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EC number: 915-730-3
CAS number: -
Skin sensitisation (OECD TG 429): Sensitising (EC3 of 6.07%)
Skin sensitisation (HRIPT, 101 subjects): Non sensitising up to 40%.
Respiratory sensitisation: Non-sensitising in absence of human data and
absence of structural alerts for respiratory sensitisation.
There are several experimental data for assessing the skin sensitisation
of the substance: In vitro skin sensitisation, LLNAs and HRIPT. The LLNA
which EC3 is used for classification and labelling will be presented
first, thereafter the HRIPT because this information will be used for
setting the assessment factors when deriving the DNEL for this endpoint.
Thereafter the result of the other LLNAs and in vitro skin sensitisation
will be presented. No records have been made for the in vitro data
because these do not aid in the risk assessment.
Key LLNA (OECDTG 429)
In the key local lymph node assay (LLNA), according to OECD TG 429,
groups of mice were topically treated on the dorsal surface of both ears
with 0 (vehicle), 2.5, 5.0, 10, 25 or 50% (v/v) OTNE in 3:1 diethyl
phthalate/ethanol daily for 3 days. On the 6thday, mice were injected
intravenously with H-thymidine and 5 hours later were euthanized. The
draining auricular lymph nodes were extracted and prepared for
determination of H-thymidine content. All mice survived to
termination. Stimulation indices at 2.5, 5, 10, 25, and 50% were 1.4,
2.4, 5.2, 28.9, and 13.5, respectively. OTNE was considered to have skin
sensitising activity with an EC3 of 6.07%, indicating the concentration
at which a stimulation index of 3 is observed. According to OECD
guideline 429, a SI ≥3 indicates sensitisation.
HRIPT in human subjects:
In addition to the LLNAs, a human repeated insult patch test is
available. In this repeated insult patch test, 0.3 ml of 40% OTNE in 3:1
diethyl phthalate/ethanol, vehicle only, or saline was applied directly
to an occluded patch, which was placed on the upper right arm of each
subject. Applications were made on Monday, Wednesday and Friday for 3
consecutive weeks. The patches remained in place for 24 hours and
application sites were scored for inflammatory effects just prior to the
next patch application. This procedure was repeated until 9 induction
applications of the test material were completed. Ten to seventeen days
after application of the last induction patch, a challenge patch was
applied to a virgin site on the left upper arm. Twenty-four hours later,
the patch was removed and sites were scored at 24, 48, and 72 hours
after application of the challenge patch. During the induction phase,
17/101 subjects experienced slight or mild erythema. These reactions
appeared to be transient in nature and did not increase in severity over
the induction phase. 4/101 subjects experienced slight or mild erythema
at the 48- or 72-hour challenge reading. None of these reactions were
considered indicative of sensitization.
Two other supporting LLNAs (OECDTG 429)
One carried out in 2008: Groups of mice were topically treated on
the dorsal surface of both ears with 0 (vehicle), 2.5, 5.0, 10, 25 or
50% (v/v) OTNE in 3:1 diethyl phthalate/ethanol daily for 3 days. On the
6th day, mice were injected intravenously with H-thymidine and 5
hours later were euthanized. The draining auricular lymph nodes were
extracted and prepared for determination of H-thymidine content. All
mice survived to termination. Stimulation indices at 2.5, 5, 10, 25, and
50% were 1.1, 1.3, 2.0, 5.6, and 11.3, respectively. OTNE was considered
to have skin sensitising activity with an EC3 of 14.2%.
One other LLNA carried out in 2005: Groups of mice were topically
treated on the dorsal surface of both ears with 0 (vehicle), 2.5, 5.0,
10, 25 or 50% (v/v) OTNE in 3:1 diethyl phthalate/ethanol daily for 3
days. On the 6th day, mice were injected intravenously with
H-thymidine and 5 hours later were euthanized. The draining auricular
lymph nodes were extracted and prepared for determination of
H-thymidine content. All mice survived to termination. Stimulation
indices at 2.5, 5, 10, 25, and 50% were 1.63, 1.44, 1.36, 2.83, and
32.42, respectively. OTNE was considered to have skin sensitising
activity with an EC3 of 25.14%.
In vitro skin sensitisation (OECD TG 442, C, B and D)
Next to these in vivo assays, 3 in vitro assays are available for OTNE.
The studies and results are presented in the sequence of the skin
sensitisation AOP. The peptide reaction is the molecular initiating
event, the effects on the cellular level are predicted with the
Keratinosens and for the effects on a tissue level the h-CLAT is used.
In summary, the DPRA is negative, the Keratinosens inconclusive,
because a negative results should be considered inconclusive when the
substance has a log Kow > 5 (OTNE's log Kow is 5.6) and the h-CLAT is
positive. The overall result of these three tests is inconclusive
because only one out of three tests show a clear negative result and one
a clear positive result. Four qualities of OTNE were used containing
91.5, 91.8, 85.1 and 86.9% substance.
Sample A Standard OTNE
Sample B Purified OTNE
Sample C in AlCl3 including standard catalyst
Sample D high concentration catalyst: AlCl3 low Iso E
Direct Peptide Reactivity Assay (DPRA): Direct Peptide
Reactivity Assay (DPRA): Four qualities of the substance (see Table in
this section) were tested for skin sensitisation potential in the Direct
Peptide Reactivity Assay. Synthetic peptides of cysteine and lysine were
reacted with each test article for 24± 2 hours. The extent of peptide
depletion was analyzed using high performance liquid chromatography
(HPLC) coupled with ultra-violet (UV) spectrometric detection. The test
articles were prepared at 100 mM concentrations. All of the requirements
for a valid assay were achieved. The depletion of cysteine or lysine in
the peptide mixtures was ≤5.74% for all the test items and therefore the
substance is negative in the DPRA.
KeratinoSens assay (2015): Four qualities (see Table in this
section) were tested for skin sensitisation potential in the
KeratinoSens assay. KeratinoSens cells were cultured for 24 hours,
treated for 48 hours, and assessed for luciferase induction and
cytotoxicity. The experiment was performed using 12 final concentrations
in the range of 2000 - 0.977 μM. After the exposure period luminescence
was determined within 30 minutes of addition of the ONE-Glo Reagent.
According to the current prediction model, the test articles Sample A
Control (SSA1), Sample B Purified Control (SSB1), Sample C ALCL3
Standard (SSC1), and Sample D ALCL3 Low ISOP (SSD1), were predicted to
be non-sensitizers. It should be noted that the partition coefficient of
OTNE has been determined as log P = 5.65 at 30°C under OECD TG 117
(HPLC). According to the specific scope and limitations of the OECD TG
442D test method, test chemicals with a Log P up to 5 have successfully
been tested, while substances with a Log P >7 fall outside of the
applicability domain. As the Log P of the test substances falls between
5 and 6, and only limited information is available on the reliability of
the assay in that range, the results should be interpreted with caution
and considered to be inconclusive
Human Cell Line Activation Test (2015): Four qualities of OTNE
(see Table in this section) were tested to the Draft OECD Guideline “In
Vitro Skin Sensitisation: Human Cell Line Activation Test (h-CLAT). For
all test articles, the mean cell viabilities for all eight
concentrations in both main tests were greater than 50%, passing the
acceptance criterion of ≥50%. Both the positive and negative controls
passed all acceptance criteria in each of the main tests. The qualities
SSA1, SSB1, SSC1 and SSD1 each produced a positive response in THP-1
human monocytic cells in the two independent main tests conducted.
Therefore, all qualities are considered to be positive in the h-CLAT.
The substance is not a respiratory sensitiser in absence of human
data indicating such effects. In addition, the respiratory sensitisation
of OTNE is assessed using the integrated evaluation strategy for
respiratory sensitisation data in the ECHA guidance (R7A, Fig. 7.3-4,
substance is a skin sensitiser;
substance does not belong to the di-isocyanates;
has no structural alerts or is structurally related to chemicals causing
respiratory sensitisation as presented in Table R.7.3-1 in the ECHA
guidance of 2008 or those provided in the following document:
Therefore OTNE is not considered to be a respiratory sensitiser.
Based on the available information, OTNE should be classified as
sensitising to the skin in accordance with the criteria outlined in the
EU CLP (1272/2008/EC and its amendments) resulting in Skin Sens. 1B /
H317: May cause an allergic skin reaction.
In absence human data indicating respiratory sensitisation and using the
ITS in the ECHA guidance (R.7a, 7.3, 2017) OTNE is not considered to be
a respiratory sensitiser in accordance with the criteria outlined in the
EU CLP (EC 1272/2008 and its amendments)
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