1,1'-(chlorophenylmethylene)bis[4-methoxybenzene]

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-01-09 to 2018-03-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

Adopted 29th July, 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1,1'-(chlorophenylmethylene)bis[4-methoxybenzene]

EC Number:

255-002-6

EC Name:

1,1'-(chlorophenylmethylene)bis[4-methoxybenzene]

Cas Number:

40615-36-9

Molecular formula:

C21H19ClO2

IUPAC Name:

1-[chloro(4-methoxyphenyl)phenylmethyl]-4-methoxybenzene

Test material form:

solid

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: 25 mg of the test item were applied directly atop the EpiDerm tissue using an application spoon avoiding compression of the test item. To ensure good contact with the skin the test item was moistened with 25 μL H2O. The test item was spread to match size of the tissue.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The EpiDerm Skin Model is a well-established organotypic, three-dimensional model of the human epidermis and is used for in vitro experiments since many years. It is known for its similarity to human skin.

Vehicle:

water

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Reconstructed Human Epidermal Model EpiDerm™ (EPI-200-SIT, MatTek)
- Tissue batch number(s): 25876

EpiDerm Kit:
The EpiDerm tissues were provided as kits (MatTek), consisting of the following components relevant for this study:
1x sealed plate containing e.g. 24 reconstructed epidermis units (area: 0.63 cm^2); each reconstructed epidermis is attached to a cell culture insert and maintained on nutritive agar for transport (Lot: 25876)
2x 24-well plates
4x 6-well plates
1x bottle of assay medium (DMEM-based medium; Lot: 012518ALB)
1x bottle of DPBS Rinse Solution (Lot: 112117ACC)
25 pieces Nylon Mesh circles (8 mm diameter, 200 µm pore)


TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C
- Temperature of post-treatment incubation (if applicable): 37 ± 1 °C


REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps:
- 3 min experiment: After 3 min of application, the first insert was removed from the 6-well plate with forceps. Using a wash bottle, the tissue was gently rinsed about 20 times with PBS (phosphate buffered saline) to remove any residual test item. Excess PBS was removed by gently shaking the insert and blotting bottom with blotting paper.
- 60 min experiment: after 60 min application, the first insert was removed from the 6-well plate with forceps. Using a wash bottle, the tissue was gently rinsed about 20 times with PBS to remove any residual test item. Excess PBS was removed by gently shaking the insert and blotting bottom with blotting paper
- 3 min and 60 min experiment: after the 3 h MTT incubation period the MTT solution was aspirated. The wells were refilled with PBS and the PBS was aspirated. The rinsing was repeated twice and the tissues were dried.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Wavelength: 570 nm
- Filter bandwidth: ± 30 nm

NUMBER OF REPLICATE TISSUES: 2

PREDICTION MODEL / DECISION CRITERIA
See Table 1 in box 'Any other information on materials and methods incl. tables'

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg (+25 µL H2O)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): 8 N

Duration of treatment / exposure:

3 and 60 min

Duration of post-treatment incubation (if applicable):

3 h

Number of replicates:

2

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

3 min exposure

Run / experiment:

Mean of two replicates

Value:

103.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

60 min exposure

Run / experiment:

Mean of two replicates

Value:

9.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: not specified
- Direct-MTT reduction: The mixture of 25 mg test item per 1 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT is determined to be 0%.
- Colour interference with MTT: The mixture of 25 mg test item per 300 μL Aqua dest. showed no colouring as compared to the solvent. Therefore NSC is determined to be 0%. The mixture of 25 mg test item per 300 μL isopropanol showed colouring as compared to the solvent but did not absorb light in the range of 570 ± 30 nm. Therefore NSC is determined to be 0%.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative & positive control: The controls confirmed the validity of the study. The mean OD570nm of the two negative control tissues was ≥ 0.8 and ≤ 2.8 for each exposure period. The mean relative tissue viability (% negative control) of the positive control was < 15% (2.5%) after 60 min treatment. The coefficient of variation (CV) (in the range of 20 – 100% viability) of replicate tissues of all dose groups was ≤ 30% (0.3% - 1.3%).

For detailed results see Table 2 and 3 in box "Any other information on results incl. tables".

Any other information on results incl. tables

Table 2: Results of 3 min experiment

Name	Negative Control		Test Item		Positive Control
Tissue	1	2	1	2	1	2
Absolute OD570	1.796	1.783	1.798	1.924	0.076	0.097
	1.790	1.780	1.755	1.919	0.079	0.098
	1.773	1.772	1.766	1.918	0.079	0.099
OD570- Blank Corrected	1.752	1.738	1.753	1.879	0.032	0.053
	1.745	1.735	1.710	1.874	0.034	0.054
	1.728	1.728	1.721	1.873	0.034	0.054
Mean OD570of 3 Aliquots (Blank Corrected)	1.742	1.734	1.728	1.875	0.033	0.053
SD OD570 of 3 Aliquots	0.012	0.006	0.022	0.003	0.002	0.001
Total Mean OD570of 2 Replicate Tissues (Blank Corrected)	1.738*		1.802		0.043
SD OD570 of 2 Replicate Tissues	0.005		0.104		0.014
Mean Relative Tissue Viability [%]	100.0		103.7		2.5
Coefficient Of Variation [%]^***	0.3		5.8		32.5

* corrected mean OD570 of the negative control corresponds to 100% absolute tissue viability.

*** coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is ≤ 30%.

Table 3: Results of 60 min experiment

Name	Negative Control		Test Item		Positive Control
Tissue	1	2	1	2	1	2
Absolute OD570	1.814	1.777	0.164	0.246	0.089	0.088
	1.819	1.793	0.167	0.248	0.092	0.089
	1.843	1.807	0.166	0.245	0.088	0.088
OD570- Blank Corrected	1.769	1.732	0.120	0.201	0.044	0.044
	1.774	1.749	0.122	0.203	0.047	0.045
	1.798	1.763	0.121	0.200	0.044	0.043
Mean OD570of 3 Aliquots (Blank Corrected)	1.781	1.748	0.121	0.201	0.045	0.044
SD OD570 of 3 Aliquots	0.015	0.015	0.001	0.002	0.002	0.001
Total Mean OD570of 2 Replicate Tissues (Blank Corrected)	1.764*		0.161		0.044
SD OD570 of 2 Replicate Tissues	0.023		0.057		0.001
Mean Relative Tissue Viability [%]	100.0		9.1		2.5**
Coefficient Of Variation [%]^***	1.3		35.3		1.8

* corrected mean OD570 of the negative control corresponds to 100% absolute tissue viability.

** mean relative tissue viability of the 60 min positive control < 15%,

*** coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is ≤ 30%.

Applicant's summary and conclusion

Interpretation of results:

Category 1B (corrosive) based on GHS criteria

Conclusions:

In this study under the given conditions the test item showed corrosive effects. The mean relative tissue viability (% negative control) was reduced below 15% after 60 min treatment but not below 50% after 3 min treatment. 4,4’-Dimethoxytrityl chloride is therefore classified as “corrosive“ in accordance with UN GHS Criteria "Category 1B."

Executive summary:

In a primary skin corrosion study conducted according to guideline OECD 431, two EpiDerm tissues per dose group were exposed to 25 mg of 4,4’-Dimethoxytrityl chloride (99.73% purity) for 60 min and 3 min each and cytotoxicity was measured in comparison to the concurrent negative controls. Irritation was scored by the method of mean relative tissue viability. The test item showed corrosive effects. The mean relative tissue viability (% negative control) was reduced below 15% (9.1%) after 60 min treatment but not below 50% (103.7%) after 3 min treatment. 4,4’-Dimethoxytrityl chloride is therefore classified as “corrosive“ in accordance with UN GHS Criteria "Category 1B."