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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:
1995
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report date:
1995

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The current study was available before LLNA become the 1st-choice method for in-vivo testing

Test material

Constituent 1
Chemical structure
Reference substance name:
Dimethoxymethane
EC Number:
203-714-2
EC Name:
Dimethoxymethane
Cas Number:
109-87-5
Molecular formula:
C3H8O2
IUPAC Name:
dimethoxymethane
Details on test material:
- Name of test material (as cited in study report): Methylal cosmetique
- Physical state: colourless liquid
- Analytical purity: 99.986%
- Impurities (identity and concentrations): Formol < 1 ppm; water 0.0139%; methanol < 1ppm
- Lot/batch No.: 99 01 10
- Storage condition of test material: At room temperature, 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, Gambais, France
- Age at study initiation: Not specified
- Weight at study initiation: On day 1, the animals had a mean body weight of 341 ± 22 g for the males and 349 ± 17 g for the females.
- Housing: During the acclimatization period and throughout the study, the animals were housed indi vidually in polycarbonate cages (48 x 27 x 20 cm) equipped with a polypropylene bottle. Calibrated and dust-free sawdust was provided as litter (SICSA, 92142 Alfortville, France). An analysis of potential residues and major contaminants is performed periodically (Laboratoire Wolff, 92110 Clichy, France). There were no contaminants in the sawdust at levels likely to have influenced the outcome of the study.
- Diet (e.g. ad libitum): During the study, the animals had free access to "106 diet" (U.A.R., 91360 Villemoisson-sur-Orge, France). Food was periodically analysed (composition and contaminants) by the supplier. There were no contaminants in the diet at levels likely to have influenced the outcome of the study.
- Water (e.g. ad libitum): Animals had free access to drinking water, filtered by a F.G. Millipore membrane (0.22 micron).
Bacteriological and chemical analysis of the water and detection of possible contaminants (pesticides, heavy metals and nitrosamines) are performed periodically. There were no contaminants in the water at levels likely to have influenced the outcome of the study.
- Acclimation period: Animals were kept for a minimum acclimatization period of 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2°C
- Humidity (%): 30 to 70%
- Air changes (per hr): THe rate wasabout 12 cycles/hour of filtred, non-recycled air.
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

IN-LIFE DATES: Not specified

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
paraffin oil
Remarks:
Batch No. 6590
Concentration / amount:
The concentration administrated for induction by intradermal route was 5% (w/w) in the vehicle. The test substance was administrated at non-irritant concentration for induction by cutaneous route.
The test substance was administrated in its original form (at 100% concentration) for the challenge.
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
paraffin oil
Remarks:
Batch No. 6590
Concentration / amount:
The concentration administrated for induction by intradermal route was 5% (w/w) in the vehicle. The test substance was administrated at non-irritant concentration for induction by cutaneous route.
The test substance was administrated in its original form (at 100% concentration) for the challenge.
No. of animals per dose:
20 treated animals (10 males and 10 females)
10 negative control animals (5 males and 5 females)
Details on study design:
A range finding test and a main study were performed. Modalities of these test are detailed in 1) and 2). Scoring of cutanous reactions scale is shown in 3).

1) RANGE FINDING TEST
A preliminary test was performed to define the concentration to be tested in the main study:
- The Minimum Irritant Concentration (M.LC.) was determinated by intradermal route on 2 animals (1 male and 1 female).
The dorsal region of the animals was clipped 24 hours before treatment. The test substance was prepared in paraffin oil. Intradermal administration of the test substance (volume 0.1 ml) at increasing concentrations of 0.1, 1, 5% (w/w) was performed in order to determine the minimum concentration which causes an irritation. An evaluation of potential cutaneous reactions was conducted 24 and 48 hours after injection.

- The Minimum Irritant Concentration (M.L.C.) and Maximum Non-Irritant Concentration (M.N.I.C.) were determinated by cutaneous route on 2 animals (1 male and 1 female).
The dorsal region of the animals was clipped 24 hours before treatment. A volume of 0.5 ml of the test substance in its original form was applied to a dry gauze pad of approximately 4 cm2 and then held in place by an occlusive dressing for 24 hours. An evaluation of potential cutaneous reactions was conducted 24 and 48 hours after removal of the gauze pads.

2) MAIN STUDY
Twenty guinea-pigs (10 males and 10 females) of Dunkin-Hatley strain, supplied by Centre d'Elevage Lebeau, Gambais, France were exposed to the test item after an acclimatization period of at least 5 days.

Calendar of the main study

Day -1: Clipping of the scapular region (4 cm x 2 cm)

2.1. INDUCTION PHASE

2.1.1. Day 1: Intradermal route
Six intradermal injections were made into the clipped area in the scapular region, using a needle mounted on a 1 ml glass syringe.
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% NaCl),
- vehicle,
- a mixture of 5O/50 (w/v) Freund's complete adjuvant diluted to 50% (v/v) with a sterile isotonic aqueous NaCI 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 5% (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% NaCl), and, the test substance at a concentration of 5% (w/w) in the vehicle.

2.1.2. Day 7: Clipping + sodium laurylsulphare
The scapular area was clipped. The animals were treated with 0.5 ml of sodium laurylsulphate (10%) in vaseline to provoke local irritation.

2.1.3. Day 8: Application covered by an occlusive dressing
A cutaneous application on the 6 injection areas (4 cm x 2 cm) of the scapular region was performed, as follows:

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 (Semes France, 54183 Heillecourt, France), which was then applied to the scapular region and held in place for 48 hours by means of an adhesive hypoallergenic dressing (Laboratoires de Pansements et d'Hygiene, 21300 Chenove, France) and an adhesive anallergenic waterproof plaster (Laboratoire des Professions Medicales, 92240 Malakoff, France). No residual test substance
was observed at removal of the dressing.

2.1.4.Day 10: Removal of dressing and scoring after one hour
One hour after removal of the occlusive dressing, cutaneous reactions were recorded.

2.1.5. From Day 10 to 22 : Rest period
A period of 12 days without treatment is included in the test.

2.1.6. Day 21: Clipping and shaving of the flanks.

2.2. CHALLENGE PHASE

2.2.1. Day 22: Challenge application
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 (CONTROL and TREATED GROUPS) received the same treatment:
- an application of 0.5 ml of the M.N.I.C. of the test substance on the posterior right flank,
- 0.5 ml of the vehicle on the posterior left flank.

This application was performed using a 1 ml plastic syringe (0.01 ml graduations, Terumo: C.M.L., 77140 Nemours, France). The test substance and vehicle were prepared on a dry gauze pad (Semes France, 54183 Heillecourt, France), then applied to a 4 cm2 (2 cm x 2 cm) clipped area of the skin. The gauze pad was held in contact with the skin for 24 hours by means of an occlusive, hypoallergenic dressing (Laboratoires de Pansements et d'Hygiene, 21300 Chenove, France) and an adhesive anallergenic waterproof plaster (Laboratoire des Professions Medicales, 92240 Malakoff, France). No residual test substance was observed at removal of the dressing.Application

2.2.2. Day 24: First scoring
2.2.3. Day 25: Second scoring, sacrifice of the animals

Twenty-four (Day 24) and 48 hours (Day 25) after removal of the dressing from the challenge application site, the both flanks of the treated and control animals were observed in order to evaluate cutaneous reactions.

3. SCORING OF CUTANEOUS REACTIONS

Twenty-four and 48 hours after removal of the dressing from the challenge application site, the 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
"0"_ No erythema,
"1"_ Very slight erythema (barely perceptible),
"2"_ Well-defined erythema,
"3"_ Moderate to severe erythema,
"4"_ Severe erythema (beet redness) to slight eschar formation (injuries in depth).

Oedema formation
"0"_ No oedema ,
"1"_ Very slight oedema (barely perceptible),
"2"_ Slight oedema (visible swelling with well-defined edges),
"3"_ Moderate oedema (visible swelling raised more than 1 millimetre),
"4"_ Severe oedema (visible swelling raised more than 1 millimetre and extending beyond the area of exposure).
Any other lesions were noted.

OTHER OBSERVATIONS

BODY WEIGHT
The animals were weighed individually on the day of allocation into the groups, on the first day of the study (day 1), then on days 8, 15 and 25.

PATHOLOGY
A macroscopic examination of the main organs was performed on the animal found dead during the study. On day 25, after the 48-hour observation period, the surviving animals were killed by CO2 inhalation in excess.
Challenge controls:
Negative control: 10 animals (5 males and 5 females)
Positive control substance(s):
yes
Remarks:
Dinitro 2,4 chlorobenzene ???????

Study design: in vivo (LLNA)

Vehicle:
not specified
Concentration:
Not applicable
Not tested
No. of animals per dose:
Not applicable
Not tested
Details on study design:
Not applicable
Not tested
Positive control substance(s):
other: Dinitro 2,4 chlorobenzene ???????
Statistics:
not specified

Results and discussion

Positive control results:
Dinitro 2,4 chlorobenzene was used to check the sensitivity of Dunkin-Hartley Guinea-pigs (Centre d'elevage Lebeau).
Under the experimental conditions and according to the Magnusson and Kligman method, Dinitro 2,4 chlorobenzene at a concentration of 1% (w/w) induced positive skin sensitization reactions in 95% of the guinea-pigs.

Positive control was not tested in the sameexperiment than methylal cosmetique.
Données page 33 à encoder si nécessaire... mais témoin

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
100%
No. with + reactions:
0
Total no. in group:
9
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 9.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
100%
No. with + reactions:
0
Total no. in group:
9
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 9.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
100%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
Dryness of the skin on 3/20 animals
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: Dryness of the skin on 3/20 animals.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
100%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 20.0.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Not applicable Not tested
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Not applicable Not tested

Any other information on results incl. tables

Not applicable

Not tested

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
No cutaneous reactions were observed 24 and 48 hours after removal of the dressing of the challenge cutaneous application of the test substance.It was only noted a moderate dryness of the skin 24 hours after removal of the dressing for 3/20 animals.

Under the experimental conditions and according to the maximization method established by Magnusson and Kligman, no cutaneous reactions attributable to the sensitization potential of the test substance, METHYLAL COSMETIQUE, were observed in guinea-pigs.

According to Council Directive 93/21/E.E.C. (27th April 1993) adapting to technical progress for the eighteenth time Council Directive 67/548/E.E.C. on the approximation of the laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances, concerning the potential sensitizing effect by skin contact, the test substance should not be classified.
Executive summary:

Thirty guinea pigs (15 males and 15 females) of Dunkin-Hartley strain, supplied by Centre d'Elevage Lebeau, Gambais, France were exposed to the test item after an acclimatisation period of at least 5 days.

Preliminary tests were performed to determine the dose in the main study. Concentration chosen for the main study was 5% (w/w) in paraffin oil as it induced only a moderate skin irritation after testing on 2 animals (1 male and 1 female). Concentration chosen for the main study was 100% as no erythema and oedema reactions were induced on the right and left flanks of 2 animals (1 male and 1 female) 24 and 48 hours after removal of the dressing.

Animals were split in two groups for the main study: 10 animals (5 males and 5 females) for negative control group and 20 animals (10 males and 10 females) were treated with the test item. The sensitization potential of the test substance was evaluated after a 10-day induction period during which time the animals were treated with paraffin oil (control group) or the test substance (treated group). On day 1, in presence of Freund's complete adjuvant, 0.1 ml of a mixture containing the test substance at a concentration of 5% (w/w) in the vehicle was administered by intradermal route. On day 8, 0.5 ml of the test substance in its original form was applied by cutaneous route during 48 hours by means of an occlusive dressing. After a period of 12 days without treatment, cutaneous applications of 0.5 ml of the vehicle (left flank) and 0.5 ml of the test substance in its original form (right flank) were administered to all

animals. The test substance and the vehicle were prepared on a dry gauze pad then applied to the skin and held in place for 24 hours by means of an occlusive dressing. Cutaneous reactions on the challenge application sites were then evaluated 24 and 48 hours after removal of the dressing. After the final scoring period, the animals were killed. Due to the absence of cutaneous reactions, no skin samples were taken from the challenge application sites from all animals.

No cutaneous reactions were observed 24 and 48 hours after removal of the dressing of the challenge cutaneous application of the test substance. It was only noted a moderate dryness of the skin 24 hours after removal of the dressing for 3/20 animals.

Under the experimental conditions and according to the maximization method established by Magnusson and Kligman, no cutaneous reactions attributable to the sensitization potential of the test substance, METHYLAL COSMETIQUE, were observed in guinea-pigs.

According to Council Directive 93/21/E.E.C. (27th April 1993) adapting to technical progress for the eighteenth time Council Directive 67/548/E.E.C. on the approximation of the laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances, concerning the potential sensitizing effect by skin contact, the test substance should not be classified.