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EC number: 245-844-2
CAS number: 23726-93-4
Skin irritation: irritating (OECD 439, GLP, K, rel. 1)Eye irritation: not irritating (similar to OECD 405, read-across, K, rel. 2)Respiratory irritation: no data available
MTT solution containing the test item remained yellow which indicated
that the test item did not directly reduce MTT.
7.3.1/1: Mean OD562 Values and Percentage Viabilities for the Negative
Control Item, Positive Control Item and Test Item
OD562 of tissues
Mean OD562 of triplicate tissues
± SD of OD562
Relative individual tissue viability (%)
Relative mean viability (%)
± SD of Relative mean viability (%)
Negative Control Item
Positive Control Item
mean viability of the negative control tissues is set at 100%
relative mean tissue viability for the positive control treated tissues
was 3.1% relative to the negative control treated tissues and the
standard deviation value of the percentage viability was 7.7%. The
positive control acceptance criterion was therefore satisfied.
mean OD562 for the negative control treated tissues was 0.923
and the standard deviation value of the percentage viability was 7.7%. The
negative control acceptance criterion was therefore satisfied.
standard deviation calculated from individual percentage tissue
viabilities of the three identically treated test item tissues was 0.6%. The
test item acceptance criterion was therefore satisfied.
the previous 22 experiments conducted from April 2014 to August 2014
using this test method, the mean OD of the positive control was 0.084 ±
0.046 and the mean percentage viability was 9.2 ± 5.3. In this same
period the mean OD of the negative control was 0.938 ± 0.102.
Although the positive control parameters for
this study were greater than ranges based on the previous 22 experiments
the positive control was considered to have reflected the ability to respond
to an irritant chemical under the conditions of the test ie the
recommended acceptance criterion for the positive control was achieved
for this test (≤40% relative to the concurrent negative control).
The negative control in this study was comparable to the negative control
achieved in the previous 22 experiments conducted with this test method.
An in vitro skin irritation study was
performed according to the OECD Guideline 439 and in compliance with
GLP, using the EPISKINTM reconstructed human epidermis model. Triplicate
tissues were treated with the test item for an exposure period of 15
minutes. At the end of the exposure period each tissue was rinsed before
incubating for 42 hours. At the end of the post exposure incubation
period each tissue was taken for MTT-loading. The maintenance medium
from beneath each tissue was transferred to pre labelled micro tubes and
stored in a freezer for possible inflammatory mediator determination.
After MTT-loading a total biopsy of each epidermis was made and placed
into micro tubes containing acidified isopropanol for extraction of
formazan crystals out of the MTT loaded tissues. At the end of the
formazan extraction period each tube was mixed thoroughly and duplicate
200 µL samples were transferred to the appropriate wells of a pre
labelled 96 well plate. The optical density was measured at 562 nm.
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 14.8% after the 15-Minute exposure period and 42
hours post-exposure incubation period.
The relative mean tissue viability for the
positive control treated tissues was 3.1% relative to the negative
control treated tissues and the standard deviation value of the
percentage viability was 7.7%. The positive control acceptance criterion
was therefore satisfied.
The mean OD562 for the negative control
treated tissues was 0.923 and the standard deviation value of the
percentage viability was 7.7%. The negative control acceptance criterion
was therefore satisfied.
The standard deviation calculated from
individual percentage tissue viabilities of the three identically
treated test item tissues was 0.6%. The test item acceptance criterion
was therefore satisfied.
Under the test conditions, test item was
classified as irritant according to the annex VI of the Regulation (EC)
No. 1272/2008 (CLP). This study is considered as acceptable and
satisfies the requirement for skin irritation endpoint.
7.3.2/1: Mean eye irritation response data of 6 animals at each
Score at time point / Reversibility
Average time for reversion
7.3.2/2: Eye irritation response data for each animal at each
Score at time
24 h (Day 1)
0 / 0 / 0 / 0 / 0 / 0
1 / 2 / 1 / 0 / 0 / 1
1 / 1 / 1 / 0 / 0 / 1
1 / 3 / 2 / 0 / 0 / 0
48 h (Day 2)
0 / 2 / 0 / 0 / 0 / 0
0 / 1 / 0 / 0 / 0 / 0
72 h (Day 3)
Average 24, 48 and 72 h
0.33 / 1.33 / 0.33 / 0 / 0 / 0.33
0.33 / 1 / 0.33 / 0 / 0 / 0.33
0.33 / 2.33 / 0.67 / 0 / 0 / 0
Average time (unit) for reversion
an eye irritation study, 0.1 g of undiluted test material was instilled
into the right eye of 6 albino rabbits while the left eye remained
untreated and served as control. The eyes were not rinsed after the
instillation of test material. Animals were observed at 24, 48 and 72 h
after instillation of test material into eyes and then on Days 5 and 7.
The reactions in the conjunctivae (redness, chemosis and discharge), the
iris and the cornea (opacity and area involved) were scored according to
the “Illustrated guide for grading eye irritation by hazardous
calculated mean score for each animal within 3 scoring times (24, 48 and
72 h) were 0.39 for redness, 0.33 for chemosis, 0.0 for iris lesions and
0.0 for corneal opacity. The observed conjunctival reactions were fully
reversible within 5 days.
the test conditions, the test material is not classified as irritating
to the eyes according to the annex I of Regulation EC No. 1272/2008
some details on test animals and experimental conditions were missing,
this study is considered as acceptable and satisfies the requirement for
eye irritation endpoint.
supporting substance is considered adequate for read-across purpose (see
Iuclid section 13 for justification).
substance has no harmonized classification according to the Regulation
(EC) No. 1272/2008 including ATP6.
Based on the available information, the
registered substance should be classified as Skin
irr. Category 2 (H315: Causes skin irritation) according to the Annex VI
of the Regulation (EC) No. 1272/2008 (CLP).
additional self-classification is proposed regarding eye irritation
according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP).
information was available regarding respiratory irritation.
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