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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Starting Date: 24 October 2018 and Experimental Completion Date: 25 October 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
29 July 2016
Deviations:
yes
Remarks:
see below (deviation was considered to have not affected the integrity or validity of the study)
Qualifier:
according to
Guideline:
other: Method B.40bis of Commission Regulation (EC) No 440/2008, of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006, 18 December 2006, of the European Parliament and of the Council on the REACH.
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Name: XTJ-785, experimental
Lot No.: 9570-2-6738
CAS No.: Not listed
Purity: >92% C1214 alcohol, propoxylated, aminated, ethyoxylated
Expiry Date: No date established

In vitro test system

Test system:
other: EpiDerm™ Reconstructed Human Epidermis Model
Source species:
human
Cell type:
other: Epithelial, derived from human skin and fromed into a stratified, cornified epithelium
Cell source:
other: not available
Source strain:
not specified
Details on animal used as source of test system:
Not applicable
Justification for test system used:
This model incorporates several features, which make it advantageous in the study of potential dermal corrosivity. The target cells are epithelial, derived from human skin, and formed into a stratified, cornified epithelium. Test items are applied to the culture surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly.
Vehicle:
unchanged (no vehicle)
Remarks:
used as supplied
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
EpiDerm™ Reconstructed Human Epidermis Model Kit
Supplier : MatTek
Date received :23 October 2018
EpiDermTM Tissues (0.63cm2) lot number : 28685
Assay Medium lot number : 1018188MSA
Upon receipt of the EpidermTM tissues, the sealed 24 well plate was stored in a refrigerator until use.


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

REMOVAL OF TEST MATERIAL AND CONTROLS
- Rinsing was achieved by filling and emptying each tissue under a constant soft stream of Dulbecco’s Phosphate Buffered Saline (DPBS) (without Ca++ Mg++) for approximately 40 seconds, to gently remove any residual test item.


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/L MTT solution
- Incubation time: 60 minutes
- Wavelength: 570 nm
- Labtech LT-4500 microplate reader and LT-com analysis software.


NUMBER OF REPLICATE TISSUES: duplicate for test item, duplicate for positive and duplicate for negative control

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: The test item was found to directly reduce MTT and therefore additional non-viable tissues were incorporated into testing for correction purposes..



Qualitity criteria:
The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:

Negative Control
- The absolute OD570 of the negative control treated tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. The mean OD570 of the two negative control tissues should be ≥ 0.8 and ≤ 2.8 for each exposure time, which ensures that the tissue viability meets the acceptance criteria.

Positive Control
- Potassium Hydroxide 8.0N solution is used as a positive control. An assay meets the acceptance criterion if mean relative tissue viability of the 60 Minute positive control is < 15%.

Coefficient of Variation
- In the range 20 and 100% viability, the Coefficient of Variation between tissue replicates should be ≤ 30%.

DECISION CRITERIA:
STEP 1
< 50% after 3 min exposure Corrosive
≥ 50% after 3 min exposure AND < 15% after 60 min exposure Corrosive
≥ 50% after 3 min exposure AND ≥ 15% after 60 min exposure Non-corrosive

STEP 2 for test items identified as corrosive in step 1
< 25% after 3 min exposure H314
Sub-category 1A

≥ 25% after 3 min exposure H314
Combination of sub-categories 1B-and-1C

Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
50 µL
Duration of treatment / exposure:
60 minutes
3 minutes
Duration of post-treatment incubation (if applicable):
3 hours
Number of replicates:
duplicate for test item
duplicate for negative control
duplicate for positive control

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
other: relative mean viability
Run / experiment:
exposure period 3 minutes
Value:
91.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: non corrosive to the skin
Irritation / corrosion parameter:
other: relative mean viability
Run / experiment:
exposure period 60 minutes
Value:
89.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: non-corrosive to the skin
Other effects / acceptance of results:
Results:
An assessment found the test item was able to directly reduce MTT. Therefore, an additional procedure using freeze killed tissues was performed. However, the results obtained showed that negligible interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the freeze killed tissues for quantitative correction of results or for reporting purposes.

Assessment of Color Interference with the MTT endpoint
The solution containing the test item did not become colored.
This was taken to indicate the test item did not have the potential to cause color interference.


Test Item, Positive Control Item and Negative Control Item.

Exposure Period Percentage Viability
Negative Control Positive Control Test Item
3 minute 100* 3.7 91.6
60 minute 100* 2.9 89.2
*The mean viability of the negative control tissues is set at 100%



Quality Criteria:
- The mean OD570 for the negative control treated tissues was 1.931 for the 3 Minute exposure period and 1.923 for the 60 Minute exposure period. The negative control acceptance criteria were therefore satisfied.
- The relative mean tissue viability for the positive control treated tissues was 0.055% relative to the negative control following the 60 Minute exposure period. The positive control acceptance criterion was therefore satisfied.
- In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

Any other information on results incl. tables

Calcul:

Relaive mean viability (%) = (mean OD570 of test item / mean OD570 of negative control) x 100

The relative mean viabilities for each treatment group were as follows:

Exposure Period

Percentage Viability

Negative Control

Positive Control

Test Item

3 minute

100*

3.7

91.6

60 minute

100*

2.9

89.2

*The mean viability of the negative control tissues is et at 100%

Mean OD570Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Tissue

Exposure Period

MeanOD570of individual tissues

Mean OD570of duplicate tissues

Standard Deviation

Coefficient of Variation
(%)

Relative Mean Viability (%)

Negative Control

3 Minutes

1.985

1.931

0.077

4.0

100*

1.876

60 Minutes

1.822

1.923

0.142

7.4

2.023

Positive Control

3 Minutes

0.076

0.071

0.007

na

3.7

0.066

60 Minutes

0.056

0.055

0.001

na

2.9

0.054

Test Item

3 Minutes

1.691

1.769

0.110

6.2

91.6

1.846

60 Minutes

1.650

1.716

0.093

5.4

89.2

1.781

OD=Optical density

*=         The mean percentage viability of the negative control tissue is set at 100%

na=        Not applicable

 

Relative mean % tissue viability = (Mean OD570 of test item / mean OD570 of negative conrol) x 100

Coefficient of variation = (standard deviation / mean OD570 of duplicate tissues) x 100


Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was considered to be non-corrosive to the skin.
Executive summary:

The purpose of this test is to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes.

Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. The test item was found to directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT‑loading. After MTT-loading each tissue was placed in 2 mL of Isopropanol for MTT extraction.

At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 mL samples were transferred to the appropriate wells of a pre-labeled 96‑well plate. The optical density (OD) was measured at 570 nm (OD570).

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The relative mean viabilities for each treatment group were as follows:

Exposure Period

Percentage Viability

Negative Control

Positive Control

Test Item

3 minute

100*

3.7

91.6

60 minute

100*

2.9

89.2

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

Quality criteria: The quality criteria required for acceptance of results in the test were satisfied.

Conclusion

The test item was considered to be non-corrosive to the skin.