Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January to March 2001
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:
2001

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
1-bromohexane
EC Number:
203-850-2
EC Name:
1-bromohexane
Cas Number:
111-25-1
Molecular formula:
C6H13Br
IUPAC Name:
1-bromohexane
Details on test material:
Producer ATOFINA
Lot number: 8-240
Description: colorless liquid
Container: one smoked glass flask
Date of receipt: 11 January 2001
Storage conditions: at room temperature and protected from light
Purity: 99.28%
Expiry date: December 2001

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
Species and sex: male and nulliparous and non-pregnant female guinea pigs.
Strain and sanitary status: Hartley Crl: (HA) BR, Caesarian obtained, Barrier sustained - Virus Antibody Free (COBS - VAF).
Number:
- one male and one female for the preliminary test,
- 30 animals (15 males and 15 females) for the main test.
Allocation of the animals to the groups: on day 1, the animals were weighted and randomly allocated to two groups: a control group of ten animals (five males and 5 females) and a treated group of 20 animals (ten males and 10 females).
Age/weight: on day 1, the animals of the main test were 1-3 months old and had a mean body weight ± standard deviation of 333 ± 16 g for the males and 329 ± 15 g for the females.
Acclimatation: at least 5 days before beginning of the study.Identification of the animals: ear-tattoo.

The conditions in the animals room were set as follows:
- temperature : 21± 2°C
- relative humidity: 30 to 70%
- light/dark cycle: 12h/12h
- ventilation: approximately 12 cycles/hour of filtered, non-recycled air.
The temperature and relative humidity were under continuous control and recording. The records were checked daily and filed. In addition to these daily checks, the housing conditions and corresponding instrumentation and equipment are verified and calibrated at regular intervals.
During the acclimation periond and throughout the study, the animals were housed individually in polycarbonate cages with stainless steel lid (48 x 27 x20 cm) equipped with a polypropylene bottle.
Dust-free sawdust was provided as litter.Sawdust is analysed by the supplier for composition and contaminant levels.

Durinf the study, the animals had free access to "106 pelleted diet". Food is analysed regularly by the supplier for composition and contaminant levels.

Drinking water filtered by a FG Millipore ùeùbrane (0.22 micron) was provided ad libitum.
Bacteriological and chemical analysesof water are performed regularly by external laboratories. These analyses include the detection of possible contaminants (pesticides, heavy metals and nitosamines).

No contaminants were known to have been present in the diet, drinking water or bedding material at levels which may be expected to have interfered with or prejudiced of the study.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
corn oil
Remarks:
Corn oil was used for intradermal injections and acetone was used for challenge application and the preliminary test
Concentration / amount:
Three injections were made into each side of the interscapular region as follows:
- for the control group
1) Freund's Complete Adjuvant at 50% (v/v) in 0.9% NaCl
2) Corn oil
3) Vehicle at 50% (w/v) in a mixture FCA/0.9% NaCl (50/50)
- for the treated group:
1) Freund's Complete Adjuvant at 50% (v/v) in 0.9% NaCl
2) Test substance at 10% (w/w) in corn oil
3) Test substance at 10% (w/w) in the mixture FCA/0.9% NaCl (50/50)
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Remarks:
Corn oil was used for intradermal injections and acetone was used for challenge application and the preliminary test
Concentration / amount:
Three injections were made into each side of the interscapular region as follows:
- for the control group
1) Freund's Complete Adjuvant at 50% (v/v) in 0.9% NaCl
2) Corn oil
3) Vehicle at 50% (w/v) in a mixture FCA/0.9% NaCl (50/50)
- for the treated group:
1) Freund's Complete Adjuvant at 50% (v/v) in 0.9% NaCl
2) Test substance at 10% (w/w) in corn oil
3) Test substance at 10% (w/w) in the mixture FCA/0.9% NaCl (50/50)
No. of animals per dose:
30 animals (15 males and 15 females) for the main test:
- 5 males and 5 females for the control group
- 10 males and 10 females for the treated group
Details on study design:
On day 1, six injections were made deep into the dermis of a clipped interscapular area, using a needle (diameter 0.50 x 16 mm) mounted on a 1 mL plastic syringe (0.01 mL graduations).
The anterior and middle pairs of injections were performed close to each other and nearest the head, while posterior pair was performed towards the caudal part of the test area.

On day 7, the interscapular area was clipped.
As the test substance was shown to be non-irritant during preliminary test, the animals were treated with 0.5 mL of sodium lauryl sulfate at the concentration of 10% (w/w) in vaseline, in order to induce local irritation.
On day 8, a pad of filter paer was fully-loaded with the undiluted test substance and was then applied to the interscapular region of the animals of the treated group. The animals of the control group received an application of the vehicle alone under the same experimental conditions.
The pad was held in place for 48 hours by means of an adhesive hypoallergenic dressing and an adhesive anallergenic waterproof plaster.
Challenge controls:
On day 22, the animals of treated and control groups received an application ot the test substance and vehicle. The filter paper of a chamber was fully-loaded with the undiluted test substance and was then applied to a clipped area of the skin of the posterior right flank of the animals.
The vehicle was applied under the same experimental conditions to the skin of the posterior left flank.
The chambers were held in contact with the skin for 24 hours by means of an adhesive anallergenic waterproof plaster.
As equivocal cutaneous reactions were noted, a second challenge application was performed after a rest period of 16 days.

On day 39, the animals of the treated and control groups received an application of the test sustence at the concentration of 25% (w/w) to the medium left flank and the vehicle to the median right flank, under the same experimental conditions as for the firts challenge application.
Positive control substance(s):
yes
Remarks:
Mercaptobenzothiazole

Results and discussion

Positive control results:
Method: Magnusson and Kligman
Number of animals: one control group of 6 animals and one treated group of 10 animals
Induction: 1% (w/w) intradermal route day 1
20% (w/w) cutaneous route day 8
Challenge application: 20% (w/w) cutaneous route day 22

Under the expirimental conditions and according to Magnusson and Kligman method, the test substance Mercaptobenzothiazole at the concentration of 20% (w/w) induced positive skin sensitization reactions in 100% guinea-pigs.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
No. with + reactions:
6
Total no. in group:
10
Clinical observations:
Discrete to moderate erythema
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. No with. + reactions: 6.0. Total no. in groups: 10.0. Clinical observations: Discrete to moderate erythema.
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
No. with + reactions:
8
Total no. in group:
10
Clinical observations:
Discrete to moderate erythema. One oedema
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. No with. + reactions: 8.0. Total no. in groups: 10.0. Clinical observations: Discrete to moderate erythema. One oedema.
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group: negative control. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: negative control. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
No. with + reactions:
20
Total no. in group:
20
Clinical observations:
Discrete, moderate or intense erythema
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. No with. + reactions: 20.0. Total no. in groups: 20.0. Clinical observations: Discrete, moderate or intense erythema.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
No. with + reactions:
20
Total no. in group:
20
Clinical observations:
Discrete, moderate or intense erythema - Oedema on 7 animals - Crust and dryness of the skin on almost all the animals
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. No with. + reactions: 20.0. Total no. in groups: 20.0. Clinical observations: Discrete, moderate or intense erythema - Oedema on 7 animals - Crust and dryness of the skin on almost all the animals.
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
No. with + reactions:
6
Total no. in group:
20
Clinical observations:
Discrete or moderate erythema
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group: test group. No with. + reactions: 6.0. Total no. in groups: 20.0. Clinical observations: Discrete or moderate erythema.
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
No. with + reactions:
4
Total no. in group:
20
Clinical observations:
Discrete or moderate erythema
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: test group. No with. + reactions: 4.0. Total no. in groups: 20.0. Clinical observations: Discrete or moderate erythema.

Any other information on results incl. tables

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

The body weight gain of the trated animals was similar of the control animals.

On removal of the dressing, no residual test substance was observed.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the experimental conditions and according to the maximization method of Magnusson and Kligman, the test substance 1-Bromohexane induces cutaneous reactions which could be attributable to delayed contatc hypersensitivity in 3/20 (15%) guinea pigs.
However, according to classification criteria laid down in Directive 93/21/EEC adapting to technical progress for the eighteenth time Council Directive 67/548/EEC, the test substance should not be considered as a skin sensitizer.
Executive summary:

After the first challenge application, a discrete or moderate erythema was noted in 8/10 animals of the control group and a discrete, moderate or intense erythema was observed in 20/20 animals of the treated group. An oedema was recorded in 1/10 animal of the control group and in 8/20 animals of the treated group.

Dryness of the skin and crusts were observed in almost all animals of the treated group.

After the second challenge application, no erythema was observed in the animals of the control group. Dryness of the skin was noted in one animal at the 24-hour reading.

In the treated group, at the 24-hour reading, a discrete or moderate erythema was noted in 4/20and 2/20 animals respectively. A discrete or moderate erythema persisted at the 48-hour reading in 2/20 and 2/20 animals respectively.

Dryness of the skin was observed in 4/20 animals at the 24-hour reading and in 5/20 animals at the 48-hour reading.

The persistent cutaneous reactions observed in 3/20 animals of the treated group after the second challenge application could be attributed to delayed contact hypersensitivity.