1,4-Benzenediamine, 2-(methoxymethyl)-

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation / corrosion

Remarks:

other: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Dec. 10, 2008 to Feb. 09, 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study well documented, comparable to guideline, GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

according to UK, OECD and EC principles of GLP

Test material

Test material information

Constituent 1

Reference substance name:

2-(methoxymethyl)benzene-1,4-diamine

EC Number:

679-526-3

Cas Number:

337906-36-2

Molecular formula:

C8H12N2O

IUPAC Name:

2-(methoxymethyl)benzene-1,4-diamine

Constituent 2

Reference substance name:

2-Methoxymethyl-p-phenylenediamine

IUPAC Name:

2-Methoxymethyl-p-phenylenediamine

Constituent 3

Reference substance name:

MBB

IUPAC Name:

MBB

Details on test material:

- Name of test material: 2-Methoxymethyl-p-phenylenediamine, MBB, WR804025 (Code # A003825)  
- TSIN: 804025
- Substance type: Pure active substance
- Physical state: White crystalline powder 
- Stability under test conditions: The substance is considered to be stable for more than 5 years if stored in dryness and darkness at room temperature- Stability in solution: The solutions of the test substance in DMSO (10% solution w/v) and water (10% solution, w/v) can be regarded as stable for 3 d.  
- Storage condition of test material: At room temperature, in dark
- Solubility: Solubility in different solvents is as follows:  
>10 weight% in water (pH 9.4)
>10 weight% in DMSO

Test system

Vehicle:

water

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 10 mg for pre-test; 10 µL of 1.83 and 6.1% test solution and 10±2 mg of neat test substance for main test 
- Concentration: Pre-test: Neat test substance; Main test: 1.83% and 6.1% w/v in sterile water and neat test substance

VEHICLE (Sterile water)
- Amount applied: 10 µL
- Concentration: As such
- Lot/batch no.: Laboratoire AGUETTANT; Batch # 3003930
- Expiry: Oct. 31, 2009

POSITIVE CONTROL (Sodium Dodecyl Sulphate):
- Amount applied: 10 µL 
- Concentration: 5.0% w/v solution in sterile water 
- Lot/batch no.: Sigma Aldrich, Article Number L4509-100G; Batch # 046K0085
- Expiry: Oct. 21, 2009

Duration of treatment / exposure:

15 min

Details on study design:

TEST DESIGN:
- Test method: Colorimetric MTT reduction assay  
- Model used: The EpiSkin Reconstituted Human Epidermal (RHE) model obtained from SkinEthic Laboratories, Nice, France consisting of adult human derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. This epidermal model consists of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
 
TEST PROCEDURE:
- Preparation of MTT stock solution: 3.0 mg/mL of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) obtained from Sigma Aldrich was prepared by adding 60 mg MTT to 20 mL of PBS (Phosphate buffered saline- tissue rinsing solution) containing Ca++Mg++ and stored in a fridge. The concentrate was diluted to 0.3 mg/mL with assay medium when required.
- Pre-test (Assessment of Direct Test Material Reduction of MTT): A pre-test was performed to assess the ability of test substance to reduce MTT to formazan directly thus mimicking dehydrogenase activity of the cellular mitochondria of viable cells. 10 mg of the solid test substance was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in maintenance medium and incubated in the dark at 37°C, 5% CO2 for 3 h. Untreated MTT solution was used as a control. If the MTT solution color turned blue/purple relative to the control, the test substance was considered to have the ability to reduce the MTT. Since the test substance was found to be capable of directly reducing MTT, in addition to the normal pre-test procedure, a functional check with 3 water killed (non-viable) tissues was employed for each test concentration. These water killed tissues (prepared by placing untreated EPISKIN tissues in 12-well plates containing 2 mL of sterile water in each well) possess no metabolic activity but absorb and bind the test substance like viable tissues. 3 water killed tissues remained untreated.  

- SkinEthic RHE tissues: Before removal from the transport plate, each tissue was inspected for any air bubbles between the agarose gel and the insert. Each epidermis unit was aseptically removed and transferred into pre labeled 12-well plates containing 2 mL of maintenance medium, pre warmed to approx. 37°C. The tissues were placed into an incubator (37°C, 5% CO2) for at least 24 h.
 
MAIN TEST (MTT viability assay):  
- Number of tissues treated with test material: 9 tissues per test concentration (3 each for relative tissue viability analysis, color correction procedure and histology)
- Number of tissues treated with negative control: 9 (3 each for relative tissue viability analysis, color correction procedure and histology)
- Number of tissues treated with positive control: 6 (3 each for relative tissue viability and histology)
- Application of test material: Test materials were applied directly to the culture surface, at air interface, so that undiluted and/or end use dilutions can be tested directly. Tissues were treated with test material (at 1.83 and 6.1% in sterile water and neat test material)/positive control/negative control. Volume of test material administered was 10 mg ± 2 mg (following wetting of the tissue surface with 5 μL of sterile water) for neat and 10 μL for diluted test material. To aid the contact between positive control and epidermis, the SDS solution was spread over the entire surface of the epidermis using a pipette tip. The plates were kept in the biological safety cabinet at room temperature for approx. 15 min. Each test substance concentration was applied to 3 non-viable tissues in addition to the above mentioned step and 3 non-viable tissues remained untreated.

- Washing: At the end of exposure period, each tissue insert was removed from the well using forceps and rinsed using a wash bottle containing PBS without Ca++ and Mg++. Each tissue was then totally submerged in 150 mL PBS and gently shaken to remove residual test substance (three times). Finally each tissue was rinsed once again using the wash bottle.

QUALITATIVE EVALUATION OF TISSUE VIABILITY: Following rinsing the tissues were transferred to a 12-well plate containing 2 mL of maintenance medium in each well and incubated at 37°C, 5% CO2 for 42 ± 2 h. After 42 h each plate was placed into a plate shaker for 15±2 min to homogenize the released mediators in the maintenance medium. Triplicate positive, negative control and test substance treated and untreated tissue at each concentration were transferred to a 12-well plate containing 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The tissues were incubated for 3 h at 37°C, 5% CO2 in air. At the end of the 3 h incubation period, each tissue was blotted on absorbent paper to dry. The tissues (excluding non-viable tissues), were visually examined and the degree of MTT staining evaluated by MTT visual scoring system.

MTT COLOR CORRECTION: Since it was possible that colored test substance may stain the tissues, three of the test material treated tissues and negative control tissues were retained for MTT correction purposes.
 
QUANTITATIVE TISSUE VIABILITY ANALYSIS: Following qualitative evaluation of tissue viability, a total biopsy of the epidermis was made using the EpiSkin biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts were placed into labeled 1.5 mL micro tubes containing 500 µL of acidified isopropanol. Each tube was plugged, mixed thoroughly in vortex mixture and refrigerated at 1-10°C until Day 6 of experiment. 200 µL duplicate samples for MTT treated groups and color correction groups were transferred to 96-well plates. 200 µL of acidified isopropanol alone were added to the two wells designated as ‘blanks’. All wells were examined and any air bubbles removed. The optical density was measured at 540 nm (without a reference filter) using the Anthos 2001 microplate reader. 

HISTOLOGY: Histological processing and interpretation was performed by Histologix Ltd, BioCity, Pennyfoot Street, Nottingham, NG1 1GF, UK on the tissue to provide supplementary information on the skin irritation potential of the test substance. Evidence of cellular damage or disruption of the tissue morphology was used to confirm the occurrence of reduced tissue viability, as determined by the MTT assay. Details on histological score and interpretation are provided in the study report.
 
CLASSIFICATION OF IRRITATION POTENTIAL: The test material was classified based on MTT viability analysis in a prediction model:

Relative MTT true viability (% negative control) ≤ 50: Irritant (I)R38; Category 2
Relative MTT true viability (% negative control) > 50: Non - irritant (NI); Not classified

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

other: Relative tissue viability based on MTT assay (%)

Basis:

mean

Time point:

other: 15 min

Score:

109.7

Remarks on result:

other: 1.83% test solution in sterile water

Results 2

Irritation parameter:

other: Relative tissue viability based on MTT assay (%)

Basis:

mean

Time point:

other: 15 min

Score:

107.8

Remarks on result:

other: 6.1% test solution in sterile water

Results 3

Irritation parameter:

other: Relative tissue viability based on MTT assay (%) based on MTT assay (%)

Basis:

mean

Time point:

other: 15 min

Score:

97.9

Remarks on result:

other: Neat test substance

Irritant / corrosive response data:

The skin irritancy of the test substance was evaluated by MTT true viability prediction model (%MTT viability > 50% for non-irritancy):
 
- The 1.83, 6.1% w/v solution in sterile water and the neat concentrations of test substance did not induce cytotoxicity in the MTT assay indicative of skin irritation. (Non-irritant)

Other effects:

- Direct reduction of MTT by test substance relative to negative control: The test substance was considered to have the ability to directly reduce MTT (darkening of the MTT solution when directly mixed with the test substance ).

However, the test substance did not cause interference with the MTT test (as determined by the water-killed (non-viable) tissues)
- MTT color correction assay: The color of the test substance was considered not to have caused interference with the MTT test.
- Histology: Negative control samples produced no significant changes in the epithelial structure and no degenerative changes were observed. Complete necrosis of all layers of each skin sample resulted from exposure to the positive control samples.

The test substance induced no significant changes in epithelial structure and no evidence of degeneration at any of the concentrations tested, confirming the absence of cytotoxicity in the MTT assay. The focal loss of granules seen for one sample exposed to the 6.1% concentration of test substance was unlikely to be related to the test substance.

Details on histological results are provided in study report.

Any other information on results incl. tables

Quality criteria: The assay establishes the acceptance criterion for an acceptable test if the mean OD₅₄₀ of the negative control tissues is ≥ 0.6 and if the tissue viability of positive control is ≤ 40% relative to the negative control treated tissue. In this study, the quality criteria required for acceptance of results in the test were satisfied.

Qualitative evaluation of tissue viability: The visual evaluation of tissue viability (MTT loaded tissues and color correction tissues) for the test material and controls are provided in the table below:

Table 1: Qualitative evaluation of tissue viability of test material, negative control and positive control (Study # 65229)

Treatment	MTT score			Color Observed (MTT Correction Tissues)
	Tissue 1	Tissue 2	Tissue 3	Tissue 1	Tissue 2	Tissue 3
1.83% w/v test material in sterile water	-	-	-	white	white	white
6.1% w/v test material in sterile water	-	-	-	white	white	white
Neat test material	-	-	-	white	white	white
Negative Control (Sterile water)	-	-	-	white	white	white
Positive Control (5% Sodium dodecyl sulfate)	++	++	++	NA	NA	NA

MTT Visual Scoring Scheme

-    = Blue tissue (viable)

+  = Blue/white tissue (semi-viable)

++ = Tissue completely white (dead)

NA = Not Applicable

 

Quantitative evaluation of tissue viability: The results of measurement of cytotoxicity (tissue viability) by means of the colorimetric MTT reduction assay for the test substance and controls are provided in the table below:

Table 2: Mean and color corrected Mean OD₅₄₀ values and the relative mean tissue viability of the test material and controls (Study # 65229) 

Treatment	Mean OD540± SD	Relative mean viability (%)	± SD of % viability
1.83% w/v test material in sterile water	0.881 ± 0.058	109.7	± 7.2
6.1% w/v test material in sterile water	0.866 ± 0.007	107.8	± 0.9
Neat test material	0.786 ± 0.026	97.9	± 3.2
Negative Control (Sterile water)	0.803 ± 0.052	100*	± 6.5
Positive Control (5% Sodium dodecyl sulfate)	0.041 ± 0.008	5.1	± 1.0

SD = Standard Deviation

* = The mean viability of the negative control tissues was set at 100%

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: expert judgment

Conclusions:

2 -Methoxymethyl-p-phenylenediamine (MBB) was classified as non-irritant to the SkinEthic Reconstituted Human Epidermal skin when applied at 1.83 and 6.1% w/v (in sterile water) and neat (% MTT viability >50%) according to the prediction model based on MTT viability analysis.

Executive summary:

The in-vitro skin irritation potential of 2-Methoxymethyl-p-phenylenediamine (MBB) was determined following methods comparable to OECD guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method).

The skin irritation potential of the test substance was determined using SkinEthic Reconstituted Human Epidermal (RHE) Model with a prediction model based on the colorimetric MTT viability assay.

The tissues were exposed to the following test concentration for a exposure period of 15 min followed by a 42±1 h post treatment incubation period:

Test material concentration: 1.83 and 6.1% w/v in sterile water and neat.

9 tissues per test concentration/negative control (sterile water) were treated. 6 tissues treated with positive control (5% Sodium Dodecyl Sulphate) were used in this study.

In this study two endpoints, cytotoxicity in the MTT assay, expressed as percentage viability of treated cultures in comparison to negative controls, as well as morphological changes identified by histological examination at the end of the treatment, were evaluated, for skin irritation potential of test substance. Tissue viability was analyzed by visual examination and evaluation of the degree of MTT stain and the color of tissue (qualitative) and the viability was measured by the colorimetric MTT reduction assay (quantitative) based on a prediction model (% MTT viability >50%: Non irritant).

A decrease in MTT reduction capacity and changes in tissue morphology were used as indicators of potential irritancy.

Compared to the corresponding negative control and solvent control, the color-corrected MTT relative viabilities of MBB treated tissues after a 15 min exposure period and a 42 ± 1 h post-treatment incubation were as follows:

1.83% w/v solution in sterile water: 109.7%

6.1% w/v solution in sterile water: 107.8%

Neat test substance: 97.9%

The test substance was considered to have the ability to directly reduce MTT (darkening of the MTT solution when directly mixed with the test substance).However, the test substance did not cause interference with the MTT test (practically no absorbance measured in the treated killed tissues compared to the untreated killed tissues). The color of the test substance was considered not to have caused interference with the MTT test.

Histological evaluation revealed that the test substance induced no epidermal effects when applied at 1.83% (score 0), 6.1% (score 0/1) and neat (score 0) compared to the negative control (score 0).

The 1.83%, 6.1% (in sterile water) and neat concentrations of MBB did not induce cytotoxicity in the MTT assay indicative of skin irritation according to the prediction model.

2 -Methoxymethyl-p-phenylenediamine (MBB) was classified as non-irritant to the SkinEthic Reconstituted Human Epidermal skin when applied at 1.83 and 6.1% w/v (in sterile water) and neat (% MTT viability >50%) according to the prediction model based on MTT viability analysis.

This in-vitro acute skin irritation test is classified as acceptable, and satisfies the guideline requirements of the OECD 439 method.