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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From September 2002 to May 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report date:
2003

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
and EC (96/54/EEC, B6, 30 July 1996)
GLP compliance:
yes
Type of study:
guinea pig maximisation test

Test material

Constituent 1
Reference substance name:
1,6- Dibromohexane
IUPAC Name:
1,6- Dibromohexane
Details on test material:
- Name of test material (as cited in study report): 1,6-dibromohexane
- Substance type: Colorless liquid
- Physical state: Liquid
- Analytical purity: 98.97%
- Purity test date: 98.97% - 9 August 2002
- Lot/batch No.: 3/98
- Expiration date of the lot/batch: January 2003
- Storage condition of test material: In dark and at room temeperature

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, L' Arbresle, France.
- Age at study initiation: The animals of the main test were 1-2 months old.
- Weight at study initiation:
* Males: 473 ± 28 g
* Females: 418 ± 14 g
- Housing: During the acclimation period and throughout the study, the animals were housed indi vidually in polycarbonate cages with stainless steel lid (48 cm x 27 em x 20 cm) equipped wi th a polypropylene bottle. Each cage contained autoclaved sawdust (SICSA, Alfortville, France). Sawdust is analysed by the supplier for composition and contaminant levels.
- Diet (e.g. ad libitum): Free access to 106 pelleted diet (UAR, Villcmoisson, Epinay-sur-Orge, France). Food is analysed regularly by the supplier for composition and contaminant levels.
- Water (e.g. ad libitum): Ad libitum - Drinking water filtered by a FG Millipore membrane (0.22 micron). Bacteriological and chemical analyses of water are performed regularly by external laboratories. These analyses include the detection of possible contaminants (pesticides, heavy metals and nitrosamines).
- Acclimation period: At least 5 days before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 30 to 70%
- Air changes (per hr): Approximately 12 cycles/hour of filtered, non-recycled air.
- Photoperiod (hrs dark / hrs light): 12 h/12 h

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
other: Intradermal application: Corn oil - Topical application: acetone
Concentration / amount:
Preliminary Test:
* Intradermal route: 25, 10 and 5% (w/w)
* Cutaneous route: 100 and 50% (w/w)
* Challenge phase: 100, 50, 25, 10, 5 and 1% (w/w)

Main Study:
* Intradermal route:
Three injections of 0.1 mL were made into each side of this interscapular region:
- Anterior: FCA at 50% (v/v) in 0.9% NaCl (Treated group) - FCA at 50% (v/v) in 0.9% NaCI (Control group)
- Middle: test item at 10% (w/w) in corn oil (Treated group) - corn oil (Control group)
- Posterior: test item at 10% (w/w) in the mixture FCA/0.9% NaCI (50/50) (Treated group) - vehicle at 50% (w/v) in a mixture FCA/0.9% NaCI (50/50) (Control group)
* Cutaneous route: filter paper fully loaded with the undiluted test item. For the control group, vehicle alone.
* Challenge phase: filter paper fully loaded with the test item at the concentration of 1% (w/w). For the control group, vehicle alone.
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: Intradermal application: Corn oil - Topical application: acetone
Concentration / amount:
Preliminary Test:
* Intradermal route: 25, 10 and 5% (w/w)
* Cutaneous route: 100 and 50% (w/w)
* Challenge phase: 100, 50, 25, 10, 5 and 1% (w/w)

Main Study:
* Intradermal route:
Three injections of 0.1 mL were made into each side of this interscapular region:
- Anterior: FCA at 50% (v/v) in 0.9% NaCl (Treated group) - FCA at 50% (v/v) in 0.9% NaCI (Control group)
- Middle: test item at 10% (w/w) in corn oil (Treated group) - corn oil (Control group)
- Posterior: test item at 10% (w/w) in the mixture FCA/0.9% NaCI (50/50) (Treated group) - vehicle at 50% (w/v) in a mixture FCA/0.9% NaCI (50/50) (Control group)
* Cutaneous route: filter paper fully loaded with the undiluted test item. For the control group, vehicle alone.
* Challenge phase: filter paper fully loaded with the test item at the concentration of 1% (w/w). For the control group, vehicle alone.
No. of animals per dose:
Main Study:
Thirty guinea pigs were allocated to two groups: a control group of five males and five females and a treated group of ten males and ten females.
Details on study design:
RANGE FINDING TESTS:
* Intradermal route:
- intradermal injections of the dosage form preparations (0. 1 mL) were performed in the interscapular region,
- cutaneous reactions were evaluated approximately 24, 48 hours and 6 days after the injections.
* By cutaneous route:
- A filter paper (approximately 8 cm2) was fully-loaded with a dosage form preparation and was then applied to the clipped area of the skin. The filter paper was held in place by means of an occlusive dressing for 48 hours,
- cutaneous reactions were evaluated 24 and 48 hours after removal of the dressing.
* Challenge phase:
- the filter paper of a chamber (Finn Chamber(R) was fully-loaded with a dosage form preparation. The chamber was then applied to the clipped area of the skin (one concentration per flank ). The chamber was held in place by means of an occlusive dressing for 24 hours,
- cutaneous reactions were evaluated 24 and 48 hours after removal of the dressings.

MAIN STUDY:
A. INDUCTION EXPOSURE
* Intradermal route:
On day 1, six injections were made deep into the dermis of a 4 cm x 2 cm clipped interscapular area, using a needle (diameter: 0.50 x 16 mm) mounted on a 1 mL plastic syringe (0.01 mL graduations).
Three injections of 0.1 mL were made into each side of this interscapular region (i.e. three pairs of sites). The sites were distributed as Anterior, Middle, Posterior. The anterior and middle pairs of injections were performed close to each other and nearest the head, while the posterior pair was perfonned towards the caudal part of the test area.
* By cutaneous route:
As the test item was shown to be irritant during the preliminary test, a topical application of sodium lauryl sulfate was not necessary on day 7.
On day 8, a pad of filter paper (approximately 8 cm2) was fully-loaded with the undiluted test item 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. On day 10, removal of the dressing.

B. CHALLENGE EXPOSURE
On day 22, the animals of treated and control groups received an application of the test item and vehicle. The filter paper of a chamber (Finn Chamber (R) was fully-loaded with the test item at the concentration of 1% (w/w) and was then applied to a clipped area of the skin of the posterior right flank of all 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.
Challenge controls:
For the Challenge phase, the animals received a filter paper of a chamber (Finn Chamber (R)) fully loaded with the vehicle and appleid to a clipped area of the skin of the posterior right flank of the animals. The chambers was held in contact with the skin for 24 hours.
Positive control substance(s):
yes
Remarks:
Mercaptobenzothiazole (20% at challenge)

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . 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
Dose level:
challenge conc. 1% in corn oil
No. with + reactions:
16
Total no. in group:
20
Clinical observations:
erythema grade 3: 1 f, grade 2 1m1 f, grade 1: 7 m, 6 f
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: challenge conc. 1% in corn oil. No with. + reactions: 16.0. Total no. in groups: 20.0. Clinical observations: erythema grade 3: 1 f, grade 2 1m1 f, grade 1: 7 m, 6 f.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
challenge: concentration 1% in corn oil
No. with + reactions:
10
Total no. in group:
20
Clinical observations:
erythema grade 1: 5m, 5 f, dryness of skin: 1 m, 4 f
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: challenge: concentration 1% in corn oil. No with. + reactions: 10.0. Total no. in groups: 20.0. Clinical observations: erythema grade 1: 5m, 5 f, dryness of skin: 1 m, 4 f.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
challenge conc. 20% mercaptobenzothiazole
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
severe skin reactions in all animals
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: challenge conc. 20% mercaptobenzothiazole. No with. + reactions: 10.0. Total no. in groups: 10.0. Clinical observations: severe skin reactions in all animals.
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
Challenge conc 20% mercaptobenzthiazole
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
severe skin reactions in all animals
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: Challenge conc 20% mercaptobenzthiazole. No with. + reactions: 10.0. Total no. in groups: 10.0. Clinical observations: severe skin reactions in all animals.

Any other information on results incl. tables

The current test is not a LLNA test but Guinea pig maximization test.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: other: Criteria lof the Council Directive 671548IEEC (and subsequent adaptations).
Conclusions:
Under our experimental conditions and according to the maximization method of Magnusson and Kligman, the test item 1,6-Dibromohexane induced delayed contact hypersensitivity in 16/20 (80%) of the animals at the 24 h observation time and 10/20 (50%) guinea pigs at the 48h observation time. It is noted that the positive controls showed unusually severe reactions in this test. As the intradermal induction dose was 10% , i.e. relatively high and the response rate was between 80% at 24 h and 50% at 48h, a classification as skin sensitizer category 1 B according to Regulation EC 286/2011 amending regulation EC 1272/2008 is applied.
Executive summary:

The potential of the test item 1,6-Dibromohexane (batch No. 0003/98) to induce delayed contact hypersensitivity was evaluated in guinea pigs according to the maximization method of Magnusson and Kligman and to OECD (No. 406, 17th July 1992) and EC (96/541EEC, B.6, 30 July 1996) guidelines. The study was conducted in compliance with the principles of Good Laboratory Practice Regulations.

Methods

Thirty guinea pigs were allocated to two groups: a control group of five males and five females and a treated group of ten males and ten females.

On day 1, three pairs of intradermal injections were performed in the interscapular region of all animals:

* Freund's complete adjuvant (FCA) diluted at 50% (v/v) with 0.9% NaCl (both groups),

* test item at the concentration of 10% (w/w) in corn oil (treated group) or vehicle alone (control group),

* test item at the concentration of 10% (w/w) in a mixture FCA/0.9% NaCl (50/50, v/v) (treated group) or vehicle at the concentration of 50% (w/v) in a mixture FCA/0.9% NaCl (50/50, v/v) (control group).

On day 8, the animals of the treated group received a topical application of the undiluted test item to the same test site, which was then covered by an occlusive dressing for 48 hours. The animals of the control group received an application of vehicle under the same experimental conditions.

On day 22, all animals of both groups were challenged by a cutaneous application of the test item at the concentration of 1% (w/w) in acetone to the right flank. The test item was maintained under an occlusive dressing for 24 hours. The vehicle was applied to the left flank under the same experimental conditions. Skin reactions were evaluated approximately 24 and 48 hours after removal of the dressing.

At the end of the study, animals were killed without examination of internal organs. No skin samples were taken from the challenge application sites.

Results

No systemic clinical signs and no deaths were noted during the study. After the challenge application, no cutaneous reactions were observed in the animals of the control group. In the treated group, at the 24 hour reading, a discrete to intense erythema was noted in 13/20, 2/20 and 1/20 animals, respectively. At the 48 hour reading, a discrete erythema persisted in 9/20 animals and appeared in 1/20 animals. The skin reactions recorded at the 48 hour reading in 10/20 animals were anributed to delayed contact hypersensitivity.

Conclusion

Under our experimental conditions and according to the maximization method of Magnusson and Kligman, the test item 1,6-Dibromohexanc (batch No. 0003/98) induces delayed contact hypersensitivity in 10/20 (50%) guinea pigs. According to the classification criteria laid down in Council Directive 67/548fEEC (and subsequent adaptations), the test item should be considered as a skin sensitizer. As the intradermal induction dose was 10% , i.e. relatively high and the response rate was between 80% at 24 h and 50% at 48h, a classification as skin sensitizer category 1 B according to Regulation EC 286/2011 amending regulation EC 1272/2008 is applied.

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