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Skin sensitisation

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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:
1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Study performed before the implementation of the REACH regulation

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): DIACETONE ALCOHOL.
- Physical state: Colourless liquid.
- Analytical purity: 99.72%.
- Lot/batch No.: 9609P0513.
- Expiration date of the lot/batch: October 1997.
- Stability under test conditions: Not reported.
- Storage condition of test material: At room temperature and protected from light.

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Centre d'Elevage Lebeau, 78950 Gambais, France.
- Age at study initiation: Approximately 3 months old.
- Weight at study initiation: 347 ± 31 g (males); 373 ± 20 g (females).
- Housing: Housed individually in polycarbonate cages.
- Diet (e.g. ad libitum): 106 diet (U.A.R., 91360 Villemoisson-sur-Orge, France), ad libitum.
- Water (e.g. ad libitum): Filtered by a F.G. Millipore membrane (0.22 micron), ad libitum.
- Acclimation period: At least 5 days before the beginning of the study.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 30 to 70
- Air changes (per hr): About 12
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
physiological saline
Concentration / amount:
25 %
Day(s)/duration:
single
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Day(s)/duration:
single
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challenge
Route:
epicutaneous, occlusive
Vehicle:
other: See Any other information on materials and methods.
Concentration / amount:
-Intradermal induction: 25% (w/w) in sterile isotonic saline solution (0.9% NaCl).
-Epicutaneous induction: Undiluted.
-Cutaneous challenge: Undiluted.

II) Challenge period: undiluted test substance
No. of animals per dose:
Control group: 5/sex.
Test substance group: 10/sex.
Details on study design:
RANGE FINDING TESTS: A preliminary test was conducted in order to determine the concentrations to be tested in the main study.


A preliminary test was conducted in order to determine the concentrations to be tested in the main study.

Preparation of the animals:
For all animals and before each treatment, the application sites were:
-clipped on Days -1 and 7 (scapular area 4 cm x 2 cm),
-clipped and shaved on Day 21 (each flank 2 cm x 2 cm).

I) Induction phase by intradermal and cutaneous routes
 
1) Intradermal route
On Day 1, six injections were made deep into the dermis of a clipped area (4 cm x 2 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:
Treatment group:
A) Anterior: Freund's complete adjuvant diluted at 50% (v/v) with sterile isotonic saline solution (0.9% NaCl)
B) Middle: test substance at 25% (w/w) in 0.9% NaCl
C) Posterior: mixture of 50/50 (w/v) of A and B
 
Control group:
A) Anterior: Freund's complete adjuvant diluted at 50% (v/v) with 0.9% NaCl
B) Middle: vehicle (0.9% NaCl)
C) Posterior: mixture of 50/50 (w/v) of A and B
 
2) 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 lauryl sulfate (10% w/w) in vaseline in order to induce local irritation. 
 
On Day 8, a topical application to the region of the intradermal injections was performed.
 
Control group:
Application of 0.5 mL of the vehicle
 
Treatment group:
Application of 0.5 mL of the test substance undiluted.
 
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. No residual test substance was observed after removal of the dressing. Cutaneous reactions were recorded one hour after removal of the occlusive dressing. 
 
II) Challenge phase
On Day 22, the animals from both groups received an application of 0.5 mL of the test substance undiluted to the posterior right flank, and 0.5 mL of the vehicle to the posterior left flank. This application was performed using a 1 mL glass syringe. The test substance and the vehicle were prepared on a dry gauze pad, then applied to a 4 square cm clipped area of the skin. The gauze pad was held in contact with the skin for 24 hours by means of occlusive, hypoallergenic dressing and an adhesive anallergenic waterproof plaster. No residual test substance was observed after removal of the dressing. 
 
Twenty-four and 48 hours after the challenge application, both flanks of the treated and control animals were observed in order to evaluate cutaneous reactions, according to the following scale:
 
Erythema and eschar formation: Grade
No erythema: 0
Very slight erythema (barely perceptible): 1
Well-defined erythema: 2
Moderate to severe erythema: 3
Severe erythema (beet redness) to slight eschar formation (injuries in depth): 4
 
Oedema: Grade
No oedema: 0
Very slight oedema (barely perceptible): 1
Slight oedema (visible swelling with well-defined edges): 2
Moderate oedema (visible swelling raised more than 1 mm): 3
Severe oedema (visible swelling raised more than 1 mm and extending beyond the area of exposure): 4
 
Any other lesions were noted. 
 
Clinical examinations: The animals were observed twice a day during the study in order to check for clinical signs and mortality.
 
Body weight: The animals were weighed individually on the day of allocation into the groups, on the first day of the study (Day 1), on Days 8 and 15 and on the last day of the study (Day 25). 
Challenge controls:
See details on study design.
Positive control substance(s):
yes
Remarks:
2,4-Dinitro Chlorobenzene

Results and discussion

Positive control results:
Under the experimental conditions and according to the Magnusson and Kligman method, the test substance 2,4-Dinitro Chlorobenzene at a concentration of 0.5% (w/w) induced positive skin sensitization reactions in 50% of the guinea-pigs.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
Undiluted
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No cutaneous reactions were observed
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: Undiluted. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: No cutaneous reactions were observed.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
Undiluted
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No cutaneous reactions were observed
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: Undiluted. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: No cutaneous reactions were observed.
Reading:
other: 1st reading: right flank
Hours after challenge:
24
Group:
negative control
Dose level:
Undiluted
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No cutaneous reactions were observed
Remarks on result:
other: Reading:
Reading:
other: 2nd reading: right flank
Hours after challenge:
48
Group:
negative control
Dose level:
Undiluted
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No cutaneous reactions were observed
Remarks on result:
other: Reading:
Reading:
other: 1st reading: left flank
Hours after challenge:
24
Group:
negative control
Dose level:
0.9% NaCl
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No cutaneous reactions were observed
Remarks on result:
other: Reading:
Reading:
other: 2nd reading: left flank
Hours after challenge:
48
Group:
negative control
Dose level:
0.9% NaCl
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No cutaneous reactions were observed
Remarks on result:
other: Reading:
Reading:
other: positive control not applied in the test
Group:
positive control
Remarks on result:
not measured/tested

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions and according to the maximization method of Magnusson and Kligman, no cutaneous reactions attributable to the sensitization potential of the test substance diacetone alcohol were observed in guinea-pigs.
Executive summary:

The skin sensitization potential of diacetone alcohol was assessed in a study performed according to OECD Guidelines for the Testing of Chemicals No. 406 and in compliance with GLP in male and female Dunkin-Hartley guinea pigs (de Jouffrey, 1997).  In the main study, 10 animals/sex comprised the diacetone alcohol test group and 5 animals/sex comprised the vehicle control group.  The intradermal induction was carried out with 0.1 mL of 25% (w/w) of diacetone alcohol in vehicle (a solution of 0.9% w/v of NaCl), and epicutaneous induction was performed with 0.5 mL of undiluted diacetone alcohol to the dorsal area under occlusive conditions.  The challenge exposure also was conducted with 0.5 mL of undiluted diacetone alcohol.  Additionally, all animals were dermally exposed to 0.5 mL of 10% w/w sodium lauryl sulphate (SDS) in vaseline 24 hours prior to topical sensitization of the skin area in order to induce local irritation (diacetone alcohol was shown to be non-irritating in the preliminary test).  Skin reactions were observed and recorded 1 hour after dermal and 24 and 48 hours after the challenge exposure, all according to the grading scale by Magnusson and Kligman.  Test and control animals displayed normal body weight gain throughout the investigation and no mortalities or clinical signs were observed.  On Day 10, following dermal induction, signs of irritation were observed at the site of application in both control and treated groups.  Following the challenge exposure, no incidences of erythema or oedema were observed, either at 24 or 48 hours, in all animals.  Under the experimental conditions and according to the maximization method of Magnusson and Kligman, no cutaneous reactions attributable to the sensitization potential of diacetone alcohol were observed in guinea-pigs.  Therefore, the results of this study demonstrated that diacetone alcohol showed no evidence of contact skin sensitization in guinea pigs.