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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation, other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13th October - 14th November 1994
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:
1995
Report Date:
1995

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Justification for not using non-LLNA method is lack of availability.
The Local Lymph Node Assay (LLNA) OECD testing guideline No. 429 was first adopted in 2002. The information on sensitization potential of the substance was generated in 1995 according the OECD guidelines No. 406, which were accepted in these years.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
99.3 % purity

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Centre d'Elevage Lebau, 78950 Gambais, France
- Age at study initiation: NDA
- Weight at study initiation: 343 ± 22 g
- Housing: Individually in polycarbonate cages (48 x 27 x 20 cm) fitted with a polypropylene bottle
- Diet (e.g. ad libitum): Animals had free access to 106 diet (U.A.R., 91360 Villemoisson-sur-Orge, France)
- Water (e.g. ad libitum): Animals had free access to drinking water, filtered by a F.G. Millipore membrane
- Acclimation period: At least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2 ºC
- Humidity (%): 30 to 70 %
- Air changes (per hr): 12 cycles / hours of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12/2

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
paraffin oil
Concentration / amount:
1, 5 and 25 % for the prelimiary study.
100 % for the main study.
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
paraffin oil
Concentration / amount:
1, 5 and 25 % for the prelimiary study.
100 % for the main study.
No. of animals per dose:
5 control and 10 test animals.
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 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 Conrtration (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 patch of approximately 4 cm 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
A. INDUCTION EXPOSURE

Intradermal route:
On day 1, six intradermal injections were made into a clipped area (4 cm x 2 cm) in the scapular region. Three injections of 0.1 ml 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% NaCI),
• 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% NaCI), 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 cm x 2 cm) 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, which was then applied to the scapular region and held in place for 48 hours by means of an adhesive hypoallergenic dressing and an adhesive anallergenic waterproof plaster. No residual test substance was observed at removal of the dressing.
One hour after removal of the occlusive dressing, cutaneous reactions were recorded.


B. CHALLENGE EXPOSURE

At the end of the rest period on day 22, the test substance was applied at the Maximum Non-Irritant 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 I ml plastic syringe. The test substance and vehicle were prepared on a dry gauze pad, then applied to a 4 cm^2 (2 cm x 2 cm) clipped area of he skin. The gauze pad was held in contact with the skin for 24 hours by means of an occlusive, hypoallergenic dressing and an adhesive anallergenic waterproof plaster.
No residual test substance was observed at removal of the dressing.
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:
5
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: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0 % test substance
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 % test substance. 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:
4
Total no. in group:
10
Clinical observations:
3 animals with very slight and 1 with well defined erythema
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 100 %. No with. + reactions: 4.0. Total no. in groups: 10.0. Clinical observations: 3 animals with very slight and 1 with well defined erythema.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
100 %
No. with + reactions:
3
Total no. in group:
10
Clinical observations:
2 animals with very slight and 1 with well defined erythema
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 100 %. No with. + reactions: 3.0. Total no. in groups: 10.0. Clinical observations: 2 animals with very slight and 1 with well defined erythema.

Any other information on results incl. tables

Very slight to well defined erythema was observed in 40% of the animals after 24 hours and 30 % after 48 hours of the removal of the dressing. Dryness of the skin was oberved in 3/10 animals at the 48 hour reading. No oedema was observed 24 and 48 hours after removal of the dressing of the challenge cutaneous application of the test substance.

Microscopic Examination

Skin of the treated right flank (100 % concentration test substance) was examined in 4/10 females showing cutaneous reactions.

Slight to moderate acanthosis, sometimes together with minimal hyperkeratosis was noted in all 4 females. This was associated with minimal mononuclear cell and minimal to slight granulocyte inflamation if the upper dermis in all 4 females; extravasation of red blood cells was noted in the upper dermis of 1/4 examined females.

No treated skin from the 4/10 examined females showed spongiosis and in the absence of the classical association of spongiosis, acanthosis and mononuclear cell infiltration of dermis, the above mentioned lesions reported in 4/10 examined females were equivocal.

Acanthosis can be seen in cases of irritative inflammatory reaction and the minimal microscopic changes noted in the 4/10 examined females might be 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 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, n-propyl bromide, 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 343 ± 22 g. The study was performed using the test substance 1-bromopropane 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 classed as a sensitizer.