Registration Dossier

Administrative data

Description of key information

Skin sensitisation in vitro (OECD TG 442D) : potential sensitising substance

Skin sensitisation in vivo (OECD TG 429) : not sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vitro
Data waiving:
study technically not feasible
Justification for data waiving:
other:
Justification for type of information:
The available in in chemico test methods are not applicable for the substance:
- "Soybean oil, epoxidized, Me ester, reaction products with propylene glycol" as the h-CLAT assay is out of the applicability domain since the log Kow value is with a range of 3.7-6.2 (pH 7) significantly above the limit Log Kow of 3.5 which is indicated in the OECD 442E guideline. False negative and false positive results can be expected due to the Log Kow.
- "Soybean oil, epoxidized, Me ester, reaction products with propylene glycol" is an UVCB the DPRA assay (OECD442C – Key event 1 OECD IATA AOP) and in silico testing (DEREK) is also out of the applicability domain.

On the basis that the test substance does not fall into the applicability domain of one or more in-vitro methods (covering at least 2 of 3 of the key events in the OECD IATA AOP) and an absence of supporting reliable data it is recommended to perform the LLNA test (OECD 429 ). In accordance with REACH Regulation (EC) 1907/2006: Annex VII section 8.3.2 column 2 the conduct of the OECD TG 429: Local Lymph Node Assay is considered justified.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 January 2017 - 03 February 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
Information from in vitro test method(s)assessing inflammatory response in keratinocytes for examining skin sensitisation potential are recognised according to Article 13(3).
Details on study design:
- Test material preparation: Batch EM97160711 of the test item was a yellow liquid. The test item was dissolved in dimethyl sulfoxide at 20 mg/ml. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.098 – 200 μg/ml (2-fold dilution series). The highest concentration of 200 μg/ml was selected based on solubility. The highest concentration of 200μg/ml formed a homogeneous suspension (stable dispersion). All other concentrations formed a clear solution. Two independent experiments were performed in triplicate.

- Positive control: Ethylene dimethacrylate glycol, the final concentration of the positive control ranged from 7.81 to 250 μM (final concentration DMSO of 1%) triplicates
- Negative control: The vehicle of the test substance, dimethyl sulfoxide (DMSO). Eighteen wells were tested per plate.

- Test system: A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSens™ cell line). Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock. Cells from this original stock can be propagated up to a maximum passage number (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.

- Cell culture
Basic medium: Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) foetalcalf serum.
Maintenance medium: Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum and geneticin (500 μg/ml).
Exposure medium: Dulbecco’s minimal supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum.

- Environmental conditions:
Humidity: 80 - 100% (actual range 65 - 90 %),
CO2: 5.0 ± 0.5% CO2 in air
Temperature: 37.0 ± 1.0°C (actual range 35.8 - 37.1°C).

-Subculturing
Confluency: 80-90% confluency when subcultured.
Maximum number of passages: 25

- Treatment
Exposure: 150 mL exposure medium + 50 μL of the 25-fold diluted test chemical and control substances.
Background measurement: Three wells per plate were left empty (no cells and no treatment) to assess background values.
Incubation time: The treated plates were incubated for about 48 hours at 37±1.0 C in the presence of 5% CO2.
Independent repeat of experiment: In total 2 experiments were performed.

- Analysis
Procedure: The assay plates were removed from the incubator and the medium is removed. Addition of 200 μL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) to each well. The plates were shaken for at least 3 minutes at room temperature. Plates with the cell lysates were placed in the luminometer to assess the quantity of luciferase (integration time one second).

- Cytotoxicity assessment
Procedure: MTT viability assay

- Interpretation
The following parameters are calculated in the KeratinoSensTM test method:
1. The maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the tested chemical and positive control
2. The EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) was obtained
3. The IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability.
Control IC50 and IC30 are calculated by linear interpolation, and the overall IC50 and IC30 are calculated as the mean of the individual repetitions.

A KeratinoSensTM prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 repetitions, otherwise the KeratinoSensTM prediction is considered negative:
1. The Imax is higher than (>) 1.5 fold and statistically significantly different as compared to the solvent (negative) control (as determined by a two-tailed, unpaired Student’s ttest)
2. The cellular viability is higher than (>) 70% at the lowest concentration with induction of luciferase activity above 1.5 fold (i.e. at the EC1.5 determining concentration)
3. The EC1.5 value is less than (<) 1000 μM (or < 200 μg/mL for test chemicals with no defined MW)
4. There is an apparent overall dose-response for luciferase induction
Positive control results:
Experiment 1: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.83 and the EC1.5 35.2 μM.
Experiment 2: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 3.43 and the EC1.5 14.4 μM.
Key result
Parameter:
other: Imax
Run / experiment:
Experiment 1
Value:
1.51
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: EC1,5 (µg/mL)
Run / experiment:
Experiment 1
Value:
196.4
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: Viability (%)
Run / experiment:
Experiment 1
Value:
> 70
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: at all test concentrations
Key result
Parameter:
other: Imax
Run / experiment:
Experiment 2
Value:
3.62
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: EC1.5 (µg/mL)
Run / experiment:
Experiment 2
Value:
115.3
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: Viability (%)
Run / experiment:
Experiment 3
Value:
> 70
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: at all test concentrations
Other effects / acceptance of results:
Two independent experiments were performed. 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 in at least one concentration.
- The EC1.5 of the positive control was between 5 and 125 μM (35.2 μM and 14.4 μM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (2.83-fold and 3.43-fold in experiment 1 and 2, respectively).
- The average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (8.5% and 6.6% in experiment 1 and 2, respectively).

Overall it is concluded that the test conditions were adequate and that the test system functioned properly.
Interpretation of results:
study cannot be used for classification
Conclusions:
Based on the results of this KeratinoSensTM assay (OECD442D) study "Soybean oil, epoxidized, Me ester, reaction products with propylene glycol" is identified as a potential sensitising substance. However, as the test method only addresses a specific key event (Key event 2 OECD IATA AOP) of skin sensitisation, currently this result should not be used in isolation to identify a skin sensitiser. Further testing is required for classification.
On the basis that the test substance does not fall into the applicability domain of one or more methods (covering at least 2 of 3 of the key events in the OECD IATA AOP, as explained in the Skin sensitisation: in vitro_Waiver), in accordance with REACH Regulation (EC) 1907/2006: Annex VII section 8.3.2 column 2 the conduct of the OECD TG 429: Local Lymph Node Assay is considered justified.
Executive summary:

Evaluation of in vitro skin sensitization potential of "Soybean oil, epoxidized, Me ester, reaction products with propylene glycol" was performed using the KeratinoSens™ assay according to OECD TG 442D. The test item was dissolved in dimethyl sulfoxide at 20 mg/ml. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.098 – 200 μg/ml (2-fold dilution series). Two independent experiments were performed. Both a positive control ( Ethylene dimethacrylate glycol 7.81 to 250 μM ) and vehicle control (DMSO) were applied in the test. A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element, was exposed to 150 mL exposure medium + 50 μL of the 25-fold diluted test chemical and control substances. The treated plates were incubated for about 48 hours at 37±1.0 C in the presence of 5% CO2. Doses were tested in triplicates.

Both the positive and the negative control produced a valid result. The test item showed no toxicity (no IC30 and IC50 value). Statistically significant induction of the luciferase activity was measured in both experiments. The maximum luciferase activity induction (Imax) was 1.51-fold and 3.62-fold in experiment 1 and 2 respectively. The EC1.5 of the test item was 196.4 and 115.3 μg/ml in experiment 1 and 2, respectively. "Soybean oil, epoxidized, Me ester, reaction products with propylene glycol" is classified as positive in the KeratinoSensTM assay.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 Jun 2017 - 06 Jul 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
On the basis that the test substance does not fall into the applicability domain of one or more in-vitro methods (covering at least 2 of 3 of the key events in the OECD IATA AOP) and an absence of supporting reliable data it is recommended to perform the LLNA test (OECD 429 ). In accordance with REACH Regulation (EC) 1907/2006: Annex VII section 8.3.2 column 2 the conduct of the OECD TG 429: Local Lymph Node Assay is considered justified.
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2010
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
2003
Guideline:
other: EC, No 440/2008, part B: "Skin Sensitization: Local Lymph Node Assay"
Version / remarks:
2008
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA:J
Remarks:
Inbred
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF
- Age at study initiation: Young adult animals (approximately 9 weeks old)
- Weight at study initiation: 18.1 to 23.7 g
- Housing: polycarbonate cages (Makrolon MIII type; height 18 cm.)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days before the commencement of dosing
- Indication of any skin lesions: health inspection was performed

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): 10 or greater air changes per hour with 100% fresh air
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark
- IN-LIFE DATES: From:14 Jun 2017 (start dosing) To: 03 jul 2017
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 25, 50, 100%
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: up to 100%
- Irritation:Erythema and eschar formation observations were performed once daily on Days 1-6 (on Days 1-3 within 1 hour after dosing). According to a numerical scoring system. Furthermore, a description of all other (local) effects was recorded.
- Systemic toxicity: Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.All the animals were examined for reaction to dosing. The onset, intensity and duration of these signs was recorded (if appropriate), particular attention being paid to the animals during and for the first hour after dosing.
- Ear thickness measurements: Ear thickness measurements were conducted using a digital thickness gauge (Kroeplin C110T-K) prior to dosing on Days 1 and 3, and on Day 6.
- Erythema scores:
Erythema and eschar formation:
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema (beet redness) to slight eschar formation (injuries in depth) 3
Severe erythema (beet redness) to eschar formation preventing grading of erythema 4

MAIN STUDY
In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 25, 50 or 100% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Aceton: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.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: the test item is regarded as a sensitiser in the LLNA if the exposure to at least one concentration of the test item resulted in an incorporation of 3H-methyl thymidine at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index.

TREATMENT PREPARATION AND ADMINISTRATION:
The dosing formulations were prepared daily and dosed within 4 hours after adding the vehicle to the test item.
The dosing formulations were kept at room temperature until dosing.
The dorsal surface of both ears was topically treated (25 μL/ear), at approximately the same time on each day. The formulation was 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.

TREATMENT PREPARATION AND ADMINISTRATION:
Positive control substance(s):
other: Historical control
Statistics:
Basketter DA, Lea LJ, Dickens A, Briggs, D, Pate I, Dearman RJ and Kimber I. A comparison of statistical approaches to the derivation of EC3 values from local lymph node assay dose responses. J Appl Toxicol 1999;19:261-266
Positive control results:
For both scientific and animal welfare reasons, no concurrent positive control group was included in the study. An extensive data base is available with reliability checks performed each half year during at least the recent 9 years showing reproducible and consistent positive results.
Key result
Parameter:
SI
Value:
1
Variability:
0.1
Test group / Remarks:
0%
Key result
Parameter:
SI
Value:
1.5
Variability:
0.4
Test group / Remarks:
25%
Key result
Parameter:
SI
Value:
1.6
Variability:
0.3
Test group / Remarks:
50%
Key result
Parameter:
SI
Value:
2.1
Variability:
0.3
Test group / Remarks:
100%
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
Mean DPM per lymph node:
- Vehicle control group: 467 +/- 35
- 25%: 707 +/- 183
- 50%: 756 +/- 136
- 100%: 975 +/- 146

DETAILS ON STIMULATION INDEX CALCULATION
The nodes were pooled for each animal. DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean.

EC3 CALCULATION
As no SI>3 was measured, the EC3 was not determined.

CLINICAL OBSERVATIONS:
No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study.

BODY WEIGHTS
Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.
Interpretation of results:
other: not classified
Remarks:
based on CLP Regulation (1272/2008/EC).
Conclusions:
Based on the results of this OECD TG 429 study "Soybean oil, epoxidized, Me ester, reaction products with propylene glycol" does not need to be classified as a skin sensitizer in accordance with the CLP Regulation (1272/2008/EC).
Executive summary:

An LLNA, skin sensitization study was performed according to OECD TG 429 and in compliance with GLP. 4 mice per test group were exposed to 0, 25, 50 and 100% w/w "Soybean oil, epoxidized, Me ester, reaction products with propylene glycol" in Acetone/Olive oil (4:1 v/v.) Historical positive control tests were performed with α-Hexylcinnamaldehyde. The animals did not show any treatment related clinical signs during the course of the study. . In this study Stimulation Indices (S.I.) of 1.5, 1.6 and 2.1 were determined with the test item at concentrations of 25%, 50% and 100% w/w in acetone/olive oil (4:1 v/v), respectively. Since the lowest dose did not elicit a SI ≥ 3, the EC3 value was not determined. Based on the results of this study, the test substance does not need to be classified as a skin sensitizer in accordance with the CLP Regulation (1272/2008/EC).

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

OECD TG 442D - KeratinoSens

Evaluation of in vitro skin sensitization potential of "Soybean oil, epoxidized, Me ester, reaction products with propylene glycol" was performed using the KeratinoSens™ assay according to OECD TG 442D. The test item was dissolved in dimethyl sulfoxide at 20 mg/ml. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.098 – 200 μg/ml (2-fold dilution series). Two independent experiments were performed. Both a positive control ( Ethylene dimethacrylate glycol 7.81 to 250 μM ) and vehicle control (DMSO) were applied in the test. A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element, was exposed to 150 mL exposure medium + 50 μL of the 25-fold diluted test chemical and control substances. The treated plates were incubated for about 48 hours at 37±1.0 C in the presence of 5% CO2. Doses were tested in triplicates.

Both the positive and the negative control produced a valid result. The test item showed no toxicity (no IC30 and IC50 value). Statistically significant induction of the luciferase activity was measured in both experiments. The maximum luciferase activity induction (Imax) was 1.51-fold and 3.62-fold in experiment 1 and 2 respectively. The EC1.5 of the test item was 196.4 and 115.3 μg/ml in experiment 1 and 2, respectively. "Soybean oil, epoxidized, Me ester, reaction products with propylene glycol" is classified as positive in the KeratinoSensTM assay.

Based on the results of this KeratinoSensTM assay (OECD442D)  study "Soybean oil, epoxidized, Me ester, reaction products with propylene glycol" is identified as a potential sensitising substance. However, as the test method only addresses a specific key event (Key event 2 OECD IATA AOP) of skin sensitisation, currently this result should not be used in isolation to identify a skin sensitiser. Further testing is required for classification. However, the available in chemico/in vitro test methods overing at least 2 of 3 of the key events in the OECD IATA AOP are not applicable for the substance: - "Soybean oil, epoxidized, Me ester, reaction products with propylene glycol" as the h-CLAT assay is out of the applicability domain since the log Kow value is with a range of 3.7-6.2 (pH 7) significantly above the limit Log Kow of 3.5 which is indicated in the OECD 442E guideline. False negative and false positive results can be expected due to the Log Kow.   - "Soybean oil, epoxidized, Me ester, reaction products with propylene glycol" is an UVCB the DPRA assay (OECD442C – Key event 1 OECD IATA AOP) and in silico testing (DEREK) is also out of the applicability domain.On the basis that the test substance does not fall into the applicability domain of one or more methods (covering at least 2 of 3 of the key events in the OECD IATA AOP, as explained in the Skin sensitisation: in vitro_Waiver), in accordance with REACH Regulation (EC) 1907/2006: Annex VII section 8.3.2 column 2 the conduct of the OECD TG 429: Local Lymph Node Assay is considered justified.

OECD TG 429 - LLNA

An LLNA, skin sensitization study was performed according to OECD TG 429 and in compliance with GLP. 4 mice per test group were exposed to 0, 25, 50 and 100% w/w "Soybean oil, epoxidized, Me ester, reaction products with propylene glycol" in Acetone/Olive oil (4:1 v/v.) Historical positive control tests were performed with α-Hexylcinnamaldehyde. The animals did not show any treatment related clinical signs during the course of the study. In this study Stimulation Indices (S.I.) of1.5, 1.6 and 2.1 were determined with the test item at concentrations of 25%, 50% and 100% w/w in acetone/olive oil (4:1 v/v), respectively. Since the lowest dose did not elicit a SI ≥ 3, the EC3 value was not determined. Based on the results of this study, the test substance does not need to be classified as a skin sensitizer in accordance with the CLP Regulation (1272/2008/EC).

Justification for classification or non-classification

Based on the results of the OECD TG 429 LNNA study, "Soybean oil, epoxidized, Me ester, reaction products with propylene glycol" does not need to be classified as a skin sensitizer in accordance with the CLP Regulation (1272/2008/EC).