Registration Dossier

Administrative data

Description of key information

BTMAOH is considered as corrosive to skin (Skin corrosion Category 1A) based on a pH ≥ 11.5 and the results of an in vitro test (OCED 431) with BTMAOH in methanol.

Any eye irritation study is waived as the registered substance is classified as corrosive to skin and therefore also classified as irritating to eyes with risk of serious damage to eyes, in accordance with the CLP Regulation.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
26 March 2018 - 30 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The substance can be produced through different process routes, yielding solutions in water or organic solvent (e.g. methanol or ethylene glycol).
It was initially unclear how to register the substance (mono-constituent or multi-constituent substance) and which manufactured substance to test to fulfil the REACH data requirements. After consultation with the ECHA helpdesk, the test program was started with the manufactured substance of the Lead registrant (solvent: methanol) in which the highest amount of solvent could be removed without causing degradation of the substance. This resulted in the selection of a solution of 56-57% BTMAOH in methanol as test substance.
During the course of the test program, and in order to aid meaningful risk assessment, after consultation with ECHA and upon ECHA's recommendation, it was considered to be more appropriate to test the water-based manufactured substance. As a consequence, some testing was performed with a BTMAOH solution in methanol, and some testing was performed with a BTMAOH solution in water.

The current entry reflects a test performed with a methanol-based test solution.
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
29 July 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test". Official Journal of the European Union No. L142
Version / remarks:
31 May 2008
Deviations:
no
GLP compliance:
yes
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
skin obtained from plastic surgery from multiple donors
Details on animal used as source of test system:
EpiDerm™ Reconstructed Human Epidermis (Lot no.: 28307, kit E and F)
Justification for test system used:
Recommended test system in international guidelines (OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model (EPI-200)
- Tissue lot number: 28307, kit E and F
- All cells used to produce Epiderm™ are purchased or derived from tissue obtained by MatTek Corporation from acredited institutions.
- The model consists of keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis.
- Cells are screened for potential biological contaminants (HIV-1, Hepatitis B, Hepatitis C, bacteria, yeast and fungi)
- Surface: 0.6 cm^2

FREEZE-KILLED TISSUES
- Model used: EpiDerm Skin Model (EPI-200)
- Tissue lot number: 27958, kit G and 27667, kit G
- Living epidermis was transferred to a freezer (≤-15°C), thawed, and then again transferred to (≤-15°C). The freeze-killed epidermis was stored at ≤ -15°C until use. Freeze-killed tissues were thawed by placing them for 1 hour at room temperature in a 6 well plate on 0.9 mL DMEM. Further use of killed tissues was similar to living tissues.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment at 37.0 ± 1.0°C. (actual range 35.7 - 36.2°C)

COLOR INTERFERENCE CHECK
To assess the color interference, 50 μL of the test item or 50 μL Milli-Q water as a negative control were added to 0.3 mL Milli-Q water. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0°C in the dark. At the end of the exposure time the mixture was shaken and it was checked if a blue / purple color change was observed.

REDUCTION OF MTT CHECK
To assess the ability of the test item to reduce MTT, 50 μL of the test item or 50 μL Milli-Q water as a negative control were added to 1 mL MTT solution (1 mg/mL) in phosphate buffered saline. The mixture
was incubated for approximately 1 hour at 37.0 ± 1.0ºC. At the end of the exposure time it was checked if a blue / purple color change or a blue / purple precipitate was observed.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2 replicates, 2 negative controls, 2 positive controls per exposure duration.
In addition, two freeze-killed tissues were treated with test item and two freeze-killed non treated tissues were used per exposure duration for the cytotoxicity evaluation with MTT.

REMOVAL OF TEST MATERIAL AND CONTROLS
- After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test item.

ACCEPTANCE OF RESULTS:
The in vitro skin corrosion test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range.
b) The mean relative tissue viability following 1-hour exposure to the positive control should be <15 %.
c) In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30%.
d) The %NSC should be ≤ 30% relative to the negative control OD.
e) The non-specific MTT reduction should be ≤ 30% relative to the negative control OD.

DECISION CRITERIA
A test item is considered corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test item considered non-corrosive (viability ≥ 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%.

A test item is considered non corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.

DATA INTERPRETATION
See Table 1 (Any other information on methods incl. tables)
Control samples:
yes, concurrent negative control
yes, concurrent positive control
other: concurrent control for MTT reduction by test item
Amount/concentration applied:
50 μL of the undiluted test item was applied on top of the tissues.
Duration of treatment / exposure:
3-minute and 1-hour
Number of replicates:
2 test item treated tissues, 2 negative control, and 2 positive control per exposure duration
2 freeze-killed tissues treated with test item and two freeze-killed negative control tissues per exposure duration
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3-minute exposure
Value:
23
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
12%
Remarks on result:
other: The test item value is corrected for the non-specific MTT reaction (1.11%)
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1-hour exposure
Value:
2.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
6.3%
Remarks on result:
other: The test item value is corrected for the non-specific MTT reaction (2.71%)
Other effects / acceptance of results:
- OTHER EFFECTS:
- Colour interference with MTT: No
- Direct-MTT reduction: Yes
The non-specific reduction of MTT by the test item was 1.11% and 2.71% of the negative control tissues after 3 minutes and 1 hour respectively.

ACCEPTANCE OF RESULTS:
The absolute mean OD570 (1.80 and 1.82 for 3-minute and 1-hour treatment, respectively) of the negative control tissues was within the acceptance limits of OECD 431 and the laboratory historical control data range The mean relative tissue viability following the 1-hour exposure to the positive control was 6.3%.
In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates for the negative control was <2%.
Interpretation of results:
Category 1A (corrosive) based on GHS criteria
Conclusions:
The in vitro skin corrosion test was performed according to OECD guideline 431 and GLP principles. Based on the results it is concluded that the test substance is corrosive under the experimental conditions described in this report.
Executive summary:

In anin vitroskin corrosion test performed according to OECD 431 and in accordance with GLP principles, the influence of the test substance on the viability of the human skin was tested using the EpiDerm Skin Model. BTMAOH in methanl (50 μL) was applied directly on top of skin tissues and after 3 minutes or 1 hour rinsed away with phosphate saline buffer. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with test item compared to the negative control tissues was 23% and 2.9%, respectively, after correction for non-specific MTT reduction. Since the mean relative tissue viability was <50% after the 3-minute treatment, it can be concluded that the test item is corrosive to the skin. Because the relative tissue viability after 3 minutes was <25% BTMAOH is classified as corrosive sub-category 1A.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (corrosive)

Eye irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In an in vitro skin corrosion test performed according to OECD 431 and in accordance with GLP principles, the influence of the test substance on the viability of the human skin was tested using the EpiDerm Skin Model. BTMAOH in methanl (50 μL) was applied directly on top of skin tissues and after 3 minutes or 1 hour rinsed away with phosphate saline buffer. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with test item compared to the negative control tissues was 23% and 2.9%, respectively, after correction for non-specific MTT reduction. Since the mean relative tissue viability was <50% after the 3-minute treatment, it can be concluded that the test item is corrosive to the skin. Because the relative tissue viability after 3 minutes was <25% BTMAOH is classified as corrosive sub-category 1A.

Justification for classification or non-classification

In view of the high pH and tissue viability <25% after 3 minutes in an in vitro skin corrosion test (OECD TG 431), BTMAOH is considered to be corrosive to skin, category 1A.

In accordance with section 3.3.2.3 of Annex 1 of the CLP Regulation, the registered substance is also classified as irritating to eyes with risk of serious damage to eyes.