Reaction mass of isopentyl salicylate and 2-methylbutyl salicylate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 8th, 2018 to October 26th, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

February 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: ECVAM DB-ALM protocol 155: KeratinoSens

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Test material

Test material information

Constituent 1

Reference substance name:

Reaction Mass of Isoamyl salicylate and 2-Methylbutyl salicylate

IUPAC Name:

Reaction Mass of Isoamyl salicylate and 2-Methylbutyl salicylate

Test material form:

liquid

In vitro test system

Details on the study design:

- Cell line used: KeratinoSens™ (Givaudan) cultured in maintenance medium (DMEM 1 g/l glucose, 9.1% non-heat inactivated foetal calf serum, 0.05% geneticin - stored at 5ºC ± 3ºC) at 37ºC, 5% CO2. Cells were exempt of mycoplasma.
- Passage number and level of confluence of cells: cells were used at passage 17 in repetition 1, passage 19 in repetition 2 and passage 21 in repetition 3.
- The cells were trypsinized according to the current working instruction IL 09. Cells suspension were adjusted to a density of 8.10^4 cells/ml in the seeding medium. 125 µl of the cell suspension at 8.10^4 cells/ml (i.e. 10^4 cells per well) were distributed in three white plates for the induction measurement and two transparent plates to assess the cytotoxicity. The seeded plates were incubated 24 hours ± 1 hour at 37 ºC, 5% CO2. The H12 wells were left without cells and allowed the measurement of blanks.
- Luminometer used: GloMax™ (Promega) MULTISKAN EX plate reader (Thermo life sciences) - reading range 0 - 3.5 units of Absorbance, linearity range 0 - 2.200 units of Absorbance
- Number of repetitions and replicates: the study was composed of three independent repetitions. For each repetition, the test item and the reference items were replicated on three independent plates for the measurement of induction and two plates for the measurement of cytotoxicity. Each repetition was performed on a different day with a fresh stock solution.
- Test chemical concentrations: Test item was diluted 100-fold (10X plate) in sterile water from a 200 mM stock solution and then diluted 25-fold in a new plate (4X plate). Finally, there was a further 4-fold dilution in the seeding plate
(1X plate). A positive control was diluted 100-fold in DMSO (Sigma Aldrich Batch no.W228613) from a 6.4 mM stock solution and then diluted 25-fold in a new plate in treatment medium and then further diluted 4-fold in the seeding plate. The test item was tested at 12 concentrations according to a geometric progression of ratio 2 from 0.98 µM to 2000 µM.
-Negative control: 6 wells of solvent control (1% DMSO in treatment medium) with cells and 1 well of solvent control without cell by culture plate. Positive control: 5 concentrations of cinnamaldehyde (SIGMA ALDRICH Batch no.W228613) on each culture plate. The concentration varies from 4 to 64 µM according to a geometric progression of ratio 2.

Description of evaluation and decision criteria used:
The test item is identified as potential skin sensitizer if the 4 following conditions are met in 2 of 2 or 2 of 3 repetitions. Otherwise, the keratinosens™ prediction is considered as negative:
1) The Imax is strictly 1.5 fold higher of the basal luciferase activity* statistically significantly to the value obtained for the negative control (as determined by a two-tailed, unpaired Student's t-test on the raw RLU values). If the Imax is exactly equal to 1.5, the test item is rated as negative and no EC1.5 value is calculated.
2) The EC1.5 value is strictly below 1000 μM (or <200 μg/ml for the test item with no defined molecular weight)
3) At the lowest concentration with a gene induction above 1.5, the cell viability must be strictly above 70% (i.e EC1.5 < IC70).
4) There is an apparent overall dose-response for luciferase induction, which is similar between the repetitions.
- Description of study acceptance criteria used:
1) Positive control:
- The gene induction must be statistically significant above the threshold of 1.5 in at least one dose.
- The EC1.5 value should be between IDEA Lab historical data: mean EC1.5 value ± 2 SD and the average induction, in each repetition, for cinnamaldehyde at 64 μM should be between 2 and 8. If the latter criterion is not fulfilled, the dose-response of cinnamaldehyde should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
2) Negative control: for each repetition, the coefficient of variation of the solvent controls (3 x 6 wells) must be less than 20%. If for one repetition the validity criteria are not met, a third repetition should be considered.

Results and discussion

Positive control results:

Imax = 8.46
EC1.5 = 7.06 (geometric mean)

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

mean

Parameter:

other: Imax

Value:

1.35

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Imax lower than 1.5, no EC1.5 is calculated

Results 2

Key result

Run / experiment:

other: Geometric mean

Parameter:

other: IC50

Value:

2 000

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS:
- The test item did not cause any precipitation or any other type of interference that might have had lead to confouding the possible sensitising effects of the test item.

DEMONSTRATION OF TECHNICAL PROFICIENCY: recommended substances for demonstrating technical proficiency with the KeratinoSens™ test method were tested to validate the method.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Average variability in yhr 3x6 control wells should be <20% (for each master plate).
- Acceptance criteria met for positive control:
1) Gene induction (luciferase activity) must be statistically significant > 1.5 in at least one dose. The luciferase activity was > in every single dose.
2) Average inductions in 3 replicates at 64 µM should be between 2-8, if this criterion is not fulfilled the test may be accepted based on dose-response: The average induction was 8.46, but there was an apparent overall dose-response for luciferase induction.
3) EC1.5 should be between 2 standard deviations of the historical mean (i.e 7-30 µM based on validation dataset: EC1.5 was 7.06 and therefore within the historical range (3-22 µM)
- Acceptance criteria met for variability between replicate measurements: yes, CV% < 20%
- Range of historical values if different from the ones specified in the test guideline: 3-22 µM

Any other information on results incl. tables

Table 1. Positive control results.

Cinnamaldehyde	4 µM	8 µM	16 µM	32 µM	64 µM	EC1.5	Imax
Rep 1	1.33	2.18	3.77	6.00	15.17	4.80	15.17
Rep 2	1.37	1.45	2.12	3.16	5.52	8.58	5.52
Rep 3	1.24	1.45	2.13	3.22	4.69	8.56	4.69
Mean	1.31	1.69	2.67	4.13	8.46	7.06*	8.46

*geometric mean

 

Table 2. Negative Control Results.

Control solvent	CV %  control solvent
Rep 1	10.5
Rep 2	8.8
Rep 3	6.8

Table 3. Test item results.

	VIABILITY		INDUCTION
ID-18/04211	IC50  µM	IC30  µM	Imax	Linear EC1.5  µM	EC1.5 Lin/Log  µM
Rep 1	53.17	44.96	1.51	30.76	30.58
Rep 2	53.85	36.30	1.11	-	-
Rep 3	71.36	53.15	1.42	-	-
Mean	-	-	1.35	-	-
Geometric mean	58.90	44.27	-	-	-

Repetition 1: Imax is slightly higher than 1.5 (1.51) at only one concentration close to the IC30. The calculated EC1.5 is lower than 1000 µM and the EC1.5 viability is higher than 70%, the repetition is therefore positive. However the corresponding positive control being higher on this repetition, a third repetition was carried out.

Repetition 2 and 3: Imax is lower than 1.5, the EC1.5 is not determined. The repetition 2 and 3 are negatives.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

EU criteria

Conclusions:

The test item showed an Imax of 1.35 and an IC50 and IC30 higher than 2000 μM in KeratinoSens™. The test item showed no sensitisation potential under test conditions, therefore, the KeratinoSens prediction is considered negative.

Executive summary:

The test method KeratinoSens™ has been performed as part of an integrated approach to support the identification of the sensitization potential of the test item according to OECD 442D, under GLP conditions.

The aim of the study is to evaluate the potential of the test item to activate the gene AKR1C2 y quantifying the luciferase induction after 48h in transformed keratinocytes. 12 concentrations of the test item ranging from 0.98 to 2000 μM were prepared by serial dilution in culture medium containing 1% DMSO and added to 96-well plates of KeratinoSens cells™, in 3 separate runs of 3 replicates each. Positive and negative controls were run in parallel, as well as a cytotoxicity assay (MTT reduction). All acceptance criteria were fulfilled for the positive and negative controls in each run. The test item showed an Imax of 1.35 and an IC50 and an IC30 higher than 2000 μM in KeratinoSens™, the mean cell viability was 58.90 and 44.27 for IC50 and IC30 respectively. Under the testing conditions the test item has no sensitisation potential.