Reaction mass of pentyl ester butanoic acid and 2-methylbutyl ester butanoic acid

Endpoint summary

Description of key information

The key studies presented in this section were conducted to recognised testing guidelines with GLP certification.

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:

key study

Study period:

24 July 2019 - 22 January 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted on 04 February 2015

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:

Name: AMYL BUTYRATE
Synonyms: Pentyl Butyrate (Sum of Isomers) Reaction mass of 2-methylbutyl butyrate and pentyl butyrate
CAS No.: 540-18-1
EC No.: 908-712-1
Description: Clear colorless to pale yellow liquid
Storage condition: At room temperature
Molecular weight: 158.24 g/mol
Specific test item requirements (handling conditions): None

Details on the study design:

VEHICLE
Based on solubility results, the selected vehicle was acetonitrile.

POSITIVE CONTROL
Name: Cinnamaldehyde
CAS No.: 104-55-2
Purity: 99.5%
Batch No: MKCG8499
Supplier: Sigma Aldrich
Molecular weight: 132.16 g/mol
Specific handling conditions: none

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 physical aspect of the formulation was colorless limpid solution and was used just after its preparation.

CONTROL SAMPLES
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.

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 vehicle specified in the reference control samples preparation section. 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 vehicle did not impact the percentage of peptide depletion.

TEST ITEM FORMULATION
Test item formulation preparation
The test item was pre-weighed and stored under appropriate conditions until ready to perform testing. It was dissolved in the selected vehicle (acetonitrile) at 100 mM. This formulation had the aspect of a colorless limpid solution.
The formulation was used just after its preparation.

Additional samples to test the neat test item
Since the test item is a multi-constituent substance, the reactivity of neat test item was evaluated.
Neat test item was tested as such (without prior dilution) by incubating it at 1:10 and 1:50 ratios with the cysteine and lysine peptides, respectively.

TEST SYSTEMS
Cysteine peptide
Peptide sequence: Ac-RFAACAA-COOH
Peptide sequence synonyms: Ac RFAACAA-OH
Molecular weight: 750.88 g/mol
Supplier: JPT Peptide Technologies GmbH
Batch No.: 111016HS_MHe_W1218
Purity: 95.99%
Storage condition: At -20°C
Description: White powder
Specific handling conditions: none

Batch number and any information relating to the characterization and integrity of the test system are documented in a certificate of analysis, which is archived in Citoxlab France files.
The cysteine peptide solution was freshly prepared at 0.667 mM in an aqueous phosphate buffer (pH 7.5) solution. The detailed preparation method is described in a Citoxlab France analytical method, specific to the DPRA test.

Lysine peptide
Peptide sequence: Ac-RFAAKAA-COOH
Peptide sequence synonyms: Ac RFAAKAA-OH
Molecular weight: 775.91 g/mol
Supplier: JPT Peptide Technologies GmbH
Batch No.: 120514HS_DW_W1218
Purity: 94.66%
Storage condition: At -20°C
Description: White powder
Specific handling conditions: none

Batch number and any information relating to the characterization and integrity of the test system are documented in a certificate of analysis, which is archived in Citoxlab France files.
The lysine peptide solution was freshly prepared at 0.667 mM in an aqueous ammonium acetate buffer (pH 10.2) solution. The preparation method is described in a Citoxlab France analytical method, specific to the DPRA test.

EQUIPMENT, REAGENTS AND COMPUTER SYSTEMS (INDICATIVE LIST)
Equipment list
High Performance Liquid Chromatography (HPLC) systems with a UV detector (220 nm)
Analytical chromatographic columns (Zorbax SB C18, 100 x 2.1 mm; 3.5 µm HPLC analytical column)
Precision electronic scales
pH meter
Small laboratory equipment [micropipettes, vortex, etc.]
Centrifuge
Fridge
Freezer

REAGENTS
Only reagents with high purity were used throughout this study. Their purity grade and the identification of the Suppliers are described in the raw data.
The list of reagents used in this study is described in a Citoxlab France analytical method procedure, specific to the DPRA test.

DESIGN OF THE DIRECT PEPTIDE REACTIVITY ASSAY
The test item was tested in one run. The run was processed as described below.

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.

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).

Reference control samples preparation
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.

Reference control C samples
Reference control C samples were prepared for each vehicle 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).

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).

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).

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

Incubation of the samples
All samples (co-elution controls, reference controls, test item and positive control samples) were then incubated during 24 (± 2) hours 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).

As no precipitate was observed, the vials were directly transferred onto the HPLC/UV system.

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.

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),
- additional neat test item 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.


DATA ANALYSIS AND CALCULATION
Calculation of the percentage peptide depletion
Each appropriate peak was integrated and the peak area for calibration standards, control and test item samples were determined. Based on the concentration of standards and their peak area, a linear calibration curve was generated. Then, the concentration of peptide was determined in each sample from absorbance at 220 nm, measuring the peak area of the appropriate peaks and calculating the concentration of peptide using the linear calibration curves. Then, for each positive control and test item replicate, the percentage depletion of peptide was determined from 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 vehicle) by using the following formula:

% depletion = [ 1 – (Peptide peak area in replicate injection / Mean peptide peak area in relevant reference control C samples) ] x 100

Then, the mean percentage depletion of the three replicates was calculated for each peptide as well as the mean of the percentage cysteine and percentage lysine depletions. Negative depletion values were considered as "Zero" for the calculation of the mean % depletion.
Peak areas and peptide concentrations are presented in the report. Standard Deviation (SD) and Coefficient of Variation (CV) were calculated and reported.

Evaluation of the possible co-elution of the test item with the lysine or cysteine peptides
In order to detect possible co elution of the test items with a peptide, chromatograms of the co-elution control samples were analyzed and compared with those of the reference control C samples.

ACCEPTANCE CRITERIA
The run was considered valid if the following criteria were fully met:
- the calibration curves should have a coefficient of determination (r2) ≥ 0.99,
- the mean peptide concentrations of the reference control A samples should be within ± 10% of the nominal concentration,
- the cinnamaldehyde depletion control samples should meet the following acceptance criteria:
- for the cysteine peptide, the mean percentage depletion value should be between 60.8 and 100% with a SD < 14.9%,
- for the lysine peptide, the mean percentage depletion value should be between 40.2 and 69.0% with a SD < 11.6%,
- the CV of the mean peptide peak area of the nine reference control B and C samples in acetonitrile must be < 15.0%.

The test item’s results were considered valid if the following criteria were fully met:
- the mean peptide concentrations of the reference control C samples prepared in the appropriate vehicle should be within ± 10% of the nominal concentration
- the maximum SD for the test item replicates should be < 14.9% for the percentage cysteine depletion value and < 11.6% for the percentage lysine depletion value.

Positive control results:

The acceptance criteria for the positive controls were satisfied.

Run / experiment:

other: 1

Parameter:

other: cysteine peptide depletion (%)

Value:

0

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Value set to zero for negative depletion

Run / experiment:

other: 2

Parameter:

other: cysteine peptide depletion (%)

Value:

3.39

Vehicle controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: 3

Parameter:

other: cysteine peptide depletion (%)

Value:

5.26

Vehicle controls validity:

valid

Positive controls validity:

valid

Run / experiment:

mean

Parameter:

other: cysteine peptide depletion (%)

Value:

2.88

Vehicle controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: 1

Parameter:

other: lysine peptide depletion (%)

Value:

0

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Value set to 0 due to negative depletion

Run / experiment:

other: 2

Parameter:

other: cysteine peptide depletion (%)

Value:

0

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Value set to 0 due to negative depletion

Run / experiment:

other: 3

Parameter:

other: cysteine peptide depletion (%)

Value:

0.1

Vehicle controls validity:

valid

Positive controls validity:

valid

Run / experiment:

mean

Parameter:

other: cysteine peptide depletion (%)

Value:

0.03

Vehicle controls validity:

valid

Positive controls validity:

valid

Run / experiment:

mean

Parameter:

other: neat test item cysteine peptide depletion (%) (calculated)

Value:

1.02

Vehicle controls validity:

valid

Positive controls validity:

valid

Run / experiment:

mean

Parameter:

other: neat test item lysine peptide depletion (%) (calculated)

Value:

0

Vehicle controls validity:

valid

Positive controls validity:

valid

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. Therefore, this vehicle was selected for the present study.

 

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, these vials were centrifuged at 400g for a period of 5 minutes at room temperature to force precipitate to the bottom of the vial. Positive control samples incubated in both cysteine and lysine peptides and reference control samples (prepared in acetonitrile:milli‑Q water and in acetonitrile) incubated in 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, the vials were directly transferred into the HPLC/UV system.

 

EVALUATION OF THE 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.

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 percentage 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 2.88%,

- for the lysine peptide, the mean depletion value was 0.03%.

The mean of the percentage cysteine and percentage lysine depletions was equal to 1.46%. Accordingly, the test item was considered to have no or minimal peptide reactivity.

Since the test item was a multi-constituent substance, the reactivity of the neat test item was also evaluated.

The mean percent depletion values for the neat test item were calculated for each peptide using the formula described in Data analysis and calculation:

- for the cysteine peptide, the mean depletion value was 1.02%,

- for the lysine peptide, the mean depletion value was 0.00%.

The mean of the percent cysteine and percent lysine depletions with the test item neat was equal to 0.51%. These results confirm those obtained with test item prepared at 100 mM (no or minimal peptide reactivity).

Therefore, the DPRA prediction is considered as negative and the test item may have no potential to cause skin sensitization. However, since precipitates were observed at the end of the incubation with the cystein peptide, the peptide depletion may be underestimated.

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 sensitization potential of the test item.

RESULTS TABLES AND FIGURES

Results for the test item and the positive control are presented in Tables 1 and 2, respectively. The peakareas for calibration curve samples are presented in Tables 3.1 and 3.2, the peptide concentrations and peak areas in reference control samples are presented in Tables 4.1 and 4.2.  

Representative chromatograms of the co-elution, reference control C and test item samples are presented for each peptide in Figures 1 to 8.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study, the DPRA prediction is considered as negative and the test item AMYL BUTYRATE, 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, AMYL BUTYRATE, to synthetic cysteine and lysine peptides. This test addresses the molecular initiating event of the skin sensitisation adverse outcome pathway (AOP) and is part of a tiered strategy for skin sensitization assessment.

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, protected from light. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with Ultra-Violet detection at 220 nm.

Peptide reactivity was reported as percentage 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 vehicle).

 

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.

 

The reactivity of the test item at 100 mM in acetonitrile was first evaluatedand the mean of the percentage cysteine and percentage lysine depletions was equal to 1.46%.

Since the test item is a multi-constituent substance, the reactivity of the neat test item was also evaluated.

The mean of the percent cysteine and percent lysine depletions with the neat test item was equal to 0.51%. Accordingly, the test item was considered to have no or minimal peptide reactivity.

 

Precipitates were observed at the end of the incubation with the cystein peptide in both assays (100 mM and neat), the peptide depletion may be thus underestimated.

Therefore, the DPRA prediction is considered as negative and the test item may have no potential to cause skin sensitization, though with limitations due to its precipitation with the cysteine peptide.

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 sensitization potential of the test item.

Under the experimental conditions of this study, the DPRA prediction is considered as negative and the test item AMYL BUTYRATE, 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:

key study

Study period:

29 May 2019 - 25 September 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted on June 2018.

Deviations:

yes

Remarks:

Test items were not filtered before treatment

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

Description Clear colorless to pale yellow liquid
Storage condition Room temperature
Molecular weight 158.24 g/mol
Purity 99.80%

Details on the study design:

TEST SYSTEM

KeratinoSens cells - the cell line KeratinoSens is stably transfected with a modified plasmid. This plasmid contains an ARE sequence from the AKR1C2 gene and a SV40 promotor which are inserted upstream of a luciferase gene. The resulting plasmid was transfected into HaCaT keratinocytes and clones with a stable insertion selected in the presence of Geneticin / G-418. Induction of luciferase gene is the endpoint evaluated and reflects the activation by the test item of the Nrf2 transcription factor in this test.

Supplier: this cell line was provided by Givaudan.
Batch: D1
Storage conditions: at -80°C.
Mycoplasm: absence of mycoplasm was confirmed.

Specific equipment:
96-well plate Luminometer with injectors and optical density reader (Varioskan Flash).

Media:
- Maintenance medium No. 1: DMEM containing GlutaMAXTM, 1000 mg/L D-Glucose, Sodium Pyruvate and supplemented with 9.1% Fetal Calf Serum (FCS) and 500 µg/mL G-418
- Maintenance medium No. 2: DMEM with 9.1% FCS without G-418,
- Treatment medium: DMEM with 1% FCS without G-418,
- Freezing medium: DMEM with 20% FCS and 10% DMSO.

STUDY DESIGN

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).
When a solution or a stable dispersion was obtained in these vehicles, the formulations were 100-fold diluted in culture medium. Then, a visual inspection of the samples was performed immediately as well as after an overnight period of incubation at 37°C to evaluate the presence or absence of precipitate/emulsion.

Method for a run of KeratinoSens assay:
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 10^4 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 10^4 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.


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.

ENDPOINT MEASUREMENTS

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.

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 preferentially (not exceeding 30 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.

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:

Mean Imax - 7.9
Mean EC1.5 - 9.1

Run / experiment:

other: Experiment 1 Run 1

Parameter:

other: Imax

Value:

1.21

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: Experiment 1 Run 2

Parameter:

other: Imax

Value:

1.33

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:

KERATINOSENS RUN (Appendices 2 and 3)
The Imax, IC30, IC50, EC1.5 and viability values obtained for cells treated with test item in each run as well as the mean and SD values are presented in Appendix 2. The viability values (%), induction values, Imax, IC30, IC50 and EC1.5 values obtained with the positive control are presented in Appendix 3. In addition the luminescence values of all negative control wells and the %CV between these values for each run are also presented in Appendix 3.

All acceptance criteria were fulfilled for the positive and negative controls in each run; both runs were therefore considered as validated.  

The two runs were 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 was observed in any test item-treated wells at the end of the 48-hour treatment period in either run,
. in both runs, a high decrease in cell viability (i.e. cell viability < 70%) was noted at concentrations ≥ 500 µM,  except at the highest tested concentration (i.e. 2000 µM) where cell viability was ≥ 90% without any test item precipitate which may have prevented the exposition of the cells to the test item, as is usually observed in this type of assay. This absence of cytotoxicity at the highest tested concentration is not considered as biologically relevant, thus the associated results are not taken into consideration in the interpretation,
. the corresponding IC30 and IC50 were not calculated but estimated between 250 and 500 µM,
. no statistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations in either run,
. moreover and for both runs, the Imax values were < 1.5 (i.e. 1.21 and 1.33 in the first and second runs, respectively) and thus no EC1.5 was calculated.  

The evaluation criteria for a negative response are met in both runs, 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 sensitization potential.

SOLUBILITY TEST

In the solubility test, and at all tested concentrations (i.e.at 100 and 200 mM), the test itemwas found soluble in DMSO and water for injections (colorless solution) whereas foaming heterogeneous emulsions were obtained when tested in culture medium. 

 

Once the test item stock formulation in DMSO was diluted in the treatment culture medium to final concentrations of 2000 µM, a slight precipitate was immediatelyobserved. This slight precipitate was not observed anymoreafter an overnight period of incubation at 37°C. Whendiluted in the treatment culture medium to final concentrations of 1000 µM, no precipitate/emulsion was noted, immediately as well as after an overnight period of incubation at 37°C. It was the same forthe test item stock formulations in water for injections, for both dilutions.

 

On the basis of these results, the test item stock formulation to be used in the main test was therefore selected to be 200 mM in DMSO.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study, the test item, AMYL BUTYRATE, 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 evaluate the potential of the test item,AMYL BUTYRATE, to activate the Nrf2 transcription factor.This test is a part of a tiered strategy for the evaluation of skin sensitisation potential.

 

The design of this study was based on the OECD Guideline No. 442D and the study was performedin compliance with Citoxlab France standard operating procedures and with the OECD Principles of Good Laboratory Practice.

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.

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. Two independent validated runs were performed as part of this study.

All acceptance criteria were fulfilled in both runs; the study was therefore considered as validated.

This study was performed testing concentrations from 0.98 to 2000 µM in culture medium containing 1% DMSO.

 

At these tested concentrations,nostatistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations in either run.

 

The evaluation criteria for a negative response are met in both runs, 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 sensitization potential.

Under the experimental conditions of this study, the test item, AMYL BUTYRATE, was negative in the KeratinoSens assay and therefore was considered to have no potential to activate the Nrf2 transcription factor

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The in vitro tests for skin sensitisation are validated, and the results are suitable for use as the basis for classification according to the UN GHS categories. The results of the tests presented in this section indicate that the registered substance does not meet the criteria for skin sensitisation in accordance with the Classification, Labelling, and Packaging (CLP) regulation (1272/2008).