Registration Dossier

Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05-07 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD test Guideline No. 431 without any deviation.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspected on 05 July 2016 / signed on 28 October 2016)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Physical state / Appearance: White solid
Specific details on test material used for the study:
- Storage condition of test material: The test item was stored dry and in the dark in a closed aluminium bag at room temperature (20 ± 10 °C). On 23 February 2017 the aluminium bag was opened and the contents were split into single use aliquots. The test item aliquots were stored in amber jars flushed with nitrogen.

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
Following the REACH up and down strategy, the EpiDerm™ Human Skin Model method was used to assess skin corrosion as recommended in the OECD test guideline No. 431.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis Model Kit
- Supplier: MatTek
- Tissue batch number(s): EpiDermTM Tissues (0.63cm2) lot number 25803
- Production Date: 03 April 2017
- Shipping date: 03 April 2017
- Delivery date: 04 April 2017
- Date received: 04 April 2017
- Date used: 06 April 2017
- Assay Medium lot number: 033017TME
- 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, in a humidified atmosphere of 5% CO2
- Temperature of post-treatment incubation: 37 °C, in a humidified atmosphere of 5% CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: rinsing was performed using a wash bottle and was achieved by filling and emptying each tissue under a constant soft stream of DPBS for approximately 40 seconds to gently remove any residual test item
- Observable damage in the tissue due to washing: No visible damage to the rinsed tissues was noted following the rinsing step..
- Modifications to validated SOP: none reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL MTT solution
- Incubation time: 3 hours at 37 °C, humidified atmosphere of 5% CO2.
- Spectrophotometer: Labtech LT 4500 microplate reader
- Wavelength: 570 nm
- Filter: without reference filter
- Filter bandwidth: filter band pass ± 10
- Linear OD range of spectrophotometer: 0.0 to 2.6 nm.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
In order to confirm the integrity of the test system a comparison was made with historical data for negative and positive controls obtained by the laboratory in the previous six months. All values for negative and positive controls were within the historical range and this was taken to indicate the adequate functioning of the test system.
- Viability: OD (540-570 nm) = 1.7 ± 0.105 and 1.739 ± 0.134 for viable and non-viable tissues, respectively (acceptance criteria: between 1.0-3.0)
- Barrier function: ET-50 = 5.36 hours and 6.71 hours for viable and no-viable tissues, respectively (acceptance criteria: between 4.77-8.72 hours)
- Morphology: Tissue viability and the barrier function test are within acceptable ranges and indicate appropriate formation of the epidermal barrier, the presence of a functional stratum corneum, a viable basal cell layer, and intermediate spinous and granular layers.
- Contamination: sterile (Long term antibiotic and antimycotic free culture)
- Reproducibility: For the previous 43 experiments conducted between August 2016 and February 2017 using this test method, the mean OD of the positive control was 0.079 ± 0.025 after 3 minutes of exposure and 0.068 ± 0.025 after 60 minutes of exposure. The mean percentage viability was 4.3 ± 1.0 after 3 minutes of exposure and 3.8 ± 1.0 after 60 minutes of exposure (The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues was ≤15% relative to the negative control treated tissues after 1 hour exposure). In this same period the mean OD of the negative control was 1.845 ± 0.239 after 3 minutes of exposure and 1.810 ± 0.235 after 60 minutes of exposure (The assay establishes the acceptance criterion for an acceptable test if the mean OD for the negative control treated tissues was ≥0.8 and ≤2.8 for every exposure time).
The historical control values given were obtained on a different microplate reader to the machine that was used in this study. They were also measured at 562nm whereas in this study the values were measured at a wavelength of 570nm. Even though the values were obtained on a different machine the values are still representative of the historical range for the laboratory and the minor difference of 8nm between 562nm and 570nm is not considered to impact upon the applicability of the historical comparison for this study.

NUMBER OF REPLICATE TISSUES: Duplicate tissues for test item, negative and positive controls

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues
- Procedure used to prepare the killed tissues : Freeze-killed tissues were prepared prior to the study by placing untreated EPIDERMTM tissues in an empty 12 well plate and storing in a freezer (-14 to -30 °C) for a minimum of 24 hours. Before use each tissue was thawed by placing in 0.9 mL of assay medium for approximately 1 hour at room temperature.
- N. of replicates : 2
- Method of calculation used: True viability = mean OD (treated viable tissues) - [OD (treated killed tissues)-OD (untreated killed tissues)]

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PR
EDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
- If the test item is corrosive with the viability less than 25% after 3 min exposure: H314; Sub-category 1A
- If the test item is corrosive with the viability greater than or equal to 25% after 3 min exposure: H314; Sub-category 1A
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 of the unformulated test item (undiluted) wetted with 25 μL of sterile water to increase tissue surface contact.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL of sterile distilled water was used as supplied.

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL of 8.0 N Potassium Hydroxide was used as supplied.
Duration of treatment / exposure:
3 and 60 minutes
Duration of post-treatment incubation (if applicable):
Not applicable
Number of replicates:
Duplicate tissues for test item, negative and positive controls

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minutes exposure
Value:
73.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
4.6%
Remarks on result:
other: corrosive to the skin
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minutes exposure
Value:
5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
4.2%
Remarks on result:
other: corrosive to the skin
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: none reported
- Direct-MTT reduction: The MTT solution containing the test item turned blue which indicated that the test item directly reduced MTT. Direct reduction was <30% relative to the negative control and therefore acceptable.
- Colour interference with MTT: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was 1.542 for the 3 Minute exposure period and 1.589 for the 60 Minute exposure period. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 4.2% relative to the negative control following the 60 Minute exposure period. The positive control acceptance criterion was therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: 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.
- Historical data: Furthermore, in order to confirm the integrity of the test system a comparison was made with historical control data for negative and positive controls obtained by the laboratory in the previous six months.
The historical control values were obtained on a different microplate reader to the machine that was used in this study. They were also measured at 562nm whereas in this study the values were measured at a wavelength of 570nm. Even though the values were obtained on a different machine the values are still representative of the historical range for the laboratory and the minor difference of 8nm between 562 nm and 570 nm is not considered to impact upon the applicability of the historical comparison for this study. All values obtained were within the historical range of the testing laboratory.

Any other information on results incl. tables

Table 7.3.1/1: Mean OD562 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Tissue

Exposure Period

Mean OD570 of individual tissues (tvt)

Mean OD570 of duplicate tissues

Corrected OD570 of tissues
(tvt-[tkt-ukt])

Standard Deviation

Coefficient of Variation

(%)

Relative Mean Viability (%)

Negative Control

3 Minutes

1.566

1.542

-

0.035

2.2

100*

1.517

60 Minutes

1.631

1.589

0.059

3.7

1.547

Positive Control

3 Minutes

0.081

0.071

-

0.014

N/A

4.6

0.061

60 Minutes

0.075

0.068

0.011

N/A

4.2

0.060

Test Item

3 Minutes

1.281

1.367

1.128

0.121

8.8

73.1

1.452

60 Minutes

0.507

0.475

0.080

0.046

9.7

5.0

0.442

 

3 minute exposure corrected mean OD570= 0.344 (tkt) – 0.105 (ukt) = 0.239

60 minute exposure corrected mean OD570= 0.521 (tkt) – 0.126 (ukt) = 0.395

3 minute exposure direct MTT reduction relative to the negative control = 15.5%
60 minute exposure direct MTT reduction relative to the negative control = 24.9%

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

Coefficient of variation = Standard deviation / Mean OD570 of duplicate tissues

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

OD = optical density

N/A = not applicable

tvt =    treated viable tissues

tkt =    treated killed tissues

ukt = untreated killed tissues

Applicant's summary and conclusion

Interpretation of results:
Category 1B (corrosive) based on GHS criteria
Remarks:
combination of category 1B-and-1C
Conclusions:
Under the experimental conditions of this study, the test substance is classified as H314 “Causes severe skin burns and eye damage”, combination of category 1B-and-1C, according to the Regulation (EC) No 1272/2008 (CLP) and to the GHS.
Executive summary:

An in vitro skin corrosion study was performed according to the OECD Guideline 431 and in compliance with GLP, using the EpiDerm™ Human Skin Model.

 

Duplicate tissues were treated with 25 mg of the undiluted test item wetted with 25 µL of sterile water to increase tissue surface contact 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 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 viability of the test item treated tissues was 73.1 and 5.0 % after 3 and 60 minutes exposure, respectively. The relative mean viability of the negative control treated tissues was 100 % after 3 and 60 minutes exposure. The relative mean viability of the positive control treated tissues was 4.6 and 4.2 % after 3 and 60 minutes exposure, respectively. The quality criteria required for acceptance of results in the test were satisfied.

Under the experimental conditions of this study, the test substance is classified as H314 “Causes severe skin burns and eye damage”, combination of category 1B-and-1C, according to Regulation (EC) No 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for skin icorrosion endpoint.