Isobutyl isobutyrate

Endpoint summary

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:

2017-2018

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

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

Sponsor’s identification Eastman IBIB
CAS No: 97-85-8
Batch No. XK16016428
Purity 98.7%
Molecular weight :144.21 g/mol
Appearance: Colorless liquid
Expiry/retest date: 09 February 2019 (2 years from receipt)
Storage conditions: Ambient temperature in the dark (15-25°C)

Details on the study design:

Preparation of Positive Control and Cysteine Peptide Depletion Samples and Co-elution Controls

Acetonitrile solutions of Eastman IBIB and solutions of the positive control were diluted with the Cysteine peptide to prepare solutions containing 0.5 mM Cysteine and 5 mM of either Eastman IBIB or the positive control. For the co-elution control, buffer solution was used in place of the Cysteine stock solution.

Preparation of Positive Control and Lysine Peptide Depletion Samples and Co-elution Controls
Acetonitrile solutions of Eastman IBIB and solutions of the positive control were diluted with the Lysine peptide to prepare solutions containing 0.5 mM Lysine and 25 mM of either Eastman IBIB or the positive control. For the co-elution control, buffer solution was used in place of the Lysine stock solution.

Incubation
The appearance of the Eastman IBIB and positive control samples in the HPLC vials was documented after preparation and then the vials placed into the autosampler of the HPLC set at 25°C for a minimum of 22 hours incubation prior to initiation of the analysis run. Prior to initiation of the run the appearance of the samples in the vials was assessed and documented again.

Analysis
The concentration of both the Cysteine and Lysine peptides in the presence of Eastman IBIB and the associated positive controls was quantified by HPLC using UV detection as detailed in the chromatographic section.

Calculations
The peak area response for the peptide in each calibration chromatogram was measured

Positive control results:

The mean depletion for psotive control in percentage was 72.3.

Parameter:

other: peptide depletion

Remarks:

Cysteine peptide depletion (%)

Value:

1.2

Vehicle controls validity:

not examined

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

Cinnamic Aldehyde

Parameter:

other: peptide depletion

Remarks:

peptide depletion (%)

Value:

0.73

Vehicle controls validity:

not examined

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

Cinnamic acid

Interpretation of results:

other: Not classified

Remarks:

Not classified

Conclusions:

The overall mean result of 0.968% depletion places Eastman Isobutyl isobutyrate in the reactivity class of “no to minimal” and hence it is predicted by DPRA to be a non skin sensitizer.

Executive summary:

In chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), following OECD TG 442C was conducted to assess the reactivity and sensitizing potential of Eastman isobutyl isobutyrate. Solutions of IBIB were successfully analysed by the validated DPRA analytical method in both Cysteine and Lysine containing synthetic peptides. The overall result of 0.968% depletion places Eastman IBIB in the reactivity class of “no to minimal” and therefore it is predicted by DPRA to be a non-skin sensitizer.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

Physical state: Liquid-Organic Liquid Solution
Storage Conditions : Room Temperature in the dark
Solvent : 1% DMSO in cell culture medium
Administration method : In cell culture medium
Concentrations tested (PM): 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98

Details on the study design:

Description of the test system:
The KeratinoSensTM cell line (test system) is an immortalized adherent cell line derived from HaCaT human keratinocytes, stably transfected with a selectable plasmid containing the luciferase gene under the transcriptional control of the Anti-oxidant Response Element (ARE) from a gene that is known to be upregulated by contact sensitisers. The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes, and the dependence of the luciferase signal in the recombinant cell line on Nrf2 has been demonstrated. This allows quantitative measurement (by luminescence detection) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic test substances.

Characterisation of the test system:
licence agreement between XCellR8 and the test method developer (Givaudan) was established in 2014. KeratinoSensTM cells were supplied and propagated. The cell culture was then adapted to animal productfree conditions by XCellR8 and reference chemicals described in the guideline and in the performance standards were used to confirm the reliability, accuracy, sensitivity and specificity values were shown to be comparable than those derived from the Validated Reference Method (VRM) KeratinoSens

Method of administration of test item:
single application of 12 concentrations of the test item was applied in cell culture medium (dilution factor of 2) with a final concentration of DMSO of 1%. The top concentration was previously determined by solubility testing

Method of administration of reference items:
single application of 5 concentrations of the positive control was applied in cell culture medium (dilution factor of 2) with a final concentration of DMSO of 1%.
A single application of culture medium with 1% DMSO was applied as the negative control

Exposure times of test items and reference items:
Cells were incubated with the test or reference item for 48 ± 2h before endpoints measurements

Number of repetitions:
Three repetitions (runs) were performed. Each repetition consisted of 3 x 96-well plates for luminescence and 2 x 96-well plates for MTT. The validity of each repetition was assessed following acceptance criteria described in section 12.1.

Overview
Preliminary testing: Determination of the top concentration by solubility testing
Day 1: Seeding cells (3 x 96-well plates for Luminescence; 2 x 96-well plate for MTT).
Day 2: 24h after seeding the test and control items were applied and plates were incubated at 370C, 5% C02, 2 95% relative humidity for 48 ± 2h.
Day 4: Evaluation of luciferase activity by luminescence (3 plates) and cell viability by MIT testing (2 plates)

Positive control results:

The positive control (cinnamic aldehyde) produces positive results, i.e. the luciferase gene induction by this control is statistically above the threshold of 1.5 in at least one of the tested concentrations

Parameter:

other: Luciferase measurements and M TT viability testing were performed

Remarks:

Luciferase measurements and M TT viability testing were performed

Negative controls validity:

valid

Remarks:

DMSO

Positive controls validity:

valid

Remarks:

Cinnamic Aldehyde

Remarks on result:

no indication of skin sensitisation

Remarks:

No EC1.5 as no concentration did induce the luciferase activity above the 1.5 threshold

Other effects / acceptance of results:

Test results are acceptable if:
- The positive control (cinnamic aldehyde) produces positive results, i.e. the luciferase gene induction by this control is statistically above the threshold of 1.5 in at least one of the tested concentrations.
-The Imax and the EC1.5 for cinnamic aldehyde is calculated and meet the following targets:
- average induction in the three replicates for cinnamic aldehyde at 32 11M is between the XCellR8 historical range (currently 1.6 and 3)
- EC1.5 value for cinnamic aldehyde is between the XCellR8 historical range (currently 6 and 39 VIM).
Note: At least one of these criteria must be met, otherwise the run is discarded. If only one criterion is met, it is recommended to check the dose-response curve of cinnamic aldehyde in order to decide on acceptability.
-CV% of blank values < 20%

Interpretation of results:

other:

Remarks:

Not classified

Conclusions:

The human skin sensitisation potential of Isobutyl Isobutyrate was assessed using the validated in vitro method, the KeratinoSens assay, adapted to fully animal-free by XCellR8, and validated in-house to determine keratinocyte activation.After 48h exposure of cells with 12 concentrations of di-tertbutyl hydroquinine, Luciferase measurements and M TT viability testing were performed.Isobutyl Isobutyrate was classified as non sensitiser.

Executive summary:

In Vitro Assessment of the Skin Sensitisation Potential of Isobutyl Isobutyrate was conducted according to OECD Test Guideline 442D (the KeratinoSens test method). After 48h exposure of cells with 12 concentrations of di-tertbutyl hydroquinine, luciferase measurements and M TT viability testing were performed. A single application of culture medium with 1% DMSO was applied as the negative control.A positive control of cinnamic aldehyde was tested. Three repetitions (runs) were performed. Solubility results of di-tertbutyl hydroquinine indicated the item was soluble in DMSO at 20 mg/ml. Testing on this study were done with subsequent dilution in cell culture medium 1% DMSO giving a top concentration of 400gg/ml. The maximum induction was observed at a test concentration of 7.813 gM, which showed an Imax value of 1.204 in repetition 1; 1.576 at 0.97711 M in repetition 2; 1.362 at 15.62511 M in repetition 3.No EC1.5 as no concentration did induce the luciferase activity above the 1.5 threshold in repletion 1 and 3 tests. Isobutyl Isobutyrate caused luciferase induction 21.5 in repetition 2, at the lowest concentration, however there was no dose-response induction and Isobutyl Isobutyrate was classified as a non-sensitiser.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The potential for isobutyl isobutyrate to cause dermal sensitization was evaluated following OECD TG 442D and

OECD TG 442C studies. Isobutyl Isobutyrate was assessed using the validated in vitro method, the KeratinoSens^TMassay. The cell line was exposed for 48h with 12 concentrations of isobutyl isobutyrate. Luciferase measurements and MTT viability testing showed non sensitizer response of isobutyl isobutyrate. Eastman IBIB showed 0.968%i depletion in the reactivity class of “no to minimal” and therefore it is predicted by DPRA to be a non-skin sensitizer.

Justification for classification or non-classification

No evidence of a sensitization response was observed when cell cultures were exposed to the test material in a in vitro OECD442D study. Based on a results of assessment,Isobutyl Isobutyrateis not classified for “Skin Sensitization” according to GHS classification system. In other key

in chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), following OECD TG 442C assay, Eastman IBIB revealed reactivity class of “no to minimal” and therefore was predicted by DPRA to be a non-skin sensitizer. The substance is not classified for sensitisation potential category.