Reaction product of 2,3-epoxypropyl neodecanoate and Benzenesulfonic acid, C10-13-sec-alkyl derivatives

Endpoint summary

Description of key information

Studies of skin sensitisation potential in chemico (DPRA), in vitro (KeratinoSens assay) an in vivo (LLNA) are available for the submission substance [reaction product of 2,3-epoxypropyl neodecanoate and benzenesulfonic acid, C10-13-sec-alkyl derivatives].

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1-29 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

4 February 2015

Deviations:

no

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

Identification: Reaction product of 2,3-epoxypropyl neodecanoate and benzenesulfonic acid, C10-13-sec-alkyl derivatives
Appearance: Brown liquid
Batch: P718261998
Storage: At room temperature
Stable under storage conditions until: 01 June 2018 (expiry date)

Details on the study design:

Solubility of the test item in an appropriate solvent was assessed before performing the DPRA. An appropriate solvent dissolved the test item completely, i.e. by visual inspection the solution had to be not cloudy nor have noticeable precipitate. The following solvents were evaluated: acetonitrile (ACN), Milli-Q water (MQ), MQ/ACN (1:1, v/v), isopropanol,
acetone, acetone/ACN (1:1, v/v) and dimethylsulfoxide (DMSO)/ACN (1:9, v/v). Test item stock solutions were prepared freshly for each reactivity assay. For the cysteine and lysine reactivity assay 103.24 mg and 60.15 mg of the Reaction product of 2,3-epoxypropyl neodecanoate and benzenesulfonic acid, C10-13-sec-alkyl derivatives were pre-weighed into a clean amber glass vial and dissolved, just before use, in 1861 μL and 1084 μL ACN, respectively, to obtain 100 mM solutions. Visual inspection of the forming of
a clear solution was considered sufficient to ascertain that the test item was dissolved. The test item, positive control and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively.

Positive control results:

The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 77.0% ± 0.0%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%). The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 48.2% ± 1.9%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).

Key result

Run / experiment:

other: Mean (%)

Parameter:

other: Cysteine depletion

Value:

10.5

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: Mean (%)

Parameter:

other: Lysine depletion

Value:

87.6

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

The validation parameters (calibration curve, mean concentration of Reference Control samples, the mean percent peptide depletion values for the positive control with its standard deviation value and the standard deviation value of the test item), were all within the acceptability criteria for the DPRA. The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 77.0% ± 0.0%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%). The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 48.2% ± 1.9%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).

Summary of results

Test material	SPCC depletion		SPCL depletion		Mean SPCC / SPCL depletion	Reactivity class
	Mean	SD	Mean	SD
Reaction product of 2,3- epoxypropyl neodecanoate and benzenesulfonic acid, C10-13-sec-alkyl derivatives	10.5%	6.4%	87.6%	0.8%	49.1%	Positive: High reactivity

Interpretation of results:

study cannot be used for classification

Conclusions:

In the cysteine reactivity assay the test item showed 10.5% SPCC depletion; in the lysine reactivity assay the test item showed 87.6% SPCL depletion. The mean of the SPCC and SPCL depletion was 49.1% and as a result the test item was considered to be positive in the DPRA and was classified in the “high reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Executive summary:

The objective of this study was to determine the reactivity of the submission substance [reaction product of 2,3-epoxypropyl neodecanoate and benzenesulfonic acid, C10-13-sec-alkyl derivatives] towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). After incubation of the test item with either SPCC or SPCL, the relative peptide concentration was determined by HPLC with gradient elution and PDA detection at 220 nm and 258 nm. SPCC and SPCL % depletion values were calculated and used in a prediction model which assigns the test material to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers. Acetonitrile was found to be an appropriate solvent to dissolve the test item. In the cysteine reactivity assay the test item showed 10.5% SPCC depletion while in the lysine reactivity assay the test item showed 87.6% SPCL depletion. The mean of the SPCC and SPCL depletion was 49.1% and as a result the test item was considered to be positive in the DPRA and classified in the “high reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. The submission subtance is therefore considered to be a potential skin sensitiser on the basis of this DPRA.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

3-24 November, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

February 2015

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

Identification: Reaction product of 2,3-epoxypropyl neodecanoate and benzenesulfonic acid, C10-13-sec-alkyl derivatives
Appearance: Brown liquid
Batch: P718261998
Storage: At room temperature
Stable under storage conditions until: 01 June 2018 (expiry date)

Details on the study design:

A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (the KeratinoSens™ cell line). For testing, cells were 80-90% confluent. One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight. The passage number used was P+8 in experiment 1 and P+4 in experiment 2.

The medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control items were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were then incubated for about 48 hours at 37C in the presence of 5% CO2. In total 2 experiments were performed.

The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 μL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 3 minutes at room temperature. Plates with the cell lysates were placed in a plate reader to assess the quantity of luciferase (integration time two seconds).

For the KeratinoSens cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT and cells were incubated for 3 hours at 37°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution to each well. After shaking, the absorption was measured at 570 nm with the plate reader.

Positive control results:

The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold at at least one concentration.
The EC1.5 of the positive control was between 5 -125 μM (50 μM and 114 μM in Experiment 1 and 2, respectively). A dose-response was observed and the induction at 250 μM was 2.71-fold in Experiment 1 and 1.79 in Experiment 2.

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: EC1.5 (uM)

Value:

7.9

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: EC1.5 (uM)

Value:

3.3

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

Both tests passed the acceptance criteria:
The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold at at least one concentration.
The EC1.5 of the positive control was between 5 -125 μM (50 μM and 114 μM in Experiment 1 and 2, respectively). A dose-response was observed and the induction at 250 μM was 2.71-fold in Experiment 1 and 1.79 in Experiment 2.
The average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (5.9% and 11% in Experiment 1 and 2, respectively).
Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

Two independent experiments were performed. The cells were in these experiments incubated with the test item in a concentration range of 0.98 -2000 μM (2-fold dilution steps) for 48 hours. Activation of the ARE-dependent pathway was assessed by measuring the luminescence induction compared to the vehicle control. In addition, the viability was assessed with an MTT assay.

Experiment 1

Precipitation was observed at the start of the incubation period in the 96-well plates at 2000 μM and at the end of the incubation period at 1000 and 2000 μM.

The test item showed toxicity: the calculated IC30 was 22 μM and the calculated IC50 was 24 μM.

A dose-related luminescence activity induction was observed after treatment with the test item: the Imax was 4.42 and the EC1.5 was 7.9 μM.

The positive control Ethylene dimethacrylate glycol caused a dose-related induction of luciferase activity. The Imax was 2.71 and the EC1.5 was 50 μM.

Experiment 2

Precipitation was observed at the end of the incubation period in the 96-well plates at 2000 μM.

The test item showed toxicity: the calculated IC30 was 21 μM and the calculated IC50 was 24 μM.

A dose-related luminescence activity induction was observed after treatment with the test item: the Imax was 3.23 and the EC1.5 was 3.3 μM.

The positive control Ethylene dimethacrylate glycol caused a dose-related induction of luciferase activity: the Imax was 1.79 and the EC1.5 was 114 μM.

Summary of results

	EC1.5 (µM)	Imax	IC30 (µM)	IC50 (µM)
Test item EXP 1	7.9	4.42	22	24
Test item EXP 2	3.3	3.23	21	24
+control EXP 1	50	2.71	-	-
+control EXP 2	114	1.79	-	-

Interpretation of results:

other: Positive result, not suitable to assign a sub-category

Conclusions:

The test item is classified as positive in the KeratinoSens assay since positive results (>1.5-fold induction) were observed at test concentrations with a cell viability of >70% compared to the vehicle control.

Executive summary:

The ability of of the test material (reaction product of 2,3-epoxypropyl neodecanoate and benzenesulfonic acid, C10-13-sec-alkyl derivatives) to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway was investigated in the KeratinoSens assay. The test material was suspended in DMSO. Two independent experiments were performed. Both experiments passed the acceptance criteria, and it is concluded that the test conditions were adequate and that the test system functioned properly. The test material showed toxicity (IC30 values of 22 μM and 21 μM and IC50 values of 24 μM and 24 μM in Experiments 1 and 2, respectively). A biologically relevant, concentration-related induction of the luciferase activity (EC1.5 values of 7.9 μM and 3.3 μM in Experiments 1 and 2, respectively) was measured in both experiments. The maximum luciferase activity induction (Imax) was 4.42 -fold and 3.23 -fold in Experiment 1 and 2 respectively. The test material is therefore classified as positive in the KeratinoSensTM assay since positive results (>1.5-fold induction) were observed at test concentrations with a cell viability of >70% compared to the vehicle control. In conclusion, Reaction product of 2,3-epoxypropyl neodecanoate and benzenesulfonic acid, C10-13-sec-alkyl derivatives is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes).

Reference 3

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19th December 2017 - 13th April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

July 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

Identification: Reaction product of 2,3-epoxypropyl neodecanoate and benzenesulfonic acid, C10-13-sec-alkyl derivatives
Appearance: Brown liquid
Batch: P718261998
Test item storage: At room temperature
Stable under storage conditions until: 01 June 2018 (expiry date)

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF
- Age at study initiation: 11 weeks
- Weight at study initiation: 17.1-24.1 g ; all animals within ± 20% of the sex mean.
- Housing: group housed within treatmnet groups
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-23
- Humidity (%): 40-43
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 15 January 2018 To: 04 March 2018

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

0.5%, 1%, 2%

No. of animals per dose:

Five females

Details on study design:

A pre-screen was conducted in order to select the highest test item concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give well-defined irritation as the most pronounced response (maximum grade 2 and/or an increase in ear thickness < 25%) and/or is the highest possible concentration that can technically be applied. Initially, two test item concentrations were tested; a 100% and 50% concentration. The highest concentration was the maximum concentration as required in the test guidelines. Based on the results of the initially treated animals, eight additional animals were treated in a similar manner with four lower concentrations (10%, 5%, 2% and 1%) at a later stage. Ear thickness measurements were conducted using a digital thickness gauge prior to dosing on Days 1 and 3, and on Day 6.

In the main study, the dorsal surface of both ears was topically treated (25 μL/ear) with the test item, at approximately the same time on each day (Days 1, 2, 3). The concentrations were stirred with a magnetic stirrer immediately prior to dosing. The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test item. On Day 6, each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) containing 20 μCi of 3H-methyl thymidine. After five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) of Euthasol®. The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in PBS. Following excision of the lymph nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (200 μm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) and then stored in the refrigerator until the next day. Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL scintillation fluid. Radioactivity measurements were performed using a scintillation counter. Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

The EC3 value was calculated using linear interpolation.

Key result

Parameter:

SI

Value:

1

Variability:

+/- 0.2

Test group / Remarks:

Vehicle control

Key result

Parameter:

SI

Value:

0.5

Variability:

+/- 0.1

Test group / Remarks:

0.5%

Key result

Parameter:

SI

Value:

1.3

Variability:

+/- 0.3

Test group / Remarks:

1.0%

Key result

Parameter:

SI

Value:

1.5

Variability:

+/- 0.5

Test group / Remarks:

2.0%

Key result

Parameter:

EC3

Remarks on result:

not determinable

Cellular proliferation data / Observations:

In the pre-screen, a 100% test item concentration caused mortality. At concentrations of 50%, 10% and 5%, variations in ear thickness during the observation period exceeded 25% from Day 1 pre-dose values, and/or clinical signs of systemic toxicity were noted. Therefore these concentrations did not meet the selection criteria. At 2% and 1%, no signs of systemic toxicity were noted and no irritation was observed and therefore the 2% concentration was selected as highest concentration for the main study.

In the main study, no mortality occurred and no clinical signs of systemic toxicity were observed. Body weights and body weight gain of the animals treated at 2% remained in the same range as controls over the study period. Marked body weight loss was noted for all animals treated at 0.5% and a higher body weight gain compared to normal was noted for all animals treated at 1%. No erythema was observed in any of the animals. The scaliness shown by one animal treated at 1% on Day 4 was considered not to have a toxicologically significant effect on the activity of the nodes. All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals. Mean DPM/animal values for the experimental groups treated with test item concentrations 0.5, 1 and 2% were 324, 868 and 994 DPM, respectively. The mean DPM/animal value for the vehicle control group was 667 DPM. The SI values calculated for the test item concentrations 0.5, 1 and 2% were 0.5, 1.3 and 1.5, respectively. The results of a separate reliability check performed with HCA confirm the sensitivity of the assay. An EC3 value of 19.2% for HCA was calculated using linear interpolation, and was within the acceptable range of 4.8 and 19.5%.

Summary of results

Group	Individual DPM	Mean DPM	Mean SI
Control	278, 1061, 1031, 338, 625	667 ± 166	1.0 ± 0.2
0.5%	306, 602, 457, 72, 181	324 ± 95	0.5 ± 0.1
1%	89, 1494, 1270, 437, 752	868 ± 222	1.3 ± 0.3
2%	435, 2092, 1189, 617, 639	994 ± 302	1.5 ± 0.5

Interpretation of results:

GHS criteria not met

Conclusions:

The SI values for the tested concentrations do not exceed 3; there is therefore no evidence that the test material IC-2 (reaction product of 2,3-epoxypropyl neodecanoate and benzenesulfonic acid, C10-13-sec-alkyl derivatives) has skin sensitising potential.

Executive summary:

The potential of IC-2 (reaction product of 2,3-epoxypropyl neodecanoate and benzenesulfonic acid, C10-13-sec-alkyl derivatives) to induce skin sensitisation was investigated in a LLNA performed according to GLP, OECD, EC and EPA OPPTS guidelines. Test item concentrations selected for the main study were based on the results of a pre-screen test. Due to mortality, signs of toxicity and variation in ear thickness seen in the pre-screen, concentrations of 0.5%, 1% and 2% were selected for the main study. In the main study, three groups of five female CBA/J mice were treated with test item concentrations of 0.5, 1 or 2% w/w on three consecutive days, by open application on the ears. Five control animals were similarly treated with the vehicle alone (Acetone/Olive oil (4:1 v/v)). Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group. All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals. Mean DPM/animal values for the experimental groups treated with test item concentrations 0.5, 1 and 2% were 324, 868 and 994 DPM, respectively. The mean DPM/animal value for the vehicle control group was 667 DPM. The SI values calculated for the test item concentrations 0.5, 1 and 2% were 0.5, 1.3 and 1.5, respectively. The SI values for the tested concentrations do not exceed 3; there is therefore no evidence that the test material IC-2 (reaction product of 2,3-epoxypropyl neodecanoate and benzenesulfonic acid, C10-13-sec-alkyl derivatives) has skin sensitising potential. The results of a separate reliability check performed with HCA confirm the sensitivity of the assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Positive results were obtained for skin sensitisation potential in the DPRA and KeratinoSens assay, indicating that the submission substance [reaction product of 2,3-epoxypropyl neodecanoate and benzenesulfonic acid, C10-13-sec-alkyl derivatives] is a potential skin sensitiser. A negative result is given in the LLNA.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

DPRA

The objective of this study was to determine the reactivity of the submission substance [reaction product of 2,3-epoxypropyl neodecanoate and benzenesulfonic acid, C10-13-sec-alkyl derivatives] towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). After incubation of the test item with either SPCC or SPCL, the relative peptide concentration was determined by HPLC with gradient elution and PDA detection at 220 nm and 258 nm. SPCC and SPCL % depletion values were calculated and used in a prediction model which assigns the test material to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers. Acetonitrile was found to be an appropriate solvent to dissolve the test item. In the cysteine reactivity assay the test item showed 10.5% SPCC depletion while in the lysine reactivity assay the test item showed 87.6% SPCL depletion. The mean of the SPCC and SPCL depletion was 49.1% and as a result the test item was considered to be positive in the DPRA and classified in the “high reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. The submission subtance is therefore considered to be a potential skin sensitiser on the basis of this DPRA.

KeratinoSens

The ability of of the test material (reaction product of 2,3-epoxypropyl neodecanoate and benzenesulfonic acid, C10-13-sec-alkyl derivatives) to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway was investigated in the KeratinoSens assay. The test material was suspended in DMSO. Two independent experiments were performed. Both experiments passed the acceptance criteria, and it is concluded that the test conditions were adequate and that the test system functioned properly. The test material showed toxicity (IC30 values of 22 μM and 21 μM and IC50 values of 24 μM and 24 μM in Experiments 1 and 2, respectively). A biologically relevant, concentration-related induction of the luciferase activity (EC1.5 values of 7.9 μM and 3.3 μM in Experiments 1 and 2, respectively) was measured in both experiments. The maximum luciferase activity induction (Imax) was 4.42 -fold and 3.23 -fold in Experiment 1 and 2 respectively. The test material is therefore classified as positive in the KeratinoSensTM assay since positive results (>1.5-fold induction) were observed at test concentrations with a cell viability of >70% compared to the vehicle control. In conclusion, Reaction product of 2,3-epoxypropyl neodecanoate and benzenesulfonic acid, C10-13-sec-alkyl derivatives is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes).

LLNA

The potential of (reaction product of 2,3-epoxypropyl neodecanoate and benzenesulfonic acid, C10-13-sec-alkyl derivatives) to induce skin sensitisation was investigated in a LLNA performed according to GLP, OECD, EC and EPA OPPTS guidelines. Test item concentrations selected for the main study were based on the results of a pre-screen test. Due to mortality, signs of toxicity and variation in ear thickness seen in the pre-screen, concentrations of 0.5%, 1% and 2% were selected for the main study. In the main study, three groups of five female CBA/J mice were treated with test item concentrations of 0.5, 1 or 2% w/w on three consecutive days, by open application on the ears. Five control animals were similarly treated with the vehicle alone (Acetone/Olive oil (4:1 v/v)). Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group. All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals. Mean DPM/animal values for the experimental groups treated with test item concentrations 0.5, 1 and 2% were 324, 868 and 994 DPM, respectively. The mean DPM/animal value for the vehicle control group was 667 DPM. The SI values calculated for the test item concentrations 0.5, 1 and 2% were 0.5, 1.3 and 1.5, respectively. The SI values for the tested concentrations do not exceed 3; there is therefore no evidence that the test material IC-2 (reaction product of 2,3-epoxypropyl neodecanoate and benzenesulfonic acid, C10-13-sec-alkyl derivatives) has skin sensitising potential. The results of a separate reliability check performed with HCA confirm the sensitivity of the assay.

Justification for classification or non-classification

Positive results were obtained for skin sensitisation potential in the DPRA and KeratinoSens assay, indicating that the submission substance [reaction product of 2,3-epoxypropyl neodecanoate and benzenesulfonic acid, C10-13-sec-alkyl derivatives] is a potential skin sensitiser; however a negative result is reported for a LLNA, which is considered to be the key study. The substance does not therefore require classification as a skin sensitiser according to CLP criteria.