reactions attributable to the sensitization potential of iso-propyl bromide in its undliuted form were observed in guinea pigs. iso-Propyl bromide is, therefore, non-sensitizing.
Executive summary:

In a CoR 1 dermal sensitization study with 2-bromopropane in paraffin oil, Dunkin-Hartley guinea pigs were tested using the method of Magnusson and Kligman. No cutaneous reactions attributable to the sensitization potential of iso-propyl bromide in its undliuted form were observed in guinea pigs.

 

In this study, 2 -bromopropane is not a dermal sensitizer.