Reaction mass of [2,2,4-trimethyl-1-(2-methylpropanoyloxy)pentan-3-yl] benzoate and [2,2,4-trimethyl-3-(2-methylpropanoyloxy)pentyl] 2-methylpropanoate and (3-benzoyloxy-2,2,4-trimethylpentyl) benzoate

Endpoint summary

Description of key information

An overall conclusion on the skin sensitization is based on in silico/in chemico/in vitro/in vivo data. Results of each study is summarised below.

In vitro studies:

Keratinosens: positive

DPRA: positive

Derek: negative

In vivo study:

LLNA: negative

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

01 Aug 2018 - 21 Sep 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE
DEREK NEXUS

2. MODEL (incl. version number)
DEREK NEXUS 6.0.1.

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
C1(=CC=CC=C1)C(=O)OC(C(C)(C)COC(=O)C(C)C)C(C)C.C2(=CC=CC=C2)C(=O)OC(C(C)(C)COC(=O)C3=CC=CC=C3)C(C)C.C(C(=O)OC(C(COC(=O)C(C)C)(C)C)C(C)C)(C)C

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
See attached QMRF
Endpoint: Skin sensitization.
Dependent variable: Data from several toxicity assays (e.g. LLNA, GPMT, HRIPT) and mechanistic studies (e.g. DPRA) are synthesised to arrive at an expert conclusion of whether compounds within the model training set are likely to be a skin sensitizer.
a. Model or submodel name: DEREK NEXUS – skin sensitization.
b. Model version: DEREK NEXUS 6.0.1.
c. Reference to QMRF: The QMRF DEREK NEXUS 6.0.1 – skin sensitization was prepared by the provider LHASA Ltd (UK).
d. Predicted value (model result): Assessment with DEREK NEXUS did not yield any skin sensitization alerts for this structure.
e. Predicted value (comments): Not relevant; the structure did not fall within one of the currently 90 skin sensitization alerts, the structure contains no misclassified or unclassified features and consequently the structure is predicted to be negative.
f. Input for prediction: See section 1.5
g. Descriptor values: Not relevant, DEREK NEXUS is a rule-based system.

5. APPLICABILITY DOMAIN
See attached QMRF
i. descriptor domain: see QMRF section 5.1
ii. structural fragment domain: For skin sensitization, which featuresmultiple alerts believed to cover most of the mechanisms and chemical classes responsible for activity, “no alerts fired” may be extrapolated to a negative prediction. All structure fragments were found in the DEREK database and consequently the structure falls within the applicability domain of DEREK’s skin sensitization end point.
iii. mechanism domain: As the prediction is “no alerts fired” none of the mechanisms for skin sensitization is predicted to be applicable to this structure.
iv. metabolic domain: Not relevant.

6. ADEQUACY OF THE RESULT
The present prediction may be used for preparing the REACH Registration Dossier on the substance for submission to ECHA, as required by Regulation (EC) 1907/2006 and related amendments.
This result can be directly used within a weight-of-evidence approach to complete the endpoint skin sensitization
Substance should not be classified according to DEREK NEXUS; however, this (Q)SAR prediction cannot be used as stand-alone for classification purposes or for covering the endpoint skin sensitization for registration under REACH.
The result is adequate to be used in a weight-of-evidence approach together with in chemico/in vitro studies to complete

Qualifier:

no guideline followed

Principles of method if other than guideline:

The objective of this study was to obtain a prediction on the potential for skin sensitization of the test item with the in silico model DEREK NEXUS. Derek Nexus (DN) is a knowledge-based expert system created with knowledge of structure-toxicity relationships and an emphasis on the need to understand mechanisms of action and metabolism. In this assessment version 6.0.1 of DEREK NEXUS was used. The current version contains 90 alerts for skin sensitisation and some alerts for photoallergenicity.
The level of likelihood of a structure being sensitizing to skin is expressed in terms of:
CERTAIN There is proof that the proposition is true.
PROBABLE There is at least one strong argument that the proposition is true and there are no arguments against it.
PLAUSIBLE The weight of evidence supports the proposition.
EQUIVOCAL There is an equal weight of evidence for and against the proposition.
EQUIVOCAL is recommended as being a “positive” result in Derek because the alert has still fired for the molecule, but there is an indication that there is(are) (an)other aspect(s) that Derek has considered in its calculation (such as physico-chemical properties).
DEREK NEXUS contains an expert-derived functionality that can provide negative predictions for skin sensitization. This functionality further evaluates those compounds which do not fire any skin sensitization alerts in DEREK NEXUS. The query compound is compared to a Lhasa reference set of skin sensitization data, producing the following outcomes:
• In compounds where all features in the molecule are found in accurately classified compounds from the reference set, a negative prediction is displayed: inactive.
• For those query compounds where features in the molecule are found in non-alerting skin sensitizers in the Lhasa reference set, the prediction remains negative and the misclassified features are highlighted to enable the negative prediction to be verified by expert assessment.
• In cases where features in the molecule are not found in the Lhasa reference set, the prediction remains negative and the unclassified2 features are highlighted to enable the negative prediction to be verified by expert assessment.
If a substance is predicted to be a skin sensitizer, its potency is predicted by DEREK NEXUS by calculating an EC3 value based on experimental data from the closest structurally-related substances

GLP compliance:

no

Specific details on test material used for the study:

Chemical name: Reaction mass of 2,2,4-trimethylpentane-1,3-diyldibenzoate and 3-benzoloxy-2,2,4-trimethylpentylisobutyrate and 1-isopropyl-2,2-dimethyltrimethylene diisobutyrate
CAS Number: Not available
Molecular weight: Not available – not a single substance

Key result

Parameter:

other: DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitization for the test item.

Remarks on result:

no indication of skin sensitisation

Interpretation of results:

study cannot be used for classification

Conclusions:

DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitization for the test item. Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be a non-sensitizer. Reaction Mass of 1-isopropyl-2,2 dimethyltrimethylene diisobutyrate and 3-benzoyloxy-2,2,4-trimethyl pentyl isobutyrate and 2,2,4-trimethylpentane-1,3-diyl dibenzoate is predicted to be not sensitizing to the skin.

Executive summary:

The objective of this study was to obtain a prediction on the potential for skin sensitization of the test item with the in silico model DEREK NEXUS. Derek Nexus (DN) is a knowledge-based expert system created with knowledge of structure-toxicity relationships and an emphasis on the need to understand mechanisms of action and metabolism. In this assessment version 6.0.1 of DEREK NEXUS was used. The current version contains 90 alerts for skin sensitisation and some alerts for photoallergenicity.

The level of likelihood of a structure being sensitizing to skin is expressed in terms of:

CERTAIN There is proof that the proposition is true.

PROBABLE There is at least one strong argument that the proposition is true and there are no arguments against it.

PLAUSIBLE The weight of evidence supports the proposition.

EQUIVOCAL There is an equal weight of evidence for and against the proposition.

DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitization for the test item. Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be a non-sensitizer. The test item is predicted to be not sensitizing to the skin.

This result is used within a weight-of-evidence approach to complete the endpoint skin sensitization.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

31 Jul 2018 - 12 Sep 2018

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))

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

Batch (Lot) Number: V046812101
Expiry date: 26 April 2019 (expiry date)
Physical Description: Clear colourless liquid
Storage Conditions: At room temperature

Details on the study design:

A. Preparation of Solutions for Cysteine Reactivity Assay

Synthetic Peptide Containing Cysteine (SPCC) Stock Solution
A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 10.0 mg of SPCC in 19.96 mL phosphate buffer pH 7.5

SPCC Reference Control Solutions
Three 0.5 mM SPCC reference control (RC) solutions (RCcysA, RCcysB and RCcysC) were prepared in amber vials by mixing 750 µL of the 0.667 mM SPCC stock solution with 250 µL ACN.

SPCC Calibration Curve
A SPCC calibration curve was prepared as described in the table below.

Preparation of SPCC Calibration Curve
SPCC calibration solutions SPCC concentration (mM) Preparation
STDcys1 0.534 1600 µL stock solution of 0.667 mM SPCC + 400 µL ACN
STDcys2 0.267 1 mL STDcys1 + 1 mL STDcys7
STDcys3 0.133 1 mL STDcys2 + 1 mL STDcys7
STDcys4 0.067 1 mL STDcys3 + 1 mL STDcys7
STDcys5 0.033 1 mL STDcys4 + 1 mL STDcys7
STDcys6 0.017 1 mL STDcys5 + 1 mL STDcys7
STDcys7 0 8 mL phosphate buffer (pH 7.5) + 2 mL ACN

Co-elution Control, Test Item and Positive Control Samples
The co-elution control (CC) samples, test item samples and the cinnamic aldehyde positive control samples (PC) were prepared as described in the table below.

Preparation of Co-elution Control, Test Item and Positive Control Samples
Sample Replicates Sample code Preparation
Co-elution control (CC) 1 CCcys-209637/A 750 µL Phosphate buffer pH 7.5 200 µL + ACN50 µL + 209637/A test solution (100 mM)
Cinnamic aldehyde (PC) 3 PCcys-1 to PCcys-3 750 µL Stock solution of 0.667 mM SPCC + 200 µL ACN + 50 µL Cinnamic aldehyde solution (100 mM in ACN)
Test item 209637/A 3 209637/A-cys-1 to 209637/A-cys-3 750 µL Stock solution of 0.667 mM SPCC + 200 µL ACN + 50 µL 209637/A test solution (100 mM)

B. Preparation of Solutions for Lysine Reactivity Assay

Synthetic Peptide Containing Lysine (SPCL) Stock Solution
A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 11.2 mg of SPCL in 21.62 mL of ammonium acetate buffer pH 10.2 followed by stirring for 5 minutes.

SPCL Reference Control Solutions
Three 0.5 mM SPCL reference control (RC) solutions (RClysA, RClysB and RClysC) were prepared in amber vials by mixing 750 µL of the 0.667 mM SPCL stock solution with 250 µL ACN.

SPCL Calibration Curve
A SPCL peptide calibration curve was prepared as described in the table below.

Preparation of SPCL Calibration Curve
SPCL calibration solutions SPCL concentration
(mM) Preparation
STDlys1 0.534 1600 µL stock solution of 0.667 mM SPCL + 400 µL ACN
STDlys2 0.267 1 mL STDlys1 + 1 mL STDlys7
STDlys3 0.133 1 mL STDlys2 + 1 mL STDlys7
STDlys4 0.067 1 mL STDlys3 + 1 mL STDlys7
STDlys5 0.033 1 mL STDlys4 + 1 mL STDlys7
STDlys6 0.017 1 mL STDlys5 + 1 mL STDlys7
STDlys7 0 8 mL ammonium acetate buffer (pH 10.2) + 2 mL ACN

Co-elution Control, Test Item and Positive Control Samples
The co-elution control (CC) samples, test item samples and the cinnamic aldehyde positive control samples (PC) were prepared as described in the table below.

Preparation of Co-elution Control, Test Item and Positive Control Samples
Sample Replicates Sample code Preparation
Co-elution control (CC) 1 CClys-209637/A 750 µL Ammonium acetate buffer pH 10.2 + 250 µL 209637/A test solution (100 mM)
Cinnamic aldehyde (PC) 3 PClys-1 to PClys-3 750 µL Stock solution of 0.667 mM SPCL + 250 µL Cinnamic aldehyde solution (100 mM in ACN)
Test item 209637/A 3 209637/A-lys-1 to 209637/A-lys-3 750 µL Stock solution of 0.667 mM SPCL + 250 µL 209637/A test solution (100 mM)

C. Sample Incubations
After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in the autosampler in the dark and incubated at 25±2.5°C. The incubation time between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample was 23.5 hours. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 30 hours.
Prior to HPLC PDA analysis the samples were visually inspected for precipitation. The samples that showed phase separation were centrifuged (at 400 g) for 5 minutes at room temperature.

D. HPLC-PDA Analysis
SPCC and SPCL peak areas in the samples were measured by HPLC PDA. Sample analysis was performed using the following systems:
System 1 (used for Cysteine Reactivity Assay):
• Surveyor MS HPLC pump (Thermo Scientific, Breda, The Netherlands)
• MPS 3C autosampler (DaVinci, Rotterdam, The Netherlands)
• LC Column oven 300 (Thermo Scientific)
• Surveyor PDA detector (Thermo Scientific)
System 2 (used for Lysine Reactivity Assay):
• Surveyor MS HPLC pump (Thermo Scientific, Breda, The Netherlands)
• HTC PAL autosampler (DaVinci, Rotterdam, The Netherlands)
• Column Oven #151006 (Grace, Worms, Germany)
• Surveyor PDA detector (Thermo Scientific)

Positive control results:

The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 71.6% ± 0.1%. 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 49.2% ± 0.5%. 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: 1

Parameter:

other: SPCC depletion

Value:

19.1

Key result

Run / experiment:

other: 1

Parameter:

other: SPCL depletion

Value:

0.1

Key result

Run / experiment:

other: 1

Parameter:

other: Mean of SPCC and SPCL depletion

Value:

9.6

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

A. Acceptability of the Cysteine Reactivity Assay
1) The correlation coefficient (r2) of the SPCC standard calibration curve was 0.9991. Since the r2 was >0.99, the SPCC standard calibration curve was accepted.
2) The mean peptide concentration of Reference Controls A was 0.510 ± 0.003 mM while the mean peptide concentration of Reference Controls C was 0.507 ± 0.008 mM. The means of Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCC Depletion.
3) The Coefficient of Variation (CV) of the peptide areas for the nine Reference Controls B and C was 1.4%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.
4) The mean area ratio (A220/A258) of the Reference Control samples was 17.38. The mean A220/A258 ratio ± 10% range was 15.64-19.12. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.
5) The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 71.6% ± 0.1%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).

B. Acceptability of the Lysine Reactivity Assay
1) The correlation coefficient (r2) of the SPCL standard calibration curve was 0.9995. Since the r2 was >0.99, the SPCL standard calibration curve was accepted.
2) The mean peptide concentration of Reference Controls A was 0.482 ± 0.012 mM while the mean peptide concentration of Reference Controls C was 0.486 ± 0.018 mM. The means of Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCL Depletion.
3) The CV of the peptide areas for the nine Reference Controls B and C was 4.3%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.
4) The mean area ratio (A220/A258) of the Reference Control samples was 16.45. The mean A220/A258 ratio ± 10% range was 14.81-18.10. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.
5) The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 49.2% ± 0.5%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).

 

Upon preparation as well as after incubation of the SPCC and SPCL test item samples, a phase separation was observed.

An overview of the individual results of the cysteine and lysine reactivity assays as well as the mean of the SPCC and SPCL depletion are presented in the table below. In the cysteine reactivity assay the test item showed 19.1% SPCC depletion while in the lysine reactivity assay the test item showed 0.1% SPCL depletion. The mean of the SPCC and SPCL depletion was 9.6% and as a result the test item was positive in the DPRA and was classified in the “lowreactivityclass” when using the Cysteine 1:10 / Lysine 1:50 prediction model. 

SPCC and SPCL Depletion, DPRA Prediction and Reactivity Classification for the Test Item

Test item	SPCC depletion		SPCL depletion		Mean of SPCC and SPCL depletion	DPRA prediction and reactivity classification
	Mean	± SD	Mean	± SD		Cysteine 1:10 / Lysine 1:50 prediction model
Test substance	19.1%	±2.1%	0.1%	±0.2%	9.6%	Positive: Low reactivity

SD = Standard Deviation.

 

Interpretation of results:

study cannot be used for classification

Conclusions:

Reaction Mass of 1-isopropyl-2,2 dimethyltrimethylene diisobutyrate and 3-benzoyloxy-2,2,4-trimethyl pentyl isobutyrate and 2,2,4-trimethylpentane-1,3-diyl dibenzoate was positive in the DPRA and was classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Executive summary:

The objective of the Direct Peptide Reactivity Assay (DPRA) was to determine the reactivity of the test substance towards model synthetic peptides containing either cysteine or lysine, and to categorize the test item in one of four classes of reactivity for supporting the discrimination between skin sensitizers and non-sensitizers.

The DPRA is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 hours incubation with the test item at 25°C. The synthetic peptides contain phenylalanine to aid in the detection. The relative peptide concentration is measured by high-performance liquid chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm. 

The test substance was positive in the DPRA and was classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. However, since phase separation was observed after the incubation period for both SPCC and SPCL, one cannot be sure how much test item remained in the solution to react with the peptides. Consequently, the percentages of SPCC and SPCL depletion might be underestimated.

This result is used within a weight-of-evidence approach to complete the endpoint skin sensitization.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

01 Aug 2018 - 21 Sep 2018

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)

GLP compliance:

yes

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

Batch (Lot) Number: V046812101
Expiry date: 26 April 2019 (expiry date)
Physical Description: Clear colourless liquid
Purity/Composition: See Certificate of Analysis
Storage Conditions: At room temperature

Details on the study design:

Dose formulations:
In the main experiments the test item was dissolved in dimethyl sulfoxide (DMSO) at 40 and 5 mg/mL (colourless) in experiment 1 and 2, respectively. From this stock 11 spike solutions in DMSO were prepared (2-fold dilution series). The stock and spike solution were diluted 25-fold with exposure medium. These solutions were diluted 4-fold in the assay resulting in final test concentrations of 400, 200, 100, 50, 25, 13, 6.3, 3.1, 1.6, 0.78, 0.39 and 0.20 µg/mL in experiment 1 and of 50, 40, 32, 36, 20, 16, 13, 10, 8.4, 6.7, 5.4 and 4.3 µg/mL in experiment 2 (final concentration DMSO of 1%).

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). The KeratinoSens™ cell line was generated by and obtained from Givaudan (Duebendorf, Switzerland). 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 from the frozen stock (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.

Basic medium
Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum (Life Technologies, Bleiswijk, The Netherlands).

Maintenance medium
Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum and geneticin (500 µg/mL).

Exposure medium
Dulbecco’s minimal (DMEM glutamax) supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.

Environmental conditions
All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 82 – 100%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.5 – 37.0°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature and humidity occurred due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Subculturing
Cells were subcultured upon reaching 80-90% confluency. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock, and were not cultured for more than 25 passages from the frozen stock (P+25).

Experimental Design

Plating of Cells
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 in the incubator. The passage number used was p+7 in experiment 1 and p+10 in experiment 2.

Treatment of Cells
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 covered with foil and then incubated for about 48 hours ± 1 h at 37±1.0oC in the presence of 5% CO2. In total 2 experiments were performed.

Luciferase Activity Measurement
The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega (Leiden, The Netherlands) 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 the TECAN Infinite® M200 Pro Plate Reader to assess the quantity of luciferase (integration time two seconds).

Cytotoxicity Assessment
For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1; Sigma, Zwijndrecht, The Netherlands) and cells were incubated for 3 - 4 hours at 37°C ± 1.0°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution (Sigma, Zwijndrecht, The Netherlands) to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.

Positive control results:

Experiment 1: The positive control EDMG caused a dose related induction of the luciferase activity.The Imax was 2.54 and the EC1.5 57 μM.
Experiment 2: The positive control EDMG caused a dose related induction of the luciferase activity.The Imax was 2.57 and the EC1.5 79 μM.

Key result

Run / experiment:

other: 1

Parameter:

other: EC1.5

Value:

8.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 1

Parameter:

other: Imax

Value:

2.64

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 1

Parameter:

other: IC30 (ug/ml)

Value:

21

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 1

Parameter:

other: IC50 (ug/ml)

Value:

24

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 2

Parameter:

other: EC1.5

Value:

8.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 2

Parameter:

other: Imax

Value:

2.06

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 2

Parameter:

other: IC30 (ug/ml)

Value:

18

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 2

Parameter:

other: IC50 (ug/ml)

Value:

20

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Both tests passed the acceptance criteria:
• The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration.
• The EC1.5 of the positive control was within two standard deviations of the historical mean (57 µM and 79 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.54-fold and 2.57-fold in experiment 1 and 2, respectively).
• Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (4.4% and 5.0% in experiment 1 and 2, respectively).

The test substance was evaluated for the ability to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway. An overview of the viability and luciferase activity induction is summarized in Table1. The results of the positive control are summarized in Table2.

Two independent experiments were performed. The cells were in these experiments incubated with the test substance in a concentration range of 0.20 – 400 µg/mL (2-fold dilution steps) in experiment 1 and of 4.3 – 50 µg/mL (1.25-fold dilution steps) in experiment 2 for 48 hours± 1 h. The 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 only observed at the start of the incubation period at 100 µg/mL and upwards in the 96-well plates.

·          The tes substance showed toxicity. The calculated IC₃₀was 21 µg/mL and the calculated IC₅₀was 24 µg/mL. 

·          A dose related luminescence activity induction was observed after treatment with the test substance. The I_maxwas 2.64 and the EC_1.58.7 µg/mL.

·          The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The I_maxwas 2.54 and the EC_1.557 µM. 

Experiment 2

·          No precipitation was observed at the start and end of the incubation period in the 96-well plates.

·          The test substance showed toxicity. The calculated IC₃₀was 18 µg/mL and the calculated IC₅₀was 20 µg/mL. 

·          A dose related luminescence activity induction was observed after treatment with the test substance. The I_maxwas 2.06 and the EC_1.58.5 µg/mL.

·          The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The I_maxwas 2.57 and the EC_1.579 µM. 

Table 1

Concentration (µg/mL)	0.20	0.4	0.8	1.6	3	6	13	25	50	100	200	400
Exp 1 luminescence	0.98	1.07	1.02	1.13	1.07	1.19	1.99^***	2.64^***	1.19	0.19	0.05	0.06
Exp 1 viability (%)	103.5	102.2	91.6	93.6	95.3	102.2	131.8	43.6	0.4	0.5	0.1	0.0
Concentration (µg/mL)	4.29	5.4	6.7	8.4	10	13	16	20	26	32	40	50
Exp 2 luminescence	1.09	1.21	1.24	1.49	1.63^***	1.84^***	1.85^***	2.03^***	2.06^***	1.34	1.14	0.56
Exp 2 viability (%)	104.7	108.6	114.6	126.2	122.6	112.9	84.1	46.5	27.4	12.3	6.5	0.8

Table 2

Concentration (µM)	7.8	16	31	63	125	250
Exp 1 luminescence	0.96	1.06	1.30	1.54^***	1.89^***	2.54^***
Exp 1 viability (%)	106.4	107.4	112.8	117.2	120.0	117.5
Exp 2 luminescence	1.11	1.07	1.25	1.38	1.84^**	2.57^***
Exp 2 viability (%)	105.6	115.0	108.4	111.4	112.1	112.9

Interpretation of results:

study cannot be used for classification

Conclusions:

Reaction Mass of 1-isopropyl-2,2 dimethyltrimethylene diisobutyrate and 3-benzoyloxy-2,2,4-trimethyl pentyl isobutyrate and 2,2,4-trimethylpentane-1,3-diyl dibenzoate is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate the ability of test substance to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSensTM assay. The study procedures described in this report were based on the most recent OECD guideline.

The test item was dissolved in dimethyl sulfoxide at 40 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 of0.20 – 400 µg/mL (2-fold dilution series). The highest test concentration was thehighest dose required in the current guideline. In the second experiment, a more narrow dose-response analysis was performed using a lower dilution factor of 1.25-fold to investigate the induction at 13 µg/mL in experiment 1 in more detail. No precipitate was observed at any dose level tested. Two independent experiments were performed.

The test substance showed toxicity (IC₃₀ values of 21 µg/mL and 18 µg/mL and IC₅₀values of 24 µg/mL and 20 µg/mL in experiment 1 and 2, respectively). A biologically relevant, dose-related induction of the luciferase activity (EC_1.5 values of 8.7 µg/mL and 8.5 µg/mL in experiment 1 and 2, respectively) was measured in both experiments. The maximum luciferase activity induction (I_max) was 2.64-fold and 2.06-fold in experiment 1 and 2 respectively. The test substance is classified as positive in the KeratinoSens^TM assay since positive results (>1.5 -fold induction) were observed at test concentrations < 200 µg/mL with a cell viability of > 70% compared to the vehicle control.

This result is used within a weight-of-evidence approach to complete the endpoint skin sensitization.

Reference 4

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

November 14 - December 4, 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)

Deviations:

no

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:

Batch (Lot) Number: V046812101
Expiry date: 26 April 2019 (expiry date)
Physical Description: Clear colourless liquid
Purity/Composition: See Certificate of Analysis
Storage Conditions: At room temperature

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan laboratories srl, Italy
- Age at study initiation: 8-12 weeks old
- Diet (e.g. ad libitum): Certified laboratory food (MP-OS-06)


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Humidity (%): 55 +/- 10 %
- Air changes (per hr): 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

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

Concentration:

The substance was tested at three concentrations (5 %, 10 % and 25 %)

No. of animals per dose:

5 animals / dose

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: The test item was soluble in acetone/olive oil based on trial preparations performed at Charles River Den Bosch and on information provided by the Sponsor.
- Irritation: A pre-screen test 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 50% and 100% concentration. At these test item concentrations, variation in ear thickness during the observation period were more than 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.
Based on the results of the initially treated animals, four additional animals were treated in a similar manner with two lower concentrations (10% and 25%). At a 25% and 10% test item concentration, no signs of systemic toxicity and very slight irritation of the ears were seen. Since the selection criteria were met, the 25% concentration was selected as highest concentration for the main study.
- Lymph node proliferation response: The assessment of lymph node proliferation and necropsy were not performed in the pre-screen test.
- Systemic toxicity; Erythema scores:
TS 1(%) animal Day 1 Day 2 Day 3 Day 4 Day 5 Day 6
bw(g) erythema erythema erythema erythema erythema erythema bw
(g)
left right left right left right left right left right left right

100 3 20.6 1 1a 1 1ab 1 1ab 1 1ab 1 1b 1 1 21.9
4 19.6 1 1a 1 1ab 1 1ab 1 1ab 1 1b 1 0 20.8

50 1 20.4 1 0a 1 1ab 1 1ab 1 1ab 1 0 0 0 21.0
2 19.5 1 0a 1 1ab 1 1ab 0 1ab 0 0 0 0 20.7

25 7 22.0 0 0 1 1 1 1 0 0 0 0 0 0 22.6
8 21.2 0 0 1 1 1 1 0 0 0 0 0 0 21.2

10 5 22.3 0 0 0 0 1 1 0 0 0 0 0 0 22.0
6 23.1 0 0 0 0 1 1 0 0 0 0 0 0 22.6

a.Ptosis, b. Hunched posture
Grading erythema and eschar formation (Left = dorsal surface of left ear; right = dorsal surface of right ear):
0 = No erythema
1 = Very slight erythema (barely perceptible)

- Ear thickness measurements:
TS 1 (%) Animal Day 1 Day 3 Day 6
left right left right left right
(mm) (mm) (mm) %2 (mm) % 2 (mm) % 2 (mm) % 2

100 3 0.175 0.170 0.200 14 0.195 15 0.250 43 0.255 50
4 0.195 0.180 0.220 13 0.215 19 0.245 26 0.250 39

50 1 0.185 0.190 0.195 5 0.200 5 0.240 30 0.245 29
2 0.180 0.175 0.195 8 0.205 17 0.240 33 0.235 34

25 7 0.220 0.215 0.240 9 0.245 14 0.240 9 0.240 12
8 0.235 0.225 0.235 0 0.240 7 0.245 4 0.245 9

10 5 0.225 0.220 0.230 2 0.230 5 0.220 -2 0.230 5
6 0.230 0.225 0.235 2 0.225 0 0.230 0 0.230 2

2 Percent increase compared to Day 1 pre-dose value. A 25% value is used as the threshold for selection for
use in the main study.


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
Animals were assigned to the study at the discretion of the coordinating biotechnician according to body weights, with all animals within ± 20% of the sex mean. Animals in poor health or at extremes of body weight range were not assigned to the study.
Before the initiation of dosing, a health inspection was performed and any assigned animal considered unsuitable for use in the study were replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions.

- Criteria used to consider a positive response: If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer.

TREATMENT PREPARATION AND ADMINISTRATION:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements.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) with the test item, at approximately the same time on each day. 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.

Positive control substance(s):

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

Statistics:

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.
If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer.

Positive control results:

A reliability check is carried out at regular intervals to check the sensitivity of the test system and the reliability of the experimental techniques as used by Charles River Den Bosch. In this study, performed in Jun 2018, females of the CBA/J mouse strain (Janvier, Le Genest-Saint-Isle, France) were checked for sensitivity to Alpha- Hexylcinnamaldehyde, technical grade (HCA).
Group % HCA mean
DPM ± SEM SI ± SEM
1 0% (AcOO) 378 ± 58 1.0 ± 0.2
2 5% 419 ± 27 1.1 ± 0.1
3 10% 754 ± 25 2.0 ± 0.1
4 25% 2076 ± 251 5.5 ± 0.7
Five females per group.

The SI values calculated for the item concentrations 5, 10 and 25% were 1.1, 2.0 and 5.5 respectively. An EC3 value of 14.3% was calculated using linear interpolation.
The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5%. The results of the 6 monthly HCA reliability checks of the recent years were 13.4, 14.1, 17.3, 9.8, 17.8 and 19.2%.
Based on the results, it was concluded that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.

Key result

Parameter:

SI

Value:

1.2

Test group / Remarks:

5% test item

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

10% test item

Key result

Parameter:

SI

Value:

1.3

Test group / Remarks:

25% test item

Key result

Parameter:

EC3

Value:

> 25

Remarks on result:

other: Based on the absence of a dose response, it is not expected that higher concentrations may lead to a SI ≥ 3.

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
group TS 1(%) animal Size nodes DPM / animal meanDPM ± SEM meanSI ± SEM
left right

1 0 1 n n 394 779 ± 98 1.0 ± 0.1
2 n n 819
3 n n 857
4 n n 949
5 n n 875

2 5 6 n n 636 949 ± 101 1.2 ± 0.1
7 n n 1020
8 n n 990
9 n n 853
10 n n 1247

3 10 11 n n 281 805 ± 169 1.0 ± 0.2
12 n n 830
13 n n 1298
14 n n 640
15 n n 974

4 25 16 n n 1083 1025 ± 158 1.3 ± 0.2
17 n n 1238
18 n n 1419
19 n n 886
20 n n 500


DETAILS ON STIMULATION INDEX CALCULATION
Mean DPM/animal values for the experimental groups treated with test item concentrations 5, 10 and 25% were 949, 805 and 1025 DPM, respectively. The mean DPM/animal value for the vehicle control group was 779 DPM. The SI values calculated for the test item concentrations 5, 10 and 25% were 1.2, 1.0 and 1.3, respectively.

EC3 CALCULATION
It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 25%. Based on the absence of a dose response, it is not expected that higher concentrations may lead to a SI ≥ 3.

CLINICAL OBSERVATIONS:
The very slight erythema of the ears as shown by two animals treated at 10% on Days 2 and/or 3 and all animals treated at 25% between Days 2 and 6 was considered not to have a toxicologically significant effect on the activity of the nodes.
No mortality occurred and no clinical signs of systemic toxicity were observed in the animals.

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

Body weights and skin reactions

group	TS^1 (%)	animal	Day 1			Day 2		Day 3		Day 4		Day 5		Day 6
			bw (g)^2	erythema^3		erythema		erythema		erythema		erythema		erythema		bw (g)
				left	right	left	right	left	right	left	right	left	right	left	right
1	0	1	22.4	0	0	0	0	0	0	0	0	0	0	0	0	23.2
		2	21.4	0	0	0	0	0	0	0	0	0	0	0	0	20.5
		3	20.4	0	0	0	0	0	0	0	0	0	0	0	0	20.2
		4	19.7	0	0	0	0	0	0	0	0	0	0	0	0	20.6
		5	20.9	0	0	0	0	0	0	0	0	0	0	0	0	18.6
2	5	6	21.4	0	0	0	0	0	0	0	0	0	0	0	0	22.2
		7	20.3	0	0	0	0	0	0	0	0	0	0	0	0	20.6
		8	22.1	0	0	0	0	0	0	0	0	0	0	0	0	21.9
		9	19.7	0	0	0	0	0	0	0	0	0	0	0	0	19.8
		10	18.9	0	0	0	0	0	0	0	0	0	0	0	0	18.7
3	10	11	22.0	0	0	1	0	1	0	0	0	0	0	0	0	22.7
		12	21.8	0	0	0	0	0	0	0	0	0	0	0	0	22.2
		13	20.3	0	0	0	1	0	0	0	0	0	0	0	0	21.1
		14	21.5	0	0	0	0	0	0	0	0	0	0	0	0	23.6
		15	20.5	0	0	0	0	0	0	0	0	0	0	0	0	20.5
4	25	16	21.8	0	0	0	0	0	0	1	1	1	1	1	1	22.8
		17	19.5	0	0	1	1	1	1	1	1	1	1	1	1	19.2
		18	21.1	0	0	1	1	1	1	1	1	1	1	1	1	21.4
		19	21.6	0	0	1	1	1	1	1	1	1	1	1	1	21.9
		20	20.8	0	0	1	1	1	1	1	1	1	1	1	1	21.9

¹  TS = test item (% w/w).

²  Body weight (grams).

³  Grading erythema and eschar formation(Left = dorsal surface of left ear; right = dorsal surface of right ear):

    0 = No erythema

    1 = Very slight erythema (barely perceptible)

Relative Size Lymph nodes, Radioactivity counts (DPM) and Stimulation Index (SI)

group	TS^1 (%)	animal	Size nodes^2		DPM^3/ animal	mean DPM ± SEM^4			mean SI ± SEM
			left	right
1	0	1	n	n	394	779	±	98	1.0	±	0.1
		2	n	n	819
		3	n	n	857
		4	n	n	949
		5	n	n	875
2	5	6	n	n	636	949	±	101	1.2	±	0.1
		7	n	n	1020
		8	n	n	990
		9	n	n	853
		10	n	n	1247
3	10	11	n	n	281	805	±	169	1.0	±	0.2
		12	n	n	830
		13	n	n	1298
		14	n	n	640
		15	n	n	974
4	25	16	n	n	1083	1025	±	158	1.3	±	0.2
		17	n	n	1238
		18	n	n	1419
		19	n	n	886
		20	n	n	500

¹  TS = test item (% w/w).

²  Relative size auricular lymph nodes (-, -- or ---: degree of reduction, +, ++ or +++: degree of enlargement, n: considered to be normal).

³    DPM= Disintegrations per minute.

⁴    SEM = Standard Error of the Mean.

Interpretation of results:

GHS criteria not met

Conclusions:

The skin sensitising potential of Reaction Mass of 1-isopropyl-2,2 dimethyltrimethylene diisobutyrate and 3-benzoyloxy-2,2,4-trimethyl pentyl isobutyrate and 2,2,4-trimethylpentane-1,3-diyl dibenzoate was assessed using murine local lymph node assay (LNNA). The substance is non-sensitiser in the LLNA study under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate whether the substance induces skin sensitization in mice after three epidermal exposures of the animals

Test item concentrations selected for the main study were based on the results of a pre-screen test. At a 100% and 50% test item concentration, variation in ear thickness during the observation period were more than 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 a 25% and 10% test item concentration, no signs of systemic toxicity and very slight irritation of the ears were seen. Since the selection criteria were met, the 25% concentration was selected as highest concentration for the main study.

 

In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 5, 10 or 25% 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 with³H-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 5, 10 and 25% were 949, 805 and 1025 DPM, respectively. The mean DPM/animal value for the vehicle control group was 779 DPM. The SI values calculated for the test item concentrations 5, 10 and 25% were 1.2, 1.0 and 1.3, respectively.

 

Since there was no indication that the test item elicits a SI≥3 when tested up to 25%, the test substance was not considered to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3)(if any) exceeds 25%. Based on the absence of a dose response, it is not expected that higher concentrations may lead to a SI≥3.

The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.

Based on these results, the test item would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Reference 5

Endpoint:

skin sensitisation, other

Remarks:

conclusion on the in vitro/in chemico/in silico studies

Type of information:

other: expert report

Adequacy of study:

supporting study

Study period:

November 2018

Reliability:

1 (reliable without restriction)

Type of study:

other: summary of available report

Specific details on test material used for the study:

Chemical name Reaction mass of 2,2,4-trimethylpentane-1,3-diyldibenzoate and 3-benzoloxy-2,2,4-trimethylpentylisobutyrate and 1-isopropyl-2,2-dimethyltrimethylene diisobutyrate
CAS number Not available
Molecular weight Not available – not a single substance
UVCB No

DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitization for the test item.

Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be a non-sensitizer. The test substance is predicted to be not sensitizing to the skin.

 

A valid DPRA test was performed according to OECD TG 442C and GLP principles. For the DPRA, the test substance was dissolved in acetonitrile at 100 mM and formed a clear solution by visual inspection.No co-elution of the test item with SPCC or SPCL was observed.In the cysteine reactivity assay the test item showed 19.1% SPCC depletion, and in the lysine reactivity assay the test item showed 0.1% SPCL depletion. The mean of the SPCC and SPCL depletion was 9.6%. As a result, the test substance was positive in the DPRA and was classified in the “lowreactivityclass” when using the Cysteine 1:10 / Lysine 1:50 prediction model. However, since phase separation was observed after the incubation period for both SPCC and SPCL,one cannot be sure how much test item remained in the solution to react with the peptides. Consequently,the percentages of SPCC and SPCL depletion might be underestimated.

 

A valid KeratinoSens^TM assay was performed according to OECD TG 442D and GLP principles. For the KeratinoSens^TM assay, the test item was dissolved in DMSO to final concentrations of 40 mg/mL and 5 mg/mL. From both stocks 11 spike solutions in DMSO were prepared. The stock and spike solution were diluted 25-fold with exposure medium. These solutions were diluted 4-fold in the assay resulting in final test concentrations. Two independent tests were performed. In the first experiment the cells were incubated with the test item in a concentration range of 0.2–400µg/mL (2-fold dilution series) for 48 hours. Precipitation was observed at the start the incubation period at concentrations of 100 µg/mL and upwards in experiment 1. Due to toxicity, the test concentrations used for the second experiment were in a concentration range of 4.3–50 µg/mL (1.25-fold dilution steps). The substance showed toxicity (IC₃₀ values of 21µg/mL and 18µg/mL and IC₅₀values of 24µg/mL and 20 µg/mL in experiment 1 and 2, respectively). A biologically relevant, dose-related induction of the luciferase activity (EC_1.5 values of 8.7µg/mLand 8.5µg/mLin experiment 1 and 2, respectively) was measured in both experiments. The maximum luciferase activity induction (I_max) was 2.64-fold and 2.06-fold in experiment 1 and 2 respectively. The test substance is classified as positive in the KeratinoSens^TM assay since positive results (>1.5-fold induction) were observed at test concentrations < 200µg/mLwith a cell viability of >70% compared to the vehicle control.

Conclusions:

In conclusion, from the current study data it can be concluded that Reaction Mass of 1-isopropyl-2,2 dimethyltrimethylene diisobutyrate and 3-benzoyloxy-2,2,4-trimethyl pentyl isobutyrate and 2,2,4-trimethylpentane-1,3-diyl dibenzoate has skin sensitizing properties, but information on skin sensitization potency is lacking in these studies. In the absence of a reliable and acceptable in vitro skin sensitization potency test, an in vivo test needs to be performed.

Executive summary:

DEREK NEXUS predicted a negative result, whereas the DPRA and the KeratinoSens^TM assay were both positive. Based on the two in vitro assays the test substance is considered to be a skin sensitizer. Due to the negative result in DEREK, no prediction is available on skin sensitizing potency. In addition, current in vitro assays lack to provide reliable information on skin sensitization potency. Therefore, performance of an additional in vitro assay (STEP 2) addressing the activation of dendritic cells would not yield additional information on skin sensitizing potency, irrespective of a negative or positive result. Therefore it is considered justified to omit further in vitro testing and perform an in vivo test to get reliable information on skin sensitizing potency for the substance.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Weight of evidence approach was used to assess the sensitisation potential of the substance. A DEREK assessment, DPRA, KeratinoSens^TM assay and local lympnh node assay (LLNA) were performed.

 

In vitro studies

In vitro Keratinosens study was conducted according to OECD TG 442D and GLP principles by Woutersen, J.A.J. (2018).

The objective of this study was to evaluate the ability of Reaction Mass of 1-isopropyl-2,2 dimethyltrimethylene diisobutyrate and 3-benzoyloxy-2,2,4-trimethyl pentyl isobutyrate and 2,2,4-trimethylpentane-1,3-diyl dibenzoate

to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway. The test item was dissolved in dimethyl sulfoxide at 40 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 of0.20 – 400 µg/mL (2-fold dilution series). The highest test concentration was thehighest dose required in the current guideline. In the second experiment, a more narrow dose-response analysis was performed using a lower dilution factor of 1.25-fold to investigate the induction at 13 µg/mL in experiment 1 in more detail. No precipitate was observed at any dose level tested. Two independent experiments were performed.

The test item showed toxicity (IC30 values of 21 µg/mL and 18 µg/mL and IC50 values of 24 µg/mL and 20 µg/mL in experiment 1 and 2, respectively). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 values of 8.7 µg/mL and 8.5 µg/mL in experiment 1 and 2, respectively) was measured in both experiments. The maximum luciferase activity induction (Imax) was 2.64-fold and 2.06-fold in experiment 1 and 2 respectively. The tet item is classified as positive in the KeratinoSensTM assay since positive results (>1.5 -fold induction) were observed at test concentrations < 200 µg/mL with a cell viability of > 70% compared to the vehicle control.

 

In vitro Direct Peptide Reactivity Assay (DPRA) was performed according to OECD TG442D and GLP principles by Reinen, J. (2018).

The objective of the DPRA was to determine the reactivity of Reaction Mass of 1-isopropyl-2,2 dimethyltrimethylene diisobutyrate and 3-benzoyloxy-2,2,4-trimethyl pentyl isobutyrate and 2,2,4-trimethylpentane-1,3-diyl dibenzoate

towards model synthetic peptides containing either cysteine or lysine, and to categorize the test item in one of four classes of reactivity for supporting the discrimination between skin sensitizers and non-sensitizers.

The DPRA is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 hours incubation with the test item at 25°C. The synthetic peptides contain phenylalanine to aid in the detection. The relative peptide concentration is measured by high-performance liquid chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm. The substance was positive in the DPRA and was classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. However, since phase separation was observed after the incubation period for both SPCC and SPCL, one cannot be sure how much test item remained in the solution to react with the peptides. Consequently, the percentages of SPCC and SPCL depletion might be underestimated.

 

Further evidence on the sensitization potential of Reaction Mass of 1-isopropyl-2,2 dimethyltrimethylene diisobutyrate and 3-benzoyloxy-2,2,4-trimethyl pentyl isobutyrate and 2,2,4-trimethylpentane-1,3-diyl dibenzoate

was obtained using QSAR model (Derek Nexus)(Barentsen, H.M., 2018). Derek Nexus is a knowledge-based expert system created with knowledge of structure-toxicity relationships and an emphasis on the need to understand mechanisms of action and metabolism. In this assessment version 6.0.1 of DEREK NEXUS was used. The current version contains 90 alerts for skin sensitisation and some alerts for photoallergenicity.

The level of likelihood of a structure being sensitizing to skin is expressed in terms of:

CERTAIN There is proof that the proposition is true.

PROBABLE There is at least one strong argument that the proposition is true and there are no arguments against it.

PLAUSIBLE The weight of evidence supports the proposition.

EQUIVOCAL There is an equal weight of evidence for and against the proposition.

Derek Nexus did not yield any alerts for skin sensitization for the test item. Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be a non-sensitizer. The test substance is predicted to be not sensitizing to the skin.

From the in vitro/in chemico/in silico data it can be concluded that the test substance has skin sensitizing properties, but information on skin sensitization potency is lacking in these studies. In the absence of a reliable and acceptable in vitro skin sensitization potency test, an in vivo test was performed.

In vivo Study

The objective of the mouse Local Lymph node Assay (LLNA) was to evaluate whether Reaction Mass of 1-isopropyl-2,2 dimethyltrimethylene diisobutyrate and 3-benzoyloxy-2,2,4-trimethyl pentyl isobutyrate and 2,2,4-trimethylpentane-1,3-diyl dibenzoate

induces skin sensitization in mice after three epidermal exposures of the animals

In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 5, 10 or 25% 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 with³H-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 5, 10 and 25% were 949, 805 and 1025 DPM, respectively. The mean DPM/animal value for the vehicle control group was 779 DPM. The SI values calculated for the test item concentrations 5, 10 and 25% were 1.2, 1.0 and 1.3, respectively.

Since there was no indication that the test item elicits a SI≥3 when tested up to 25%, the test item was not considered to be a skin sensitizer. It was established that the EC3 value(the estimated test item concentration that will give a SI =3)(if any) exceeds 25%. Based on the absence of a dose response, it is not expected that higher concentrations may lead to a SI≥3.

 

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the result of the skin sensitisation studies conducted, the test item is not classified for skin sensitisation according to the CLP Regulation No. 1272/2008.