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Description of key information

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.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 29 to June 6, 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Reliability 1 is assigned because the study is conducted according to OECD TG 429 in compliance with GLP, without deviations that influence the quality of the results.
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
-Source: Jackson Laboratories, Bar Harbor, ME 04609
-Age at study initiation: 8-9 weeks
-Weight at study initiation: 18-23 g
-Housing: 5/cage
-Diet (e.g. ad libitum): ad libitum
-Water (e.g. ad libitum): ad libitum
-Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
-Temperature (ºC): 21.7-27.7
-Humidity (%): 28-68
-Air changes (per hour):
-Photoperiod (hrs dark/hrs light): 12/12
Vehicle:
other: 3:1 diethyl phthalate/ethanol
Concentration:
2.5%, 5.0%, 10%, 25% and 50% (v/v)
No. of animals per dose:
5
Details on study design:
The purpose of the study was to determine if the test material would induce a hypersensitivity response in mice as measured by the proliferation of lymphocytes in the draining lymph nodes. A total of 45 CBA/J female mice were divided into 9 groups consisting of 5 animals in each group. Groups included 1 control (vehicle) group (diethyl phthalate/ethanol in a ratio of 3:1 ratio); 3 positive control groups and 5 test groups (2.5%, 5.0%, 10%, 25% and 50% (v/v) OTNE with the vehicle). The control group was treated with the vehicle only. Animals were checked daily for mortality on Day 1 to 6, for clinical observations immediately prior to dose administration, immediately post-dose on Days 1-3 and then once daily on Days 4-6, and for erythema and oedema using the Draize scoring system. Animals were also weighed daily on Days 1-6. A volume of approximately 25 µl of 5 different concentrations of OTNE (%v/v) in a vehicle of 3:1 ethanol:diethyl phthalate was applied topically to the dorsum of each ear lobe (left and right) once per day for three consecutive days. There were 24±2 hours between applications of test material. Three days after the third topical application (Day 6) all mice were injected intravenously into a tail vein with 250 µl of sterile saline containing approximately 20 µCi [3]H-thymidine. Five hours after the intravenous injection, the animals were sacrificed. The draining auricular lymph nodes were removed. At removal, the number of nodes collected per animal was recorded and the nodes were examined for size/appearance and the data recorded. A single cell suspension was prepared from the lymph nodes of each mouse. Cells were washed twice with phosphate buffered saline (PBS) and precipitated with 5% trichloroacetic acid (TCA) overnight at 2-8ºC. The pellets were recovered by centrifugation and resuspended in 1 ml of TCA and transferred to a vial containing scintillation fluid. An additional 1 ml of TCA was used to rinse the tube and it was also transferred to the scintillation fluid. Incorporation of the [3]H-thymidine was measured in a beta-scintillation counter. The mean DPM per node for each group was evaluated. Increases in [3]H-thymidine incorporation relative to the vehicle-treated control were derived for each group and recorded as stimulation indices (SI). The criterion for a positive response is that one or more concentrations of a test material elicits a 3-fold or greater increase in isotope incorporation relative to the vehicle control. Individual DPM values were analyzed by log transformation (base 10) of the data. If the data indicated that the test material was positive, the EC3 (estimated concentration of the test material required to produce a 3-fold increase in the draining lymph node cell proliferative activity) was calculated by the following formula: EC3 = c+[(3-d)/(b-d)](a-c) where the data points lying immediately above and below the SI value of 3 have the coordinates (a,b) and c,d) respectively. Body weight data were also evaluated.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The evaluation of the equality of means for body weight data was made by a one-way analysis of variance using the F distribution to assess statistical significance. If statistically significant differences between the means were found, a Dunnett's test was used to determine the degree of significance from the control means.
Positive control results:
Stimulation indices at 5, 15, and 35% were 5.2, 4.4, and 3.8, respectively. Mean DPM at 0 (vehicle), 5, 15, and 35% was 1694, 8892, 7372, and 6422, respectively.
Key result
Parameter:
EC3
Value:
6.07
Parameter:
SI
Remarks on result:
other: Stimulation indices at 2.5, 5, 10, 25, and 50% were 1.4, 2.4, 5.2, 28.9, and 13.5, respectively. EC3 is calculated to be 6.07%
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM at 0 (vehicle), 2.5, 5, 10, 25, and 50% were 1694, 2412, 3994, 8726, 48992, and 22879, respectively.
Interpretation of results:
other: Sensitising Category 1B
Remarks:
according to EU CLP (EC 1272/2008 and its amendments)
Conclusions:
Under the conditions of this study, OTNE was considered to have skin sensitising activity with an EC3 of 6.07% and a NOEC of 2.5%.
Executive summary:

In a local lymph node assay 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 [3]H-thymidine and 5 hours later were euthanized. The draining auricular lymph nodes were extracted and prepared for determination of [3]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. A NOEC of 2.5% was derived.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Introduction

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 [3]H-thymidine and 5 hours later were euthanized. The draining auricular lymph nodes were extracted and prepared for determination of [3]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 [3]H-thymidine and 5 hours later were euthanized. The draining auricular lymph nodes were extracted and prepared for determination of [3]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 [3]H-thymidine and 5 hours later were euthanized. The draining auricular lymph nodes were extracted and prepared for determination of [3]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

SSA1 (91.5%)

Sample B Purified OTNE

SSB1 (91.8%)

Sample C in AlCl3 including standard catalyst

SSC1 (85.1%)

Sample D high concentration catalyst: AlCl3 low Iso E

SSD1 (86.9%

 Iso E concentration in % 91.5  91.9  85.1  86.9
         

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.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

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. 7.3-2, 2014).

1)     The substance is a skin sensitiser;

2)     The substance does not belong to the di-isocyanates;

3)     OTNE 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: http://ec.europa.eu/health/scientific_committees/docs/annex6_respiratory.pdf

Therefore OTNE is not considered to be a respiratory sensitiser.

Justification for classification or non-classification

Based on the available information, OTNE should be classified as sensitising to the skin in accordance with the criteria outlined in the EU CLP Regulation (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, 2014) 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)