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Description of key information

The submission substance [Epoxypropyl neodecanoate, oligomeric reaction products with cyclohexane-1,2 -dicarboxylic anhydride and propylidenetrimethanol] has been tested for skin sensitisation potential in vitro (DPRA). Further testing in vitro was not technically feasible due to the physicochemical properties of the substance. Consequently in vivo testing (LLNA) has been performed in order to provide a reliable conclusion on the skin sensitisation potential of the substance.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
March - June 2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
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: Epoxypropyl neodecanoate, oligomeric reaction products with cyclohexane-1,2-dicarboxylic anhydride and propylidenetrimethanol
Appearance: Clear yellow resin
Batch: CVR 83297
Test item storage: At room temperature protected from light
Stable under storage conditions until: 27 December 2018 (expiry date)
Details on the study design:
No correction for the purity/composition of the test item was performed. 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). Epoxypropyl neodecanoate, oligomeric reaction products with cyclohexane-1,2-dicarboxylic anhydride and propylidenetrimethanol stock solutions were prepared freshly for each reactivity assay. For both the cysteine and lysine reactivity assay 224.8 mg of Epoxypropyl neodecanoate, oligomeric reaction products with cyclohexane-1,2-dicarboxylic anhydride and propylidenetrimethanol was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1754 ¿L ACN (Fisher Chemicals, Loughborough, England) to obtain a 100 mM solution. 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 SPCL Depletion for the positive control cinnamic aldehyde was 63.1% ± 1.6%. 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:
1.4
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:
2
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
no 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 74.9% ± 1.4%. 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 63.1% ± 1.6%. 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

Epoxypropyl neodecanoate,

oligomeric reaction products with

cyclohexane-1,2-dicarboxylic

anhydride and

propylidenetrimethanol

1.4%

 2.4%

 2.0%

 0.7%

1.7% 

No or minimal reactivity

Interpretation of results:
study cannot be used for classification
Conclusions:
The results of this DPRA do not indicate any skin sensitisation potential for the test material [Epoxypropyl neodecanoate, oligomeric reaction products with cyclohexane-1,2-dicarboxylic anhydride and propylidenetrimethanol] based on a lack of lysine and cysteine peptide depletion. The result should, however, be viewed with caution due to precipitation of the test material in the assay system.
Executive summary:

The objective of this study was to determine the reactivity of the test material [Epoxypropyl neodecanoate, oligomeric reaction products with cyclohexane-1,2-dicarboxylic anhydride and propylidenetrimethanol] towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). After incubation of the test material with either SPCC or SPCL, the relative peptide concentration was determined by HPLC with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which allows assigning the test chemical to one of four reactivity classes used to support the discrimination between sensitizers and nonsensitizers. The study procedures were based on the most recent OECD guidelines. Acetonitrile (ACN) was found to be an appropriate solvent to dissolve the test item and was therefore used in this Direct Peptide Reactivity Assay (DPRA). 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. In the cysteine reactivity assay the test item showed 1.4% SPCC depletion while in the lysine reactivity assay the test item showed 2.0% SPCL depletion. The mean of the SPCC and SPCL depletion was 1.7% and as a result the test item was classified in the ¿no or minimal reactivity class¿ when using the Cysteine 1:10 / Lysine 1:50 prediction model. The results of this DPRA do not indicate any skin sensitisation potential for the test material [Epoxypropyl neodecanoate, oligomeric reaction products with cyclohexane-1,2-dicarboxylic anhydride and propylidenetrimethanol] based on a lack of lysine and cysteine peptide depletion.  The result should, however, be viewed with caution due to precipitation of the test material in the assay system.

Endpoint:
skin sensitisation: in vitro
Data waiving:
study technically not feasible
Justification for data waiving:
other:
Justification for type of information:
The objective of this study was to evaluate the ability of the test material [Epoxypropyl neodecanoate, oligomeric reaction products with cyclohexane-1,2-dicarboxylic anhydride and propylidenetrimethanol] to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens(TM) assay. An initial solubility test was performed; the test material showed precipitation in Dulbecco's Modified Eagle's Medium up to very low concentrations and was therefore considered to be not suitable for testing in the KeratinoSensTM assay. No main assay was performed.
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
2015
Deviations:
yes
Remarks:
The main assay was not performed due to the very low solubility of the test material in the culture medium.
GLP compliance:
yes
Type of study:
activation of keratinocytes
Specific details on test material used for the study:
Identification: Epoxypropyl neodecanoate, oligomeric reaction products with cyclohexane-1,2-dicarboxylic anhydride and propylidenetrimethanol
Appearance: Clear yellow resin
Batch: CVR 83297
Storage: At room temperature protected from light
Stable under storage conditions until: 27 December 2018 (expiry date)

[Epoxypropyl neodecanoate, oligomeric reaction products with cyclohexane-1,2-dicarboxylic anhydride and propylidenetrimethanol] dissolved well in DMSO but the 100 -fold dilution in DMEM showed precipitation up to low concentrations of 15.6 ¿M. At lower concentrations (7.8 ¿M) the judgement of precipitation was not possible. It was concluded that the substance did not dissolve in DMEM at concentrations needed to get relevant results for the skin sensitization endpoint.

Interpretation of results:
study cannot be used for classification
Conclusions:
An initial solubility test was performed; [Epoxypropyl neodecanoate, oligomeric reaction products with cyclohexane-1,2-dicarboxylic anhydride and propylidenetrimethanol] showed precipitation in Dulbecco's Modified Eagle's Medium up to very low concentrations and was therefore considered to be not suitable for testing in the KeratinoSensTM assay.
Executive summary:

The objective of this study was to evaluate the ability of the test material [Epoxypropyl neodecanoate, oligomeric reaction products with cyclohexane-1,2-dicarboxylic anhydride and propylidenetrimethanol] to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens(TM) assay. An initial solubility test was performed; the test material showed precipitation in Dulbecco's Modified Eagle's Medium up to very low concentrations and was therefore considered to be not suitable for testing in the KeratinoSensTM assay. No main assay was performed.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 June - 26 July 2017
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)
Version / remarks:
May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
March 2003
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA): DA
Specific details on test material used for the study:
Identification: Epoxypropyl neodecanoate, oligomeric reaction products with cyclohexane-1,2-dicarboxylic anhydride and propylidenetrimethanol
Appearance: Clear yellow resin
Batch: CVR 83297
Storage: At room temperature protected from light
Stable under storage conditions until: 27 December 2018 (expiry date)
Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
Species: Mouse
Strain: CBA/J
Condition: Inbred, SPF-Quality
Source: Janvier, Le Genest-Saint-Isle, France
Number of Animals: 20 Females (nulliparous and non-pregnant). Five females per group.
Age at the Initiation of Dosing: Young adult animals (approximately 8 weeks old) were selected.
Weight at the Initiation of Dosing: 18.9-22.6 g.

On arrival and following assignment to the study, animals were group housed (up to 5 of the same dosing group together) in polycarbonate cages (Makrolon) containing sterilized sawdust as bedding material. Target temperatures of 18 to 24°C with a relative target humidity of 40-70% were maintained. The actual daily mean temperature during the study period was 22°C with an actual daily mean relative humidity of 47-68%. A 12-hour light/12-hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

Pelleted diet and drinking water were provided ad libitum throughout the study.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
10%, 25%, 50%
No. of animals per dose:
5 female
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. Two test item concentrations were tested; a 25% and 50% concentration. The highest concentration was the maximum concentration required in the test guidelines.

In the main study, three groups of five animals were treated with one concentration (10%, 25% or 50%). The highest concentration was selected from the pre-screen test. One group of five animals was treated with the vehicle. The dorsal surface of both ears was topically treated (25 ¿L/ear) at approximately the same time on each day (Days 1, 2, 3). The formulations 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 material.

On Day 6, each animal was injected via the tail vein with 0.25 mL sterile phosphate buffered saline (PBS) containing 20 ¿Ci 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 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 nodes, a single cell suspension of lymph node cells was prepared in PBS by gentle separation through stainless steel gauze (200 ¿m). Cells were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate DNA, cells were exposed to 5% trichloroacetic acid and stored overnight. Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and radioactivity measured by LSC.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Not required
Positive control results:
The results of a separate reliability check (Test Facility Study No. 518176) are reported. The check was performed in June 2017 using 5%, 10% and 25% hexyl cinnamic aldehyde. SI values of 1.2, 2.3 and 5.6 are reported (EC3: 13.2%), confirming the sensitivity of the assay.
Key result
Parameter:
SI
Value:
1.4
Variability:
standard deviation 0.4
Test group / Remarks:
10%
Key result
Parameter:
SI
Value:
1.4
Variability:
standard deviation 0.3
Test group / Remarks:
25%
Key result
Parameter:
SI
Value:
2.6
Variability:
standard deviation 0.5
Test group / Remarks:
50%
Key result
Parameter:
EC3
Value:
> 50
Remarks on result:
other: EC3 could not be calculated as all SI values are <3
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
Mean dpm/animal values for the experimental groups treated with concentrations of 10, 25 and 50% were 566, 583 and 1076, respectively. The mean dpm/animal value for the vehicle control group was 414. The SI values calculated for concentrations of 10, 25 and 50% were 1.4, 1.4 and 2.6, respectively.

EC3 CALCULATION
An EC3 could not be calculated (>50%).

CLINICAL OBSERVATIONS:
In the pre-screen, very slight irritation of the ears was shown by animals treated at 50% at Days 3 and 4, no further erythema was noted. Variations in ear thickness during the observation period were less than 25%. In the main study, no mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Alopecia between the ears and neck was noted for all animals treated at 50% between Days 4 and 6, which was considered not to have a toxicologically significant effect on the activity of the nodes. In the main study, very slight or well defined erythema of the ears was shown by several animals treated at 25% and all animals treated at 50% between Days 2 and 5 but was considered not to have a toxicologically significant effect on the activity of the nodes.

BODY WEIGHTS:
Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

The majority of auricular lymph nodes were considered normal in size, except for the nodes in one animal treated at 25% and all animals treated at 50%, which were considered enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Interpretation of results:
GHS criteria not met
Conclusions:
As all SI values were <3, the EC3 was calculated to be >3. CLP criteria for classification are not met.
Executive summary:

The objective of this study was to evaluate whether the test material [Epoxypropyl neodecanoate, oligomeric reaction products with cyclohexane-1,2-dicarboxylic anhydride and propylidenetrimethanol] shows skin sensitisation potential in mice after three epidermal exposures (LLNA, OECD 429). Test material concentrations selected for the main study were based on the results of a pre-test. In the main study, three experimental groups of five female CBA/J mice were treated with concentrations of 10, 25 or 50% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but 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. Activity was expressed as the number of disintegrations per minute (dpm) and a stimulation index (SI) was subsequently calculated for each group. The majority of auricular lymph nodes were considered normal in size, except for the nodes in some of the animals treated at 25% and all animals treated at 50%, which appeared larger in size as compared to the other treated groups. No macroscopic abnormalities of the surrounding area were noted for any of the animals. Mean dpm values for the experimental groups treated with concentrations of 10, 25 and 50% were 566, 583 and 1076, respectively. The mean dpm value for the vehicle control group was 414. SI values calculated for the test material concentrations of 10%, 25% and 50% were therefore 1.4, 1.4 and 2.6, respectively. As all of the SI values were <3, the test material was not considered to have skin sensitisation potential. The EC3 value exceeds 50%. A six-month reliability check with alpha-hexylcinnamaldehyde confirmed the sensitivity of the assay.

Endpoint:
skin sensitisation, other
Remarks:
Weight of evidence assessment and justification for testing strategy
Type of information:
other: Weight of evidence assessment
Adequacy of study:
other information
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Weight of evidence assessment
Qualifier:
no guideline required
Principles of method if other than guideline:
The objective of this expert review was to reach an overall conclusion on the skin sensitisation endpoint, based on all available relevant information including in silico / in chemico / in vitro data.
GLP compliance:
no
Type of study:
other: Expert review
Interpretation of results:
study cannot be used for classification
Conclusions:
This expert review concludes that, as the performance of further in vitro tests is technically not feasible or scientifically not justified, a final conclusion on skin sensitising properties and potency cannot be made for the submission substance. Consequently, the performance of in vivo testing (LLNA) is appropriate.
Executive summary:

The objective of this expert review was to reach an overall conclusion on the skin sensitisation endpoint, based on all available relevant information including in silico / in chemico / in vitro data. As the submission substance [Epoxypropyl neodecanoate, oligomeric reaction products with cyclohexane-1,2 -dicarboxylic anhydride and propylidenetrimethanol] is a UVCB, (Q)SAR assessment is not feasible. A preliminary solubility test showed that the KeratinoSens assay was not suitable for this substance. Performance of a U-SENS assay was considered not to be scientifically justified due to the surface activity of the substance, which could potentially give a false positive result. A DPRA was performded and shows mean depletion of synthetic peptides containing either cysteine or lysine of 1.7%; the substance was therefore considered to be negative in this assay and classified in the ¿no or minimal reactivity class¿ when using the Cysteine 1:10 / Lysine 1:50 prediction model. Based solely on the outcome of the DPRA and following ECHA guidance, no conclusion on skin sensitisation can be made. As the performance of further in vitro tests is technically not feasible or scientifically not justified, a final conclusion on skin sensitising properties and potency cannot be made. Consequently, the performance of in vivo testing (LLNA) is appropriate.

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

(Q)SAR

(Q)SAR analysis is not possible as the substance is a UVCB

In vitro testing

The results of the DPRA do not indicate any skin sensitisation potential for [Epoxypropyl neodecanoate, oligomeric reaction products with cyclohexane-1,2-dicarboxylic anhydride and propylidenetrimethanol] based on a lack of lysine and cysteine peptide depletion.  The result should, however, be viewed with caution due to precipitation of the test material in the assay system. A KeratinoSens assay could not be performed due to the very low solubility of the substance in the culture medium. Performance of a U-SENS assay was considered not to be scientifically justified due to the surface activity of the substance, which could potentially give a false positive result.

Expert review

Due to technical difficulties associated with in vitro testing and the uncertain results of the DPRA, an expert review of the testing strategy for skin sensitisation concluded that in vivo testing was appropriate. An LLNA was therefore performed.

In vivo testing

An LLNA performed with [Epoxypropyl neodecanoate, oligomeric reaction products with cyclohexane-1,2-dicarboxylic anhydride and propylidenetrimethanol] using concentrations of 10, 25 and 50% w/w reports SI values of 1.4, 1.4 and 2.6, respectively. As all of the SI values were <3, the substance was not considered to have skin sensitisation potential.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the results of the LLNA, the substance [Epoxypropyl neodecanoate, oligomeric reaction products with cyclohexane-1,2-dicarboxylic anhydride and propylidenetrimethanol] does not show skin sensitisation potential and does not require classification for skin sensitisation according to the CLP Regulation.