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Description of key information

The potential of 4,4’-Dimethoxytrityl chloride (target substance) to induce skin irritation/corrosion was evaluated in two suitable in vitro test methods (OECD 439 and OECD 431). Based on the results, 4,4’-Dimethoxytrityl chloride (99.73% purity) must be considered as corrosive to skin is therefore classified as Skin Corr. 1B, H314 in accordance with the CLP criteria. Thus, an eye irritation study did not need to be conducted in accordance with REACH Regulation (EC) No 1907/2006, Annex VII, column 2 of information requirement section 8.2.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017-10-18 to 2018-01-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted 28 July 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human, i.e. the epidermis.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™-Standard Model (EPI-200-SIT, MatTek)
- Tissue batch number(s): 25864

EpiDerm Kit:
The EpiDerm™ tissues were provided as kits (e.g. EPI-200-SIT, MatTek), consisting of the following components relevant for this study:
1x sealed 24-well plate containing e.g. 24 reconstructed epidermis units (area: 0.63 cm2); each reconstructed epidermis is attached to a cell culture insert and maintained on nutritive agar for transport (Lot No.: 25864)
2x 24-well plates
8x 6-well plates
1x bottle of assay medium (DMEM-based medium, Lot No.: 120717TMB)
1x bottle of DPBS Rinse Solution (Lot No.:092817MGKA)
1x 1 vial 5% SDS Solution (TC-SDS-5%)
25 pieces Nylon Mesh circles (8 mm diameter, 200 μm pore)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 +/-1 °C for the first 35 +/- 1 min, afterwards the plates were placed under the sterile flow until 60 +/- 1 min incubation time of the first dosed tissue was over.
- Temperature of post-treatment incubation (if applicable): 37 +/-1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: After 60 +/- 1 min incubation time the tissues were washed by filling and emptying the inserts 15 times with DPBS using a constant stream in about 1.5 cm distance from the tissue surface, staggered again in e.g. one-minute intervals. Subsequently, the inserts were completely submerged three times in 150 mL DPBS and shaken to remove rests of the test item. Finally, the inserts were rinsed once from the inside and the outside with sterile DPBS. Excess DPBS was removed by blotting the bottom with blotting paper.


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

NUMBER OF REPLICATE TISSUES: 3


PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after 15 minutes exposure and 42 h post-treatment incubation is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability after 15 minutes exposure and 42 h post-treatment incubation is greater than 50%.
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 DPBS

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30µl DPBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µl
- Concentration (if solution): 5% SDS solution
Duration of treatment / exposure:
60 ± 1 min
Duration of post-treatment incubation (if applicable):
42 h post-incubation
Number of replicates:
3 tissues per dose group
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean of three tissues
Value:
3.2
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, OD570 nm ≥ 0.8 and ≤ 2.8 (1.767)
- Acceptance criteria met for positive control: Yes, mean relative tissue viability of the three positive control tissues is ≤ 20% (2.9%)
- Acceptance criteria met for variability between replicate measurements: Yes, standard deviation (SD) of relative tissue viability obtained from each three concurrently tested tissues is ≤ 18%. (0.7-7.5%)

For detailed results see Table 1 in box "Any other information on results incl. tables".

Results of the Pre-Experiments:

The mixture of 25 mg test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT was determined to be 0%.

The mixture of 25 mg of the test item per 300 μL isopropanol showed colouring detectable by unaided eye-assessment. Therefore, the absorption of the chemical in isopropanol was measured in the range of 570 ± 30 nm.

Table 1: Result of the Test Item 4,4’-Dimethoxytrityl chloride

Name

Negative Control

Positive Control

Test Item

Tissue

1

2

3

1

2

3

1

2

3

Absolute OD570

1.911

1.656

1.728

0.101

0.083

0.104

0.126

0.093

0.089

1.911

1.672

1.722

0.103

0.086

0.107

0.127

0.096

0.088

OD570(Blank Corrected)

1.864

1.609

1.681

0.053

0.035

0.057

0.078

0.045

0.042

1.864

1.625

1.675

0.056

0.039

0.060

0.080

0.049

0.041

OD570(Blank Corrected)
- NSCliving Corrected

 -

 -

0.078

0.045

0.042

0.080

0.049

0.041

Mean OD570of the Duplicates (Blank Corrected)

1.864

1.617

1.678

0.055

0.037

0.059

0.079

0.047

0.042

Total Mean OD570of 3 Replicate Tissues (Blank Corrected)

1.719*

0.050

0.056

TODTT

 -

 -

0.056

SD OD570

0.129

0.012

0.020

Relative Tissue Viability [%]

108.4

94.0

97.6

3.2

2.2

3.4

4.6

2.7

2.4

Mean Relative Tissue Viability [%]

100.0

2.9**

3.2

Mean Relative Tissue Viability [%] -
NSCliving Corrected

 -

 -

3.2

SD Tissue Viability [%]***

7.5

0.7

1.2

CV [% Viabilities]

7.5

23.0

36.2

* Blank-corrected mean OD570 nmof the negative control corresponds to 100% absolute tissue viability.

**Mean relative tissue viability of the three positive control tissues is  20%.

*** Standard deviation (SD) obtained from the three concurrently tested tissues is ≤ 18%.

Interpretation of results:
study cannot be used for classification
Conclusions:
In conclusion, in this in vitro skin irritation study (OECD 439), 4,4’-Dimethoxytrityl chloride is considered to be irritant to the skin (UN GHS Category 2 or 1). Further testing is required.
Executive summary:

In a primary dermal irritation study conducted according to OECD guideline 439, the EpiDerm™-Model (EPI-200-SIT,) was topically exposed to 4,4’-Dimethoxytrityl chloride (99.73% purity) for 60 min and 42 h post incubation period. Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with DPBS. The mean relative tissue viability (% negative control) was ≤ 50% (3.2%) after 60 min treatment and 42 h post-incubation. Based on this result, 4,4’-Dimethoxytrityl chloride is considered to be irritating to the skin in accordance with UN GHS "Category 1 or 2" and further testing is required.

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
Reference:
Composition 0
Qualifier:
according to
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. certificate)
Test material information:
Composition 1
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.
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
Irritation / corrosion parameter:
% tissue viability
Remarks:
3 min exposure
Run / experiment:
Mean of two replicates
Value:
103.7
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Remarks:
60 min exposure
Run / experiment:
Mean of two replicates
Value:
9.1
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
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".

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%.

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."

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (corrosive)

Eye irritation

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The potential of 4,4’-Dimethoxytrityl chloride (target substance) to induce skin irritation/corrosion was evaluated in two suitable in vitro test methods (OECD 439 and OECD 431). Based on the results, 4,4’-Dimethoxytrityl chloride (99.73% purity) must be considered as corrosive to skin is therefore classified as Skin Corr. 1B, H314 in accordance with the CLP criteria. Thus, an eye irritation study did not need to be conducted in accordance with REACH Regulation (EC) No 1907/2006, Annex VII, column 2 of information requirement section 8.2.

Justification for classification or non-classification

Based on the results from the in vitro skin irritation/corrosion tests the target substance is considered to cause severe skin burns and eye damage and classification as Skin Corr 1B, H314 is warranted.

Moreover, no further testing to assess the eye irritating potential was needed and the substance will also be classified as Eye Dam 1, H318.