Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 Sep 1997-2 Oct 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.4100 (Skin Sensitisation)
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
99.5% purity

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:David Hall Limited, Burton-on-Trent, Staffordshire, UK.
- Age at study initiation: 8-12 weeks old.
- Weight at study initiation: 300- 358 g
- Housing: singly or in pairs in solid-floor polypropylene cages furnished with woodflakes.
- Diet : Animals had free access to food (Guinea Pig FD1 Diet, Special Diets Services Limited, Witham, Essex, UK)
- Water : Animals had free access to drinking water.
- Acclimation period: At least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 2 ºC
- Humidity (%): 44 to 68 %
- Air changes (per hr): 15 changes per hour.
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
arachis oil
Concentration / amount:
For Intradermal induction: 1, 5, 10 and 25 % w/v (the prelimiary study).
For Topical induction: 75, 50 and 25 % v/v (the prelimiary study).
For Topical challenge: 75, 50 % v/v
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
arachis oil
Concentration / amount:
For Intradermal induction: 1, 5, 10 and 25 % w/v (the prelimiary study).
For Topical induction: 75, 50 and 25 % v/v (the prelimiary study).
For Topical challenge: 75, 50 % v/v
No. of animals per dose:
20 test; 10 control
Details on study design:
RANGE FINDING TESTS:
A preliminary test was performed to determine the concentration to be used in the main study.

By intradermal route:
Determination of Maximum Non-Irritant Concentration (M.N.I.C.)
- On day 0 before treatment, the shoulder region 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 causes mild to moderate irritation,
- evaluation of the potential cutaneous reactions, 24, 48, 72 hours and after 7 days after injection.

By topical route:
Determination of Maximum Non-Irritant Concentration (M.N.I.C.):
- Shoulder region of the animals was clipped again
- Topical administration of the test substance undiluted at increasing concentrations was applied performed in order to determine the minimum
concentration which causes mild to moderate irritation undiluted was applied to a filter paper patch of approximately 40 mmx20 mm and then held in place by an occlusive dressing of strip of surgical adhesive tape covered by an overlapping length of aluminium foil that also were secured with a
strip of elastic adhesive bandage wound in a double layer around the torso of each animal for 48 hours,
- potential cutaneous reactions were evaluated 1 and 24 hours after removal of the pads.


MAIN STUDY
A. INDUCTION EXPOSURE

Intradermal route:
On day 0, intradermal injections were made into a clipped area (4 cm x 6 cm) in the shoulder region. Three injections of 0.1 ml were injected into
each side of the mid-line of animal, as follows:

Control group:
• Freund's complete adjuvant plus distilled water in the ratio 1:1
• Vehicle,
• A 50% w/v formulation of vehicle in Freund's Complete Adjuvant/distilled water 1:1

Treated group:
• Freund's complete adjuvant plus distilled water in the ratio 1:1
• A 10% w/v solution of the test substance in the vehicle,
• A 10% w/v emultion of the test material in a 1:1 preparation of Freund's Complete Adjuvant plus distilled water

Topical route:
On day 7, the shoulder area was clipped again. The test substance was held in place with a strip of surgical adhesive tape covered with an overlapping length of aluminium foil and further secured with a strip of elastic adhesive bandage wound.

Control group:
• Nothing was applied to the filter paper.

Treated group:
• Application of the undiluted test substance.

The test substance and the vehicle were prepared on a dry gauze pad, which was then applied to the region and held in place for 48 hours by means
of an adhesive dressing.
The degree of erythema and oedema was evaluated 1, 24 and 48 hr after dressing removal.


B. CHALLENGE EXPOSURE

At the end of the test period on day 21, the test substance was applied at the Maximum Non-Irritant Concentration (M.N.I.C.) i.e.
in its original form and one lower concentration.

On day 21, 2 animals received an application of the M.N.I.C. of the test substance and one lower concentration on the clipped flanks under occlusive
dressings for an exposure period of 24 hr.
The test substance was applied on a patch, then applied to the shorn clipped area of the skin. The gauze pad was held in place with a strip of surgical adhesive tape. The patched were occluded with an overlapping bandage wound in a double layer around the torso of each animal.
After 24 hours, the dressing was cut. The chllenge sites were swabbed with cotton wool soaked in diethyl ether.
24 and 48 hours after challenge dressing removal, the degree of erythema and oedema was quantified.
Any other reactions were also recorded.

Positive control substance(s):
no

Study design: in vivo (LLNA)

Statistics:
% of animals showing a reaction Allerginicity Level Classification

0 - 8 I Very weak
9 - 28 II Weak
29 - 64 III Moderate
65 - 80 IV Strong
81 - 100 V Very Strong

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0% test substance
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 0% test substance. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0% test substance
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 0% test substance. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
75%
No. with + reactions:
3
Total no. in group:
20
Clinical observations:
3 animals with very slight erythema grade 1
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 75%. No with. + reactions: 3.0. Total no. in groups: 20.0. Clinical observations: 3 animals with very slight erythema grade 1.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
75%
No. with + reactions:
2
Total no. in group:
20
Clinical observations:
2 animals with very slight erythema grade 1
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 75%. No with. + reactions: 2.0. Total no. in groups: 20.0. Clinical observations: 2 animals with very slight erythema grade 1.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
50%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No clinical reactions were noted
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: 20.0. Clinical observations: No clinical reactions were noted.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
50%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No clinical reactions were noted
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: 20.0. Clinical observations: No clinical reactions were noted.

Any other information on results incl. tables

Very slight erythema was observed in 15% of the animals after 24 hours and 15 % after 48 hours of the removal of the dressing.

No oedema was observed 24 and 48 hours after removal of the dressing of the challenge topical application of the test substance.

Applicant's summary and conclusion

Interpretation of results:
other: mild sensitiser
Conclusions:
Under experimental conditions and according to the maximization method of Magnusson and Kligman, no cutaneous reactions possibly attributable
to the sensitization potential of the test substance, Bromochloromethane, in its original form were observed in guinea-pigs
Executive summary:

A skin sensitization study according to the maximisation method of Magnusson and Kligman was performed on female Dunkin-Hartley guinea-pigs weighing 300 -358 g. The study was performed using the test substance Bromochloromethane and in accordance with OECD 406 and to GLP standard.

No cutaneous reactions possibly attributable to the sensitization potential of the test substance were observed and the material is therefore not classified as a sensitizer.