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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no deviations from standard test guidelines and no methodological deficiences, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
Freund's complete adjuvant test
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male/female
Details on test animals and environmental conditions:
Number: 30 animals (15 males and 15 nulliparous and non-pregnant females)
Allocation of the animals to the groups: on day-1, the animals were weighted and randomly allocated to two groups. A control group 1 consisting of ten animals (5 males and 5 females) and a treated group 2 consisting of 20 animals (ten males and ten females).
Weight: on day-1, the animals were approximatively three months old and had a mean body weight ± standard deviation of 359±19 g for the males and 355±21 g for the females.
Acclimatization: at least five days before the beginning of the study.
Identification of the animals: ear-tattoo.

During the acclimatization period and troughout the study, the conditions in the animal room were set as follows:
- temperature: 21±2°C
- relative humidity: 30 to 70 %
- light/dark cycle: 12h/12h
- ventilation: about 12 cycles/hour of filtered, non-recycled air.
The temperature and relative humidity were recorded continuously and records retained.
The housing conditions were checked monthly.
During the acclimatization period and throughout the study, the animals were housed individually in polycarbonate cages (48x27x20 cm) equipped with a polypropylene bottle.
Dust-free sawdust was provided as litter.
Bacteriological analysis of the sawdust and detection of possible contaminants were performed periodically.

All the animals had free access to 106 diet.
Each batch of food is analysed by the supplier for composition and contaminant levels.

Drinking water filtered by a FG Millipore membrane (0.22 µm) was provided ad libitum.
Bacteriological and chemical analyses of water are performed regularly. These analyses include the detection of possible contaminants (pesticides, heavy metals and nitroamines).
It was verified that no contaminants in the diet, drinking water or bedding material at levels likely to influence the outcome of the study were present.
Route:
intradermal and epicutaneous
Vehicle:
paraffin oil
Remarks:
Batch 7353 from Coopérative Pharmaceutique Française
Concentration / amount:
A preliminary test was conducted in order to determine the concentrations to be tested in the main study.

By intradermal route:
- 24 hours before treatment, the dorsal region of the animals were clipped,
- the test substance was prepared in an appropriate vehicule,
- intradermal administrations of the test substance (0.1 ml) at different concentrations were performed in the dorsal region between the shoulders,
- cutaneaous reactions were evaluated approximately 24, 48 hours and six days after injection.

By cutaneous route:
- 24 hours before treatment, both flank regions of the animals were clipped,
- if necessary, the test substance was prepared in an appropriate vehicule,
- the test substance (0.5 ml for each concentration) was applied to a dry gauze pad of approximately 4 cm2 which was held in place by an occlusive dressing for 24 hours,
- cutaneaous reactions were evaluated approximately 24, 48 hours after removal of the dressing.

Criteria for selection of concentrations
The following criteria were used:
- the concentrations should be well-tolerated systemically and locally,
- intradermal injections should cause moderate irritant effect (no necrosis or ulceration of the skin),
- topical application for the induction should cause at most weak or moderate skin reactions or be the maximal practicable concentration,
- topical application for the challenge should be the highest concentration which does not cause irritant effect.
Route:
epicutaneous, occlusive
Vehicle:
paraffin oil
Remarks:
Batch 7353 from Coopérative Pharmaceutique Française
Concentration / amount:
A preliminary test was conducted in order to determine the concentrations to be tested in the main study.

By intradermal route:
- 24 hours before treatment, the dorsal region of the animals were clipped,
- the test substance was prepared in an appropriate vehicule,
- intradermal administrations of the test substance (0.1 ml) at different concentrations were performed in the dorsal region between the shoulders,
- cutaneaous reactions were evaluated approximately 24, 48 hours and six days after injection.

By cutaneous route:
- 24 hours before treatment, both flank regions of the animals were clipped,
- if necessary, the test substance was prepared in an appropriate vehicule,
- the test substance (0.5 ml for each concentration) was applied to a dry gauze pad of approximately 4 cm2 which was held in place by an occlusive dressing for 24 hours,
- cutaneaous reactions were evaluated approximately 24, 48 hours after removal of the dressing.

Criteria for selection of concentrations
The following criteria were used:
- the concentrations should be well-tolerated systemically and locally,
- intradermal injections should cause moderate irritant effect (no necrosis or ulceration of the skin),
- topical application for the induction should cause at most weak or moderate skin reactions or be the maximal practicable concentration,
- topical application for the challenge should be the highest concentration which does not cause irritant effect.
No. of animals per dose:
A control group 1 consisting of ten animals (5 males and 5 females) and a treated group 2 consisting of 20 animals (ten males and ten females).
Details on study design:
Preparation of the animals
For all animals and before each treatment, the applications sites were:
- clipped on days 1 and 7 (scapular area 4x2 cm),
- clipped and shaved on day 21 (each flank 2x2 cm).

Intradermal route
On day 1, six injections wera made deep into the dermis of a clipped area (4x2 cm) in the dorsal region between the shoulders, using a needle mounted on a 1 ml glass syringe.

Three injections of 0.1 ml were made into each side of this shoulder region, as follows:

Injection sites* Treated group Control group
Anterior 1: FCA diluted at 50% (v/v) 1: FCA diluted at 50% (v/v)
with 0.9% NaCl with 0.9% NaCl

Middle 2: test substance at 25% (w/w) 2: vehicule
in paraffin oil

Posterior mixture of 50/50 (w/v) mixture of 50/50 (w/v)
of 1 and 2 of 1 and 2

*: three pairs of sites
FCA: Freund's complete adjuvant


Cutaneous route
On day 7, the scapular area was clipped. As the test substance was shown to be non-irritant during the preliminary tests, the animals were treated with 0.5 ml of sodium laurylsulfate (10 % w/w) in vaseline in order to induce local irritation.
On day 8, a topical application to the region of the intradermal injections (4x2 cm) was performed.

Control group: application of 0.5 ml of the vehicle.
Treated group: application of 0.5 ml of the test substance at the chosen concentration.

The test substance and the vehicle were prepared on a dry gauze pad which was then applied to the dorsal region between the shoulders and held in place for 48 hours by means of an adhesive hypoallergenic dressing and an adhesive anallergenic waterproof plaster.
On removal of the dressing, if present, any residual test substance was removed by means of a dry or a moistened gauze pad.
Cutaneous reactions were recorded one hour after removal of the occlusive dressing.
Challenge controls:
Challenge phase
On day 22, the animals from both groups received an application of 0.5 ml of the test substance at concentration of 25 % w/w to the posterior right flank, and 0.5 ml of the test substance at a concentration of 100 % to the posterior left flank.
This application was performed using a 1 ml plastic syringe.
The test substance and the vehicle were prepared on a dry gauze pad, then applied to a 4 cm2 (2x2 cm) clipped area of the skin. The gauze pad was held in contact with the skin for 24 hous by means of an occlusive, hypoallergenic dressing and an adhesive anallergenic waterproof plaster.
On removal of the dressing, if present, any residual test substance was removed by means of a dry or a mointened gauze pad.
Positive control substance(s):
yes
Remarks:
Test substance: 2,4-dinitrochlorobenzene
Positive control results:
Nulber of animals: 10 males and 10 females.
Under the experimental conditions and according to Magnusson and Kligman method, 2,4-dinitrochlorobenzene at a concentration of 1 % w/w induce positive skin sensitization reactions in 100 % of the guinea-pigs.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
0.5 ml at a concentration of 25% (w/w) on the right flank and 100% (w/w) on the left flank
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No clinical signs
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 0.5 ml at a concentration of 25% (w/w) on the right flank and 100% (w/w) on the left flank. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: No clinical signs.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
0.5 ml at a concentration of 25% (w/w) on the right flank and 100% (w/w) on the left flank
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
Dryness of the skin for 8 animals (4 males and 4 females)
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 0.5 ml at a concentration of 25% (w/w) on the right flank and 100% (w/w) on the left flank. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: Dryness of the skin for 8 animals (4 males and 4 females).

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

The body weight gain of the tretad animals was normal when compared to that of the control animals.

Challenge application : no residual test substance was observed after removal of the dressing.

No erythema or oedema was observed. Dryness of the skin was noted in 8/20 animals at the 48 -hour reading only, on the left flank (test substance undiluted). These skin reactions were attributed yo a slight irritant effect of the test substance.

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under experimental conditions and according to the maximization method of Magnusson and Kligman, no cutaneous reactions attributable to the sensitization potential of n-Amyl bromide at a concentration up to 100% (w/w) were observed in guinea-pigs.
Therefore, n-Amyl bromide should not be considered as sensitizing.
Executive summary:

The skin sensitising potential of the test material to guinea-pigs was determined by performing a maximisation test, in accordnace with standardised guidelines OECD 406 and EU Method B.6. Ten test animals and five control animals were exposed to 25% w/w and 100% w/w concentrations of the test material in paraffin oil. No signs of skin sensitisation were observed at either 24 or 48 hours post exposure.

The test material is determined to be non-sensitising and is unclassified according to Regulation 1272/2008.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:
Migrated from Short description of key information:
The skin sensitising potential of the test material to guinea-pigs was determined by performing a maximisation test, in accordnace with standardised guidelines OECD 406 and EU Method B.6. Ten test animals and five control animals were exposed to 25% w/w and 100% w/w concentrations of the test material in paraffin oil. No signs of skin sensitisation were observed at either 24 or 48 hours post exposure.

Justification for selection of skin sensitisation endpoint:
The two studies available are based on two different OECD Guidelines (429 and 406). The tests were conducted with concentrations up to 100% in each case.
Standard deviation of desintegration per minute numbers are very hight, variing from 8 to 34%.
Method with direct cutaneous contact and observation of skin reaction is retained.

Justification for classification or non-classification