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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
other: the study was performed with the sulfate salt of 1,4-diamino-2-methoxymethylbenzene
Adequacy of study:
key study
Study period:
From June 29, 2005 to Nov. 7, 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study well documented, followed guideline, GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes
Remarks:
OECD Principles of Good Laboratory Practice
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Reference substance name:
337906-37-3
Cas Number:
337906-37-3
IUPAC Name:
337906-37-3
Constituent 2
Reference substance name:
-
EC Number:
474-270-7
EC Name:
-
IUPAC Name:
474-270-7
Constituent 3
Reference substance name:
1,4-diamino-2-methoxymethyl-benzene sulfate (1:1)
IUPAC Name:
1,4-diamino-2-methoxymethyl-benzene sulfate (1:1)
Details on test material:
- Name of test material: 1,4-diamino-2-methoxymethyl-benzene sulfate (1:1); Methoxymethyl-PPD-Sulfat (Code # A012220)
- TSIN: 801337
- Molecular formula: C8H12N2O × H2SO4
- Molecular weight: 250.28
- Substance type: Pure active substance
- Physical state: Cream- rosy powder
- Stability under test conditions: Test substance was considered to be stable for more than 5 yrs when stored in dryness and darkness.
- Stability in solutions: The test substance was found to have excellent stability as 10 % solution, w/v, pH 5.1 in the presence of L(+)-ascorbic acid (acting as antioxidant substance).
- Storage condition of test material: At room temperature, protected from light and moisture
- Solubility: The solubility of test substance in different solvents is a follows:
Water: > 10 weight% (pH:1.5)
Acetone: 0.03 weight%
Acetone/water (1:1): 1.7 weight%
DMSO: >10 weight%

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Centre d'Elevage Janvier, route des Chenes Secs, 53940 Le Genest Saint Isle, France.
- Age at study initiation: Approx. 9 weeks
- Weight at study initiation: 19-24 g. The weight variation of the animals was minimal and did not exceed 20 % of the mean weight.
- Housing: Animals were housed in groups of 5 of the same dose group in plastic cages (265 x 160 x 130 mm). Dust-free sawdust made from spruce tree wood was used for bedding. The bedding was analysed at least twice a year for chemical and bacterial contaminants
- Diet: Pelleted complete mouse diet, ad libitum. Diet was sterilized by irradiation and analysed for chemical and bacterial contaminants.
- Water: Softened and filtered (0.2 µm) mains drinking water, ad libitum (via bottles). Water was analysed at least once a year for chemical contaminants and at least twice a year for bacterial contaminants by Laboratoire Sante Environnement Hygiene de Lyon.
- Acclimation period: 9 d (between animal arrival and the start of treatment). Allocation of animals to treatment groups was performed during the acclimatization period by computer-generated randomization.
No known contaminants were present in the bedding, the diet or water at levels which might have interfered with achieving the objective of the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Humidity: 55±15 %
- Air changes: Minimum 15 air changes/hour
- Photoperiod: 12 hours light (artificial)/12 hours dark
Environmental conditions were within the target ranges throughout the study.

IN-LIFE DATES: From: July. 7, 2005 To: July 12, 2005

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
Dose concentration: 0, 5, 15, 50 and 150 mg/mL
Positive control (25% HCA in DMSO): 250 mg/mL
No. of animals per dose:
Five female animals/dose group
Details on study design:
RANGE FINDING TESTS: No range-finding study was performed

MAIN STUDYANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay (LLNA)
- Criteria used to consider a positive response: A positive response was defined as a 3-fold or greater increase in isotope incorporation relative to the vehicle.

TREATMENT PREPARATION AND ADMINISTRATION:
- Treatment preparation: The test substance was prepared as a solution in the vehicle at the following concentrations: 0.5 %, 1.5 %, 5.0 % and 15.0 % (w/v). The positive control was prepared as a 25.0 % (v/v) solution in DMSO. The formulations were prepared on each treatment day and used within 5 h after preparation.
- Rationale for the choice of test material concentrations: The concentrations were chosen by the Study Sponsor on the basis of the maximum solubility in the vehicle.
- Administration of the test preparations: On Days 0, 1 and 2, the animals received 25 µL of the test formulation or vehicle on the dorsal surface of each ear. A hair dryer was used for 1 minute to dry the application sites.

OBSERVATIONS:
- Morbidity/mortality: All animals were observed at least once daily.
- Clinical signs: The animals were observed daily. On treatment days, the animals were examined before and at least once after dosing to detect any clinical signs or reaction to treatment (especially on the application sites).
- Body weight: All animals were weighed on Days -1 and 5
- Evaluation of cell proliferation: On Day 5, 250 µL of phosphate buffered saline containing 21.2 µCi of [3H] methyl thymidine was injected intravenously (using Harvard type P44 infusion pumps) to each experimental mouse. 5 h later, the animals were sacrificed by carbon dioxide inhalation and the draining auricular lymph nodes were collected and weighed. A single cell suspension of lymph node cells for each animal was prepared. Cells were washed twice with PBS and precipitated with ice cold 5 % trichloroacetic acid (TCA). Approx. 18 h later the pellets were resuspended in 1 mL of TCA and transferred into the scintillation cocktail. [3H]-methyl thymidine incorporation was measured by liquid scintillation counting in a TRI-CARB 2700TR counter (Packard). The Instrument Performance Assessment (IPA) was tested by counting special background, (3)H and (14C) IPAstandards
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
No statistical analyses were performed.

Results and discussion

Positive control results:
Mean DPM: 3476±1609
Mean Stimulation Indices (SI): 7.2 (The mean SI is greater than 3 thus validated the experimental conditions of OECD 429)
EC3 value: The EC3 value was not calculated (single value).
Mean lymph node weight: The ratio of mean lymph node weights relative to the vehicle was 2.0

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
The mean stimulation indices at the concentrations of 0.5 %, 1.5 %, 5 % and 15 % in DMSO were 1.1, 1.2, 2.2 and 6, respectively. The response was thus considered to be positive. The EC3 value of the free base (MBB) was calculated from the sulfate salt by using the conversion factor to account for the different molecular weight. EC3 value SO4-salt: 7.11% 7.11% = 7.11 g/100 ml x 0.61 (conversion factor) = 4.3 g /100 ml = 4.3% EC3 value free base: 4.3%. Based on this calculation, 2-Methoxy-Methyl-phenylenediamine is considered a moderate skin sensitiser according to the relative skin sensitisation potency classification scheme published in the ECETOC technical report on contact sensitization.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM: 529, 595, 1077 and 2875 at 0.5, 1.5, 5, and 15% test substance concentration respectively.

Any other information on results incl. tables

Mortality: No mortality was observed during the study.

Clinical signs: Oily fur around the ears was noted in group 6 animals after treatment on Day 0. Orange traces were noted in the bedding of Group 5 and 6 animals from Days 1 to 3. The color was stronger in the sixth than in the fifth group. There were no clinical signs in the other treated animals during the study.

Body weight: There were no treatment-related effects on body weight. Individual animal body weight data is provided in the study report.

Ratio of mean lymph node weights relative to the vehicle: The ratio of mean lymph node weights relative to the vehicle was 0.9, 0.9, 1.1 and 1.2 at the concentrations of 0.5%, 1.5%, 5.0% and 15.0%, respectively.

Applicant's summary and conclusion

Interpretation of results:
other: moderate skin sensitiser
Remarks:
Criteria used for interpretation of results: expert judgment
Conclusions:
2-METHOXY-METHYL-P-PHENYLENEDIAMINE SULFATE is a moderate skin sensitizer under the defined experimental conditions, with a calculated EC3 value of 7.11 %.
Executive summary:

The skin sensitizing potentialof 2 -Methoxymethyl-p-phenylenediamine sulphate (Methoxymethyl-PPD-Sulfat) was determined following OECD 429 guideline (Skin Sensitisation: Local Lymph Node Assay).

A total of 30CBA/J mice (approx. 9 weeks old)weighing 19 -24 g were acclimated for 9 days for this study. The feed used was Pelleted complete mouse diet (ad libitum). Animals were housed in a group of 5 animals in plastic cages containing dust-free sawdust as bedding material. Animals were maintained under standard laboratory conditions (temperature: 22± 2°C, humidity: 55±15 %; artificial light: 12 -hour cycle; air changes: 15 air changes per hour). 

The test substance was prepared at different test concentrations (i.e. 0, 5, 15, 50 and 150 mg/L) in the vehicle (DMSO). The vehicle group received DMSO. Positive control animals received 25% alpha-Hexylcinnamic aldehyde (HCA) in vehicle. The animals received 25 µL of the appropriate formulations or vehicle on dorsal surface of each ear on Days 0 to 2. On Day 5, 250 µL of phosphate buffered saline containing 21.2 µCi of [3H] methyl thymidine was injected intravenously to each experimental mouse. 5 h later, the animals were sacrificed by carbon dioxide inhalation and the draining auricular lymph node were collected and weighed. A single cell suspension of lymph node cells for each animal was prepared. Cells were precipitated with ice cold 5% trichloroacetic acid. Approx. 18 h later the pellets were resuspended in 1 mL of TCA and transferred into the scintillation cocktail. [3H]-methyl thymidine incorporation was measured by liquid scintillation counting in a TRI-CARB 2700TR counter (Packard).

The positive control alpha-Hexylcinnamic aldehyde (HCA) at a concentration of 25.0% in DMSO induced a 7.2 fold increase in isotope incorporation in the draining auricular lymph nodes relative to the vehicle. The response was thus considered to be positive.

2 -Methoxymethyl-p-phenylenediamine sulphate was positive in the local lymph node assay, since there was more than a 3 -fold increase in isotope incorporation in the draining auricular lymph nodes relative to the vehicle control group. The mean stimulation indices were 1.1, 1.2, 2.2 and 6 at the concentrations of 0.5%, 1.5% 5% and 15%, respectively. The EC3 value of the free base (MBB) was calculated from the sulfate salt by using the conversion factor to account for the difference in molecular weight.

EC3 value SO4 -salt: 7.11%

7.11% = 7.11 g/100 ml x 0.61 (conversion factor) = 4.3 g /100 ml = 4.3%

EC3 value free base (MBB): 4.3%.

Based on this calculation, 2 -Methoxymethyl-p-phenylenediamine considered a moderate skin sensitiser according to the relative skin sensitisation potency classification scheme published in the ECETOC technical report on contact sensitisation.

2-METHOXY-METHYL-P-PHENYLENEDIAMINE SULFATE is a moderate skin sensitizer under the defined experimental conditions, with a calculated EC3 value of 7.11 %.

This skin sensitizationtestis classified as acceptable, and satisfies the guideline requirements of the OECD 429 method.

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