Reaction mass of diethyl adipate and diethyl glutarate and diethyl succinate

Endpoint summary

Description of key information

Based on in vitro, in chemico and in silico data considered in a weight of evidence approach, reaction mass of diethyl adipate and diethyl glutarate and diethyl succinate is not classified as skin sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 29 September 2017 to 01 February 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))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- batch No.of test material:CY71353002
- Expiration date of the lot/batch: 01 June 2019
- Purity test date: 01 June 2017
- Analytical purity: 98.2%
- Commercial name of the registered substnace: Innroad Protect

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated (2-8 °C)
- Stability under test conditions: Not assessed
- Solubility and stability of the test substance in the solvent/vehicle: Not assessed. The formulation was a colorless liquid and was used just after its preparation.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not assessed

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was pre-weighed and stored under appropriate conditions until ready to perform testing. It was diluted in the selected vehicle (acetonitrile) at 100 mM.

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:

1. TEST AND CONTROL ITEMS

1.1) Vehicle
Based on solubility results, the retained vehicle was acetonitrile.
As several test items were assayed concurrently with this vehicle, its results were shared.

1.2) Positive control
The positive control was cinnamaldehyde (CAS No. 104-55-2), batch No. MKBX8146V, supplied by Sigma Aldrich. Its molecular weight was 132.16 g/mol and the purity of the batch used was 98.9%.

As several test items were assayed concurrently, the results of the positive control were shared.
The positive control was pre-weighed and stored under appropriate conditions until ready to perform testing. It was dissolved in acetonitrile at 100 mM. The formulation was a colorless liquid and was used just after its preparation.

1.3) Co-elution control samples
In order to detect possible co-elution of the test item with a peptide, co-elution control samples were prepared by incubating the test item formulation with each buffer used to dilute the peptides. Cysteine or lysine peptides were not added to these samples.

1.4) Reference control samples
For each peptide, the analytical batch included reference control samples (sub-categorized in reference control A, B or C samples). All these control samples were prepared in triplicate and at the nominal concentration of 0.500 mM in the solvent specified in § Reference control samples preparation. These samples were used to:
- reference control A: check the accuracy of the calibration curve for peptide quantification,
- reference control B: check the stability of the peptide during analysis,
-reference control C: check that the solvent did not impact the percentage of peptide depletion.

1.5) Test item formulation preparation
The test item was pre-weighed and stored under appropriate conditions until ready to perform testing. It was diluted in the selected vehicle (acetonitrile) at 100 mM. This formulation was a colorless liquid and was used just after its preparation.


2. DESIGN OF THE DIRECT PEPTIDE REACTIVITY ASSAY

The test item was tested in one run. The run was processed as described below.

2.1) Preparation of the samples
The following samples were prepared in triplicate except for the co-elution control samples for which only one sample was prepared per peptide buffer.

2.1.1) Co-elution control samples preparation
For the co-elution control with cysteine peptide:
50 µL of test item formulation was incubated with 750 µL of cysteine peptide dilution buffer (without cysteine peptide) and 200 µL of acetonitrile.

For the co-elution control with lysine peptide:
In parallel, 250 µL of test item formulation was incubated with 750 µL of lysine peptide dilution buffer (without lysine peptide).

2.1.2) Reference control samples preparation
2.1.2.1) Reference control A and B samples
In a vial, acetonitrile was added to a volume of peptide solution (cysteine or lysine) to achieve a nominal concentration of 0.500 mM.

2.1.2.2) Reference control C samples
Reference control C samples were prepared for each solvent used to dissolve the test and positive control items.

For the reference control C prepared with cysteine peptide:
50 µL of vehicle (acetonitrile) was incubated with 750 µL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 µL of acetonitrile.

For the reference control C prepared with lysine peptide:
In parallel, 250 µL of vehicle (acetonitrile) was incubated with 750 µL of lysine peptide solution (at 0.667 mM in ammonium acetate buffer at pH 10.2).

2.1.3) Cinnamaldehyde (positive control) depletion control samples preparation
For the reactivity of cinnamaldehyde with cysteine peptide:
50 µL of cinnamaldehyde at 100 mM in acetonitrile was incubated with 750 µL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 µL of acetonitrile.

For the reactivity of cinnamaldehyde with lysine peptide:
In parallel, 250 µL of cinnamaldehyde at 100 mM in acetonitrile was incubated with 750 µL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2).

2.1.4) Test item samples preparation
For the reactivity of test item with cysteine peptide:
50 µL of test item formulation was incubated with 750 µL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 µL of acetonitrile.

For the reactivity of test item with lysine peptide:
In parallel, 250 µL of test item formulation was incubated with 750 µL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2).

2.2) Incubation of the samples
All samples (co-elution controls, reference controls, test item and positive control samples) were then incubated during 24 (± 2) hours for the lysine peptide or 26 hours and 53 minutes for the cysteine peptide (see § Study plan adherence), at 25°C and protected from light before injection into the HPLC/UV system.

At the end of the incubation period, a visual inspection of the samples was performed prior to HPLC analysis to detect precipitate or phase separation (see § Results).

Samples presenting precipitate were centrifuged at 400g for a period of 5 minutes at room temperature and only supernatants were then injected onto the HPLC/UV system. Otherwise, the vials were directly transferred onto the HPLC/UV system.

2.3) Preparation of the calibration curve samples
One set of calibration standards was prepared with each analytical sequence by spiking each peptide (lysine and cysteine) in separate solutions of 20% acetonitrile:peptide dilution buffer to obtain at least six different concentration levels ranging from 0.0167 to 0.534 mM. A dilution buffer blank was also included in the standard calibration curve.
The calibration curves were defined by the relationships between the peak area signal of the peptide versus the nominal concentration. These curves were obtained by using the appropriate mathematical model.

2.4) HPLC/UV analysis of the samples
The study samples were assayed in batches using HPLC/UV analysis.
For each peptide, the analytical sequence included at least:
- one blank sample (peptide dilution buffer),
- one calibration curve injected at the beginning of the analytical batch,
- three reference control A samples,
- the co-elution control sample,
- three reference control B samples,
- reference control C sample (replicate 1),
- positive control sample (replicate 1),
- test item study sample (replicate 1),
The injection order of the reference control C, positive control and all test item study samples were reproduced identically for replicate 2 and then replicate 3:
- three reference control B samples.
The HPLC/UV method used for the samples analysis is described in CiToxLAB France internal analytical method.

Positive control results:

The mean of the percent cysteine and percent lysine depletions of Cinnamaldehyde was 77.61% which corresponds to Hight reactivity.

Key result

Parameter:

other: mean of the percent cysteine and percent lysine depletions (%)

Value:

1.62

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 acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The study was therefore considered to be valid.

SOLUBILITY RESULTS

The test item was found soluble at 100 mM in acetonitrile (without sonication step). Therefore, this vehicle was retained.

 

EVALUATION OF THE PRESENCE OF PRECIPITATE AT THE END OF THE INCUBATION WITH PEPTIDES

At the end of the incubation period, a visual inspection of all samples (co-elution controls, reference controls, test item and positive control samples) was performed prior to HPLC analysis.

 

As precipitate was observed in the test item samples incubated with the cysteine peptide, these vials were centrifuged at 400gfor a period of 5 minutes at room temperature to force precipitate to the bottom of the vial. Positive control samples incubated with both peptides and reference control samples prepared in acetonitrile with the cysteine peptide were also centrifuged at the same conditions to force precipitate to the bottom of the vials. Only supernatants were then injected into the HPLC/UV system.

For the other samples (i.e.test item samples incubated with the lysine peptide, and reference control samples prepared in acetonitrile with the lysine peptide and co-elution samples prepared with the lysine and cysteine dilution buffer), the vials were directly transferred into the HPLC/UV system.

 

EVALUATION OF THE RESULTS

Analysis of the chromatograms of the co-elution samples indicated that the test item did not co-elute with either the lysine or the cysteine peptides. As a result, the mean percent depletion values were calculated for each peptide using the formula described in § Data analysis and calculation:

.            for the cysteine peptide, the mean depletion value was 2.61%,

.            for the lysine peptide, the mean depletion value was 0.63%.

 

The mean of the percent cysteine and percent lysine depletions was equal to 1.62%. However,it is to be noted thatprecipitate was observed at the end of the incubation with the cysteine peptide.As a consequence,the corresponding peptide depletion may be underestimated.

Since the mean of the percent cysteine and percent lysine depletions was < 6.38%, the test item was considered to have no or minimal reactivity. Therefore, the DPRA prediction is considered as negative and the test item is likely not to have any potential to cause skin sensitization, though with limitations due to test item precipitation with the cysteine peptide.

Interpretation of results:

other: The DPRA prediction is considered as negative and the test item is likely not to have any potential to cause skin sensitization.

Conclusions:

Under the experimental conditions of this study, the DPRA prediction is considered as negative and the registered substance Reaction mass of diethyl adipate and diethyl glutarate and diethyl succinate was considered to have no or minimal peptide reactivity, though with limitations due to its precipitation with the cysteine peptide.

Executive summary:

The objective of this study was to evaluate the reactivity of the test item, Reaction mass of diethyl adipate diethyl glutarate and diethyl succinate,

to synthetic cysteine and lysine peptides. This test is part of a tiered strategy for skin sensitization assessment. The design of this study was based on the OECD Guideline 442C and the study was performed in compliance with the OECD Principles of Good Laboratory Practice.

Methods

 The reactivity of the test item was evaluated in chemico by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides.The method consisted of the incubation of a diluted solution of cysteine or lysine with the test item for 24 hours. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with Ultra-Violetdetection at 220 nm.

Peptide reactivity was reported as percent depletion basedon the peptide peak area of the replicate injection and the mean peptide peak area in the three relevant reference control C samples (in the appropriate solvent).

 

Results

The test item was diluted at 100 mM inacetonitrile.

 

The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The study was therefore considered to be valid.

 

Analysis of the chromatograms of the co-elution samples indicated that the test item did not co-elute with either the lysine or the cysteine peptides. As a result, the mean percent depletion values were calculated for each peptide:

.            for the cysteine peptide, the mean depletion value was 2.61%,

.            for the lysine peptide, the mean depletion value was 0.63%.

 

The mean of the percent cysteine and percent lysine depletions was equal to 1.62%. However, it is to be noted that precipitate was observed at the end of the incubation with the cysteine peptide. As a consequence, the corresponding peptide depletion may be underestimated.

 

Since the mean of the percent cysteine and percent lysine depletions was < 6.38%, the test item was considered to have no or minimal reactivity. Therefore, the DPRA prediction is considered as negative and the test item is likely not to have any potential to cause skin sensitization, though with limitations due to test item precipitation with the cysteine peptide.

 

Conclusion

 

Under the experimental conditions of this study, the DPRA prediction is considered as negative and the test item Reaction mass of diethyl adipate diethyl glutarate and diethyl succinate was considered to have no or minimal peptide reactivity, though with limitations due to its precipitation with the cysteine peptide.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 17 August to 12 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- batch No.of test material:CY71353002
- Expiration date of the lot/batch: 01 June 2019
- Purity test date: 01 June 2017
- Analytical purity: 98.2%
- Commercial name of the registered substnace: Innroad Protect

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated (2-8 °C)
- Stability under test conditions: Not assessed
- Solubility and stability of the test substance in the solvent/vehicle: Not assessed. The formulation was a colorless liquid and was used just after its preparation.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not assessed

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: On the basis of solubility results, the test item was diluted in DMSO at 200 mM. It was then diluted in DMSO by serial dilutions, using a dilution factor of 2 in the first two runs or using a dilution factor of 1.41 in the third run (in order to check a potential dose-response relationship), to obtain a total of 12 concentrations in a 96-well plat.; this 96 well plate was called "Master plate 100x". Subsequently, each formulation of the Master plate 100x was 25-fold diluted in treatment medium in another 96 well plate called "Master plate 4x" taking care to adjust all wells to the same DMSO level.
All formulations were prepared within 4 hours before use, and kept at room temperature and protected from light until use.

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:
The test item was tested in two independent runs using cells from a different passage number. A third run was performed since the results of the first two runs were not concordant. The plates were processed as described below in the § Method.

1. Solubility assay
A solubility assay was performed prior the first treatment in order to select the vehicle (among DMSO, water for injections or treatment culture medium).

Since the test item was found soluble in DMSO at 200 mM, this stock formulation was diluted in treatment culture medium to the final concentration of 2000 µM. Then, a visual inspection of the sample was performed to evaluate the presence or absence of test item precipitate/emulsion.

2.Method for a run of KeratinoSens assay
2.1 Cell seeding for testing
- Cells were grown using general culture procedures up to 80-90% confluence,
- the day prior to treatment, cells were washed twice with D-PBS containing 0.05% EDTA, harvested, re-suspended in Maintenance medium No. 2 and counted using Trypan Blue dye. Cell concentration was adjusted to a density of 8 x 104 cells/mL,
- cells were then distributed into four 96-well plates (three white plates and one transparent plate), by adding 125 µL (representing 1 x 104 cells) per well taking care to avoid sedimentation of the cells during seeding,
- after seeding, the cells were grown for 24 (± 1) hours in the 96-well microtiter plates prior to test item addition.

2.2 Treatment
- After the 24-hour growing period, the medium was removed by aspiration and replaced by 150 µL of treatment medium,
- from the Master plate 4x, a volume of 50 µL was added to each well of the three white assay plates and 50 µL to the transparent plate for the cytotoxicity evaluation,
- all plates were covered by a sealing membrane to avoid evaporation of volatile test items and to avoid cross-contamination between wells,
- the plates were then incubated for 48 (± 2) hours at 37°C, 5% CO2, 90% humidity.

2.3 Endpoint measurements
2.3.1 Microscopic observation to evaluate the presence or absence of precipitate - transparent plate
After the 48 (± 2) hours incubation period, the presence or absence of precipitate/emulsion was determined in each well by microscopic inspection.
2.3.2 Luminescence flash signal to evaluate induction signal - white plates
- After incubation, the supernatants from the white assay plates were discarded,
- the cells were washed once with D-PBS,
- a volume of 20 µL of passive lysis buffer was added to each well and the cells were incubated for 20 (± 2) minutes at room temperature and under orbital shaking,
- the plates containing the passive lysis buffer were then placed in the luminometer for reading using the following program:
- 50 µL of the luciferase substrate was added to each well,
- 1 second after this addition, the luciferase signal was integrated for 2 seconds.
2.3.3 Absorbance signal to evaluate the cytotoxicity - transparent plate
- For the cell viability assay plate, the medium was replaced by 200 µL of treatment medium,
- a volume of 27 µL of a MTT solution at 5 mg/mL in D-PBS was then added to each well of the transparent 96-well plate,
- the plates were covered with a sealing membrane and returned at 37°C in the incubator in humidified atmosphere for 4 hours (± 10 minutes),
- at the end of the incubation period, the medium was removed and a volume of 200 µL of a 10% SDS solution was added to each well,
- the plates were covered with a sealing membrane and placed at 37°C in the incubator in humidified atmosphere for an overnight period to extract the formazan from cells,
- after the overnight incubation, the absorption of each well was determined at 600 nm using the plate reader.

Positive control results:

All acceptance criteria were fulfilled for the positive control in the three different runs.

Key result

Parameter:

other: Imax

Value:

1.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
All acceptance criteria were fulfilled for the positive and negative controls in the three different runs. The three runs was therefore considered to be valid.

1.  Solubility test

In the solubility test, the test item was found soluble in DMSO at 200 mM. Therefore, this vehicle was selected for the preparation of the test item stock formulations.

No precipitate or emulsion was observed once the test item stock formulation was diluted in the treatment culture medium to a final concentration of 2000 µM.

  

2.   Keratinosens run

2.1      First run

All acceptance criteria were fulfilled for the positive and negative controls. The run was therefore considered to be valid.

The criterion "the average induction (I_max) in the three replicate plates for the positive control at 64 µM should be between 2 and 8" was not fulfilled (i.e.I_maxof 8.93). However, since a clear dose-response with increasing luciferase activity at increasing concentrations was obtained for the positive control, this was considered not to have any impact on the validity of the results of this run.

 

This run was performed using the following concentrations: 0.98, 1.95, 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µM in culture medium containing 1% DMSO.

At these tested concentrations:

.            no precipitate/emulsion was observed in any test item-treated wells at the end of the 48-hour treatment period,

.            no noteworthy decrease in cell viability was noted (i.e.cell viability > 70%), thus noIC₃₀or IC₅₀was calculated,

.            no statistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations. Moreover, the I_maxvalue was<1.5 (i.e.1.38).

The evaluation criteria for a negative response were met in this first run.

2.2      Second run

All acceptance criteria were met for the positive and negative controls, this run was therefore considered to be valid.

 

During the second run, the same concentrations were used as those tested during the first run and the following results were obtained:

.            noprecipitate/emulsion was observed in any test item-treated wells at the end of the 48-hour treatment period,

.            no noteworthy decrease in cell viability was noted (i.e. cell viability > 70%), thus noIC₃₀or IC₅₀was calculated,

.            gene-fold inductions above the threshold of 1.5 were noted at concentrations≥ 500 µM with an apparent dose-response relationship. Moreover, a statistical significance was obtained at the highest tested dose-level of 2000 µM in comparison to the negative control,

.            the I_maxwas 1.91,

.            in this second run, statistically non-significant inductions above 1.5-fold (i.e.inductions of 1.6 and 1.7 at concentrations of 500 and 1000 µM, respectively) were followed by a higher concentration with a statistically significant induction (i.e.induction of 1.9 at 2000 µM). Thus, the parameters used for the EC_1.5extrapolation were corrected manually using the formula described in the § Results analysis and was equal to 350.73 µM.

The evaluation criteria for a positive response were met in this second run.

 

Since the results of the first two runs were not concordant (first run considered as negative and second run considered as positive), a third run was performed using a narrower range of concentrations (dilution factor of 1.41).

 

2.3      Third run

All acceptance criteria were met for the positive and negative controls, this run was therefore considered to be valid.

 This run was performed using the following concentrations: 45.67, 64.39, 90.79, 128, 180.5, 264.5, 358.9, 506, 713.5, 1006, 1418 and 2000 µM in culture medium containing 1% DMSO.

At these tested concentrations:

.            no precipitate/emulsion was observed in any test item-treated wells at the end of the 48-hour treatment period,

.            no noteworthy decrease in cell viability was noted (i.e.cell viability > 70%), thus noIC₃₀orIC₅₀was calculated,

.            nostatistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations. Moreover, the I_maxvalue was<1.5 (i.e.1.35).

The evaluation criteria for a negative response were met in thisthirdrun.

 

Interpretation of results:

other:  Since two of the three runs performed were considered as negative, the final outcome is therefore negative.

Conclusions:

Under the experimental conditions of this study, the registered substance Reaction mass of diethyl adipate and diethy succinate and diethyl glutarate was negative in the KeratinoSens assay and therefore was considered to have no potential to activate the Nrf2 transcription factor.

Executive summary:

The objective of this study was to evaluatethe potential of the registered substance Reaction mass of diethyl adipate and diethyl succinate and diethyl glutarate (commercial name INNROAD PROTECT), to activate the Nrf2 transcription factor.This test is a part of a tiered strategy for the evaluation of skin sensitisation potential.Thus, data generated with the present Test Guideline (based on OECD Guideline No. 442D) should be used to support the discrimination between skin sensitizers and non‑sensitizers in the context of an integrated approach to testing and assessment.

This in vitro test uses the KeratinoSens cell line, an immortalized and genetically modified Human adherent HaCaT keratinocyte cell line. The KeratinoSens cell line is stably transfected with a plasmid containing a luciferase gene under the transcriptional control of the SV40 origin of replication promoter. This promoter is fused with an ARE sequence. Sensitizers with electrophilic properties provoke the dissociation of Keap-1 from the transcription factor Nrf2. The free Nrf2 binds to the ARE sequence contained in the plasmid and therefore induces transcription of firefly luciferase.

 

Methods

 The KeratinoSens cells were first plated on 96-well plates and grown for 24 hours at 37°C. Then the medium was removed and the cells were exposed to the vehicle control or to different concentrations of test item and of positive controls. The treated plates were then incubated for 48 hours at 37°C. At the end of the treatment, cells were washed and the luciferase production was measured by flash luminescence.In parallel, the cytotoxicity was measured by a MTT reduction test and was taken into consideration in the interpretation of the sensitisation results. Three independent runs were performed as part of this study.

 

Results

 For each run, the test item was diluted in DMSO at 200 mM.

 First run

All acceptance criteria were fulfilled for the positive and negative controls. The run was therefore considered to be valid.

This run was performed using the following concentrations: 0.98, 1.95, 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µM in culture medium containing 1% DMSO.

At these tested concentrations:

- no precipitate/emulsion was observed in any test item-treated wells at the end of the 48-hour treatment period,

- no noteworthy decrease in cell viability was noted (i.e.cell viability > 70%), thus no IC₃₀or IC₅₀was calculated,

-  no statistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations. Moreover, the I_maxvalue was<1.5 (i.e.1.38).

The evaluation criteria for a negative response were met in this first run.

 

Second run

All acceptance criteria were met for the positive and negative controls, this run was therefore considered to be valid.

During the second run, the same concentrations were used as those tested during the first run and the following results were obtained:

- no precipitate/emulsion was observed in any test item-treated wells at the end of the 48-hour treatment period,

- no noteworthy decrease in cell viability was noted (i.e.cell viability > 70%), thus noIC₃₀ or IC₅₀was calculated,

-  gene-fold inductions above the threshold of 1.5 were noted at concentrations≥ 500 µM with an apparent dose-response relationship. Moreover, a statistical significance was obtained at the highest tested dose-level of 2000 µM in comparison to the negative control,

-  the I_maxwas 1.91 and the calculated EC_1.5was 350.73 µM.

The evaluation criteria for a positive response were met in this second run.

 

Sincethe results of the first two runs were not concordant (first run considered as negative and second run considered as positive), a third run was performed using a narrower range of concentrations (dilution factor of 1.41).

 

Third run

All acceptance criteria were met for the positive and negative controls, this run was therefore considered to be valid.

This run was performedusing the following concentrations: 45.67, 64.39, 90.79, 128, 180.5, 264.5, 358.9, 506, 713.5, 1006, 1418 and 2000 µM in culture medium containing 1% DMSO.

At these tested concentrations:

-  no precipitate/emulsion was observed in any test item-treated wells at the end of the 48-hour treatment period,

-  no noteworthy decrease in cell viability was noted (i.e.cell viability > 70%), thus no IC₃₀ or IC₅₀was calculated,

- no statistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations. Moreover, the I_maxvalue was<1.5 (i.e.1.35).

The evaluation criteria for a negative response were met in this third run.

 

Discussion

 No geometric mean IC₃₀or IC₅₀was calculated since the cell viability was > 70% in all runs.

Since two of the three runs performed were considered as negative, the final outcome is therefore negative.This negative result can be used to support the discrimination between skin sensitizers and non‑sensitizers in the context of an integrated approach to testing and assessment. It cannot be used on its own to conclude on a skin sensitisation potential

 

Conclusion

Under the experimental conditions of this study, the registered substance Reaction mass of diethyl adipate and diethyl succinate and diethyl glutarate was negative in the KeratinoSens assay and therefore was considered to have no potential to activate theNrf2 transcription factor. 

Reference 3

Endpoint:

skin sensitisation, other

Remarks:

QSAR prediction for skin sensitisation (CAESAR)

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

01/02/2018

Reliability:

2 (reliable with restrictions)

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:

Please refer to the OPRF and QMRF reports attached in section Attached Justification.

Guideline:

other: REACH Guidance on QSARs R.6

Principles of method if other than guideline:

Skin sensitisation of Reaction mass of diethyl adipate and diethyl glutarate and diethyl succinate was predicted with CAESAR model implemented in the VEGA tool

Parameter:

other: QSAR

Remarks on result:

no indication of skin sensitisation

Remarks:

The registered substance is predicted 'negative'

The registered substance is predicted to be a non-sensitiser by the skin sensitization model CAESAR.

Interpretation of results:

other:

Conclusions:

Reaction mass of diethyl adipate and diethyl glutarate and diethyl succinate was predicted non sensitizer.

Executive summary:

Skin sensitisation of Reaction mass of diethyl adipate and diethyl glutarate and diethyl succinate was predicted with the Skin Sensitization model CAESAR 2.1.6, which is implemented in the QSAR tool VEGA (version 1.1.4.).

Reaction mass of diethyl adipate and diethyl glutarate and diethyl succinate

is predicted to be "non sensitiser". This prediction is reliable, it is generated by a scientifically valid model and lies within the applicability domain (please refer also to the QPRF and QMRF attached in the section 'Attached Justification').

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Three studies were performed to assess, in a weight of evidence approach, the sensitizing potential of the registered substance:

- In an in vitro assay, GLP and OECD 442D compliant (Klimish 1; Chevallier, 2017), the registered substance was negative in the KeratinoSens assay and therefore was considered to have no potential to activate the Nrf2 transcription.

- In an in chemico assay , GLP and OECD 442C compliant (Klimish 1; Chevallier, 2018), the DPRA prediction is considered as negative and the registered substance was considered to have no or minimal peptide reactivity.

- In a silico assay performed using the QSAR tool VEGA (version 1.1.4.) (Klimish 2), Reaction mass of diethyl adipate and diethyl glutarate and diethyl succinate was predicted to be "non sensitizer”.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on a weight of evidence approach using in vitro, in chemico and in silico data, Reaction mass of diethyl adipate and diethyl glutarate and diethyl succinate is not classified as a skin sensitiser according to the Regulation (EC) No. 1272/2008 (CLP).