Registration Dossier

Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 October 2016 - 01 December 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 431 (In vitro human skin corrosion)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Name: ETHYLMETHYLAMINE (EMA)
Synonyms: N-Ethylmethylamine, EMA
Batch No.: E310971456
Description: colorless liquid
Storage conditions: at room temperature and protected from light
Analytical purity: 99.79%
Expiry date: 12 July 2018.

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: human epidermal keratinocytes
Cell source:
foreskin from multiple donors
Vehicle:
unchanged (no vehicle)
Details on test system:
Species: human reconstructed epidermis (tissues).
Supplier: Episkin Laboratories, Lyon, France.
Selection: at receipt, the medium quality and temperature indicators were checked to ensure the good quality of the tissues before use.
Storage conditions: the living tissues were kept at room temperature from receipt at CiToxLAB France until required.
Description: the EpiskinTM model consists of an airlifted, living, multilayered epidermal tissue construction (surface 0.38 cm2), reconstructed from normal human epidermal keratinocytes for 13 days and produced in polycarbonate inserts in a serum-free and chemically defined medium. The model features a normal ultra structure and is functionally equivalent to human in vivo epidermis.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
- Concentration (if solution): original form

NEGATIVE CONTROL: 0.9% NaCl.
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): 0.9% NaCl

POSITIVE CONTROL: glacial acetic acid.
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): original form.
Duration of treatment / exposure:
Exposure period of 3, 60 and 240 minutes, followed by rinsing.
Duration of post-treatment incubation (if applicable):
Not applicable.
Number of replicates:
Duplicate tissues for each timepoint and tested substance (test item, negative control, positive control)

Test animals

Species:
other: reconstituted human epidermis
Strain:
other: not applicable
Details on test animals and environmental conditions:
Episkin TM Model Kit (0.38 cm2 tissues) supplied by SkinEthic Laboratories, Nice, France.
Medium and Incubation T°C: 37°C
Dates of experimental phase: from 03 November 2016 to 01 December 2016.

Test system

Type of coverage:
other: not applicable (in vitro)
Preparation of test site:
other: not applicable (in vitro)
Vehicle:
unchanged (no vehicle)
Controls:
other: in vitro negative and positive controls
Number of animals:
Not applicable
Details on study design:
PRELIMINARY TEST:
Test for direct MTT reduction with the test item and Test for the detection of the coloring potential of the test item.

MAIN TEST:
Treatment
The test item, positive and negative controls were applied on duplicate tissue. As the test item was found to have direct MTT reducing properties in the preliminary test, MTT reducing test item and negative control were applied to two water-killed tissues for each exposure time.
A volume of 50 µL was evenly applied to the surface of each tissue, using a positive displacement pipette, taking care to spread the items over the whole tissue surface area without damaging the tissue sample.
Plates were incubated at room temperature as follows: positive control for 4 hours (± 10 minutes); test item and negative control for 3 minutes (± 5 seconds), 1 hour (± 5 minutes) and 4 hours (± 10 minutes).
The positive control incubated for 4 hours (± 10 minutes) acted as control for all incubation times.

Rinsing of tissues
For all treated tissues (test item-treated, positive and negative control) at the end of the designated incubation period, each tissue insert was removed from the well of the treatment plate and rinsed with D PBS.
Rinsing was achieved by gently filling and emptying each tissue insert 12 times with 2 mL D-PBS and the surface of each tissue was swept with a cotton-bud to gently remove any residual items.

MTT viability assay
formazan transformation by viable cells, after 3-hour MTT incubation.

Optical density measurement
The OD was measured at a wavelength of 570 nm.

SCORING SYSTEM:
Data analysis for MTT reducing substances
Since the test item may directly reduce MTT, the percentage of OD due to non specific MTT reduction was calculated and subtracted to obtain the viability percentage.

- Non-Specific MTT reduction (NSMTT) calculation:
cOD 0.9%NaCl-treated Killed tissues = ODNK – ODblank
cOD Test Item-treated Killed tissues = ODTIK – ODblank

NSMTT = [(mean cODTIK – mean CodNK) / mean cODNC] x 100

If the NSMTT is 0% < NSMTT = 50% relative to the negative control (living tissues), the corrected OD value of the test item-treated water-killed tissues is subtracted from the mean cOD of the test item treated living tissues to obtain the true amount of MTT reduction (i.e. the MTT reduction produced by metabolic conversion only).

- True MTT metabolic conversion (TODTI):
TODTI = [mean cODTI – (mean cODTIK – mean cODUK)]

If the NSMTT is < 0% relative to the negative control, the corrected OD value of the water-killed tissues were not subtracted from the mean cOD of the test item-treated living tissues.

After calculation of each TODTI (i.e. for each treatment exposure), relative mean viability was calculated as follows:
Relative mean viability = (TODTI / mean cODNC) x 100

Acceptance criteria for negative and positive controls
The main test was considered as valid if the following criteria were fully met:
- the mean cOD of the negative control should be between 0.600 and 1.500,
- the relative mean viability of the positive control should be = 20% of the relative mean viability of the negative control,
- in the range 20-100% viability and for ODs = 0.3, the difference of viability between the two tissue replicates should not exceed 30%.

Interpretation: see below

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Value:
19
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 3 min. Reversibility: no data not applicable. Remarks: 100% = negative control. (migrated information)
Irritation / corrosion parameter:
% tissue viability
Value:
16
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 240 min. Reversibility: no data not applicable. Remarks: 100% = negative control. (migrated information)
Irritation / corrosion parameter:
% tissue viability
Value:
14
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 60 min. Reversibility: no data not applicable. Remarks: 100% = negative control. (migrated information)

Applicant's summary and conclusion

Interpretation of results:
Category 1A (corrosive) based on GHS criteria
Conclusions:
The test item tested in its original form, is considered to be corrosive to the skin.

According to these results, the classification of the test item is the following:
Corrosive Category 1A (HS: H314) (UN GHS 2013 and Regulation 1272/2008/EEC)
Executive summary:

The objective of this study was to evaluate the corrosive potential of the test item using the EpiskinTMreconstructed skin model.

This study was conducted in compliance with CiToxLAB France’s standard operating procedures and the principles of Good Laboratory Practices.

Method

Preliminary tests were performed to detect the ability of the test item to interfere with cell viability measurements by directly reducing MTT or by color interference.

Following the preliminary tests, the corrosive potential of the test item was tested in the main assay. Test item, and negative and positive controls were applied on duplicate tissues and incubated at room temperature as follows: positive control for 4 hours (± 10 minutes); test item and negative control for 3 minutes (± 5 seconds), 1 hour (± 5 minutes) and 4 hours (± 10 minutes).

At the end of the designated incubation period, each tissue insert was rinsed with D-PBS and placed into fresh assay medium pre-filled well. Then, they will be incubated with a MTT solution (0.3 mg/mL) for 3 hours (± 15 minutes) at +37°C, 5%CO2in humidified atmosphere. At the end of the MTT incubation period, the tissues were removed from their plastic insert. Any tissue discoloration was evaluated with the naked eye (discolored surface area and intensity).

Then, for each tissue, the epidermis was separated from the collagen matrix and both parts were put into acidified isopropanol overnight to extract the formazan (reduced MTT) out of the MTT-loaded tissues. At the end of the extraction period, the optical density of each extract was measured at 570 nm.

 

Relative viability values were calculated for each tissue and expressed as percentages of the negative control tissues viability which was set at 100% (reference viability).

Results

Preliminary tests

In the preliminary tests, the test item was found to have direct MTT reducing properties but no coloring properties.

 

Main test

All acceptance criteria for the negative and positive controls were fulfilled (Table 1), therefore the study was considered to be valid.

The true MTT metabolic conversion relative mean viabilities of the test item were:

.  19% for the 3 minutes exposure,

.   14% for the 1 hour exposure,

.    16% for the 4 hours exposure.


Discussion

The NSMTT values relative to the negative control (living tissues) were equal to 25% for the 3 minutes exposure, 9% for the 1 hour exposure and -1% for the 4 hours exposure.

These NSMTT values were highly heterogeneous between the different treatment exposure periods, and non-coherently distributed, since values decreased over time.

This could be explained by:

.  the high volatility of the test item. Despite the use of sealer, the test item could have evaporated and remained in the well in a gaseous state, thus reducing over time the quantity of liquid test item in contact with tissues,

.  the corrosive potential of the test item. A pink coloration of the medium (normally pale yellow) was noted at the end of treatment periods. This indicated the passage of the test item through the tissues and their collagen membrane.

Both phenomena could have induced a strong time-related decrease in the amount of test item remaining inside the tissues, explaining the non-consistency of the values of NSMTT over time.

 

Since at all exposure periods the mean viabilities were < 35%,the results met the criteria for a corrosive response.

 

Conclusion

The test item, tested in its original form, is considered to be corrosive to the skin.

According to these results, the classification of the test item is the following:

. Corrosive Category 1A (HS: H314)(UN GHS 2013 and Regulation 1272/2008/EEC)