Reaction mass of 2-methoxy-6-methylocta-1,5-diene and 2-methoxy-6-methylocta-2,5-diene

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 17 November 2017 Experimental completion date: 21 December 2017

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Please see any other information on materials and methods section

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

Skin sensitisers have been reported to induce genes that are regulated by the antioxidant response element (ARE). The KeratinoSens™ test is a method for which validation studies have been completed followed by an independent peer review conducted by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM). The KeratinoSens™ test method was considered scientifically valid to be used as part of an IATA (Integrated Approach to Testing and Assessment), to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling. The method cannot be used on its own, neither to sub-categorise skin sensitisers into subcategories lA and lB as defined by the UN GHS, for authourities implementing these two optional subcategories, nor to predict potency for safety assessment decisions. However, depending on the regulatory framework a positive result may be used on its own to classify a chemical int o UN GHS category 1.

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 2-methoxy-6-methylocta-1,5-diene and 2-methoxy-6-methylocta-2,5-diene

EC Number:

950-170-3

Molecular formula:

C10H18O

IUPAC Name:

Reaction mass of 2-methoxy-6-methylocta-1,5-diene and 2-methoxy-6-methylocta-2,5-diene

Specific details on test material used for the study:

Test Item Name: PG-RAW-0004
Supplier Code: PG-RAW-0004
Supplier batch/lot number: RDRW004-3
CAS number: 2109705-93-1; 2111193-86-1
Purity: 95.5%
Expiry Date: 01 Sep 19
Physical state: Clear liquid
Storage Conditions: Refrigerated

In vitro test system

Details on the study design:

Method of administration of test item:
Per plate, single application of 12 concentrations (2000, 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.813, 3.906, 1.953, 0.977 μM) 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:
Per plate, 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% and single application of culture medium with 1% DMSO was applied as the negative control (6 wells per plate).
One well per plate is left empty (no cells).

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.

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); 10,000 cells per well, p14.
Day 2: 24h after seeding the test and control items were applied and plates were incubated at 37°C, 5% CO2, 2: ≥ 95% relative humidity for 48 ± 2h.
Day 4: Evaluation of luciferase activity by luminescence (3 plates) and cell viability by MTT testing (2plates)

Data Analysis
XCellR8 Forms F0056 A (version 01) and B (version 04): Data Analysis for KeratinoSensTM were used to analyse data. These forms are Microsoft Excel workbooks containing formulae to process the raw data as described in SOP L0057. The spreadsheets have been validated in-house (July 2017).
The following parameters were calculated in the KeratinoSensTM test method:
• the maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the test item and positive control;
• the EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5- fold threshold (i.e. 50% enhanced luciferase activity);
• For each concentration showing > 1.5-fold luciferase activity induction, statistical significance is calculated (e.g. by a two-tailed Student’s t-test), comparing the luminescence values for the three replicate samples with the luminescence values in the solvent (negative) control wells to determine whether the luciferase activity induction is statistically significant (p <0.05). The lowest concentration with > 1.5-fold luciferase activity induction is the value determining the EC1.5 value. It is checked in each case whether this value is below the IC30 value, indicating that there is less than 30% reduction in cellular viability at the EC1.5 determining concentration.
• The percentage of viability as compared to the Negative control

Results and discussion

Positive control results:

Positive Control (PC) (Cinnamic aldehyde) induced ≥1.5-fold in at least one concentration. The average induction of cinnamic aldehyde was 1.91

In vitro / in chemico

Results

Run / experiment:

mean

Parameter:

other: EC1.5

Value:

11.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

Solubility Assessment
The test concentrations of PG-RAW-0004 used in the KeratinoSensTM test were selected on the basis of solubility test carried out prior to the study:
Solubility of PG-RAW-0004 (MW: 154) in cell culture medium (confirmed up to 30.8mg/ml (200mM)); subsequent dilution in cell culture medium-1% DMSO giving a top concentration of 2000µM).


Discussion
The human skin sensitisation potential of PG-RAW-0004 was assessed using validated in vitro method: the KeratinoSensTM test to determine keratinocyte activation. The method was adapted to animal product-free 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. The adapted method showed full concordance with the Validated Reference Method (VRM) – the KeratinoSensTM standard protocol. We recently obtained clarification from the European Chemicals Agency (ECHA) that data using the adapted method may be used in REACH submissions, provided that the Performance Standards data, demonstrating equivalence with the VRM, is included in the dossier.

In this study, PG-RAW-0004 was classified as a sensitiser to human skin.

The sensitisation potential of PG-RAW-0004 was quantified by calculating 2 parameters known as the EC1.5 and the IMAX value. The meanings of these are as follows:

• The EC1.5 value means the Effective Concentration (EC) of test item that caused an induction of luciferase activity of greater than 1.5-fold over untreated controls. If at least one concentration induces luciferase activity to >1.5, then the product is classified as a skin sensitiser. (Note: this classification also requires the cell viability measured by MTT to be greater than 70%).

PG-RAW-0004 did cause luciferase induction >1.5 in all 3 repetitions. Therefore, PG-RAW-0004 was classified as a sensitiser.


• The IMax value is the maximum-fold induction observed within the concentration range tested. Although the KeratinoSensTM test is not validated to predict potency, the IMAX value can provide a useful tool for a very preliminary comparison of sensitisation potential between test items. As shown in Section 13.2, the maximum induction was observed at a test concentration of 2000µM, which showed an Imax value of 1904.107 in repetition 1; 35.506 at 2000µM in repetition 2; 2358.35 at 1000µM in repetition 3. For reference, during test validation, sensitising proficiency chemicals produced Imax values of up to 36-fold over untreated controls.

All of the formal acceptance criteria of the tests were met.

Any other information on results incl. tables

Determination of the skin sensitisation potential of PG-RAW-0004

Sensitisation Potential of the test item repetition 1

REP 1	Test item concentration (µM)
	0.977	1.953	3.906	7.813	15.625	31.250	62.5	125	250	500	1000	2000
Mean of fold induction	0.941	0.902	1.156	1.274	1.434	1.478	2.186	3.061	5.489	14.129	101.988	1904.107
SD	0.039	0.045	0.049	0.085	0.150	0.273	0.091	0.219	0.945	0.920	9.893	104.463
Viability %	118.13	108.40	96.08	96.65	94.49	95.13	107.09	112.29	130.72	136.18	157.23	140.53
Imax	1904.107 at 2000µM
EC1.5	32.22µM
IC50	>2000µM as viability at 2000µM was 140.53%
IC30	>2000µM as viability at 2000µM was 140.53%

Sensitisation Potential of the test item repetition 2

REP 2	Test item concentration (µM)
	0.977	1.953	3.906	7.813	15.625	31.250	62.5	125	250	500	1000	2000
Mean of fold induction	1.144	1.165	1.254	1.434	1.350	1.293	1.581	1.801	2.656	4.082	8.710	35.506
SD	0.064	0.125	0.212	0.183	0.191	0.127	0.165	0.316	0.283	0.442	0.879	2.429
Viability %	96.15	86.87	96.06	100.59	97.83	99.59	102.22	118.07	128.96	161.25	210.83	229.39
Imax	35.506 at 2000µM
EC1.5	53.74µM
IC50	>2000µM as viability at 2000µM was 229.39%
IC30	>2000µM as viability at 2000µM was 229.39%

Sensitisation Potential of the test item repetition 3

REP 3	Test item concentration (µM)
	0.977	1.953	3.906	7.813	15.625	31.250	62.5	125	250	500	1000	2000
Mean of fold induction	0.836	1.107	1.048	1.223	1.472	1.908	3.404	6.756	26.094	338.892	2358.350	0.010
SD	0.040	0.202	0.073	0.081	0.115	0.098	0.303	0.713	1.935	35.363	123.870	0.003
Viability %	95.90	88.13	90.31	103.20	100.58	103.66	110.91	134.53	152.64	164.49	158.82	0.43
Imax	2358.35 at 1000µM
EC1.5	16.63µM
IC50	1687µM
IC30	1561µM

Determination criteria for the skin sensitisation potential ofPG-RAW-0004
	REP1	REP2	REP3
Does at least one concentration ofTest Iteminduce luciferase activity>1.5-fold:	Yes	Yes	Yes
Does the first concentration inducing luciferase activity above 1.5, have a viability above 70%:	Yes	Yes	Yes
Does EC1.5value occur at a concentration <1000µM (or <200µg/ml)	Yes	Yes	Yes
Does the test item induce the luciferase in a dose-dependent manner	Yes	Yes	Yes
Classification	Sensitiser	Sensitiser	Sensitiser

Assay Acceptane Criteria (Mean of the 3 repetitions)

Criteria	Result	Pass or Fail
1-Positive Control (PC) (Cinnamic aldehyde) induction>1.5-fold in at least one concentration	Yes	Pass
2-Average induction of PC at 32µM is [1.6-3.0]	Yes (2.11)	Pass
3-EC1.5value is [6-39µM]	Yes (11.50)	Pass
4-CV% of blank values < 20%	Yes (13.06%)	Pass

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

PG-RAW-0004 was classified as sensitiser

Executive summary:

The human skin sensitisation potential of PG-RAW-0004 was assessed using the validated in vitro method, the KeratinoSens^TMassay,adapted to fully animal-free by XCellR8,and validated in-house to determine keratinocyte activation.

 

After 48h exposure of cells with 12 concentrations of PG-RAW-0004,Luciferase measurements andMTT viability testing were performed.

PG-RAW-0004 was classified as sensitiser