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EC number: 695-748-3
CAS number: 65286-55-7
Skin irritation/corrosion: The substance was observed to be corrosive in
the in vitro EPIDERMTM Skin Corrosion Test performed
according to OECD Guideline 431 and EU Method B.40bis (Warren, 2017).
Eye irritation: The study does not need to be conducted because the
substance is classified as skin corrosion, leading to classification as
serious eye damage (category 1).
Direct MTT Reduction
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 no 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
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
Test item, positive control item and negative control item
Mean OD562 values and viabilities for the negative control, positive
control and test item are given below:
MeanOD570of individual tissues
Mean OD570of duplicate tissues
Coefficient of Variation
Relative Mean Viability (%)
OD: Optical density
*: The mean percentage viability of the negative control
tissue is set at 100%
na: Not applicable
The relative mean viabilities of the test item treated tissues were as
3 minutes exposure: 75.7%
60 minutes exposure: 12.3%
The mean OD570 for the negative control treated tissues was 1.423
for the 3‑Minute exposure period and 1.567 for the 60‑Minute exposure
period. The negative control acceptance criteria were therefore
The relative mean tissue viability for the positive control
treated tissues was 0.9% relative to the negative control following the
60‑Minute exposure period. The positive control acceptance criterion was
In the range 20 to 100% viability the Coefficient of Variation
between the two tissue replicates of each treatment group did not exceed
30% with the exception of the test item treated 3 minute exposure group
which was 31.8% However, as the relative tissue viability of this group
was considerably higher than 50% this failure would not affect the
overall classification of corrosivity. This acceptance criterion was
therefore not fully satisfied and is reported as a deviation.
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
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:
*The mean viability of the negative control tissues is set at 100%.
The quality criteria required for acceptance of results in the test were
satisfied with the exceptionthat the Coefficient of Variation of the
test item treated 3 minute exposure group exceeded the 30% limit. This
is reported as a deviation.
The test item was considered to be corrosive: UN GHS H314 Combination of
sub-categories 1B and 1C.
As the test substance is found to be corrosive to the skin, no eye
irritation study is performed.
Based on the available data and the criteria of the CLP Regulation the
substance is classified as corrosive to the skin, category 1B.
Therefore, the substance is also classified for serious eye damage
category 1, based on the criteria of the CLP Regulation.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
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