Neodecanoic acid, iron salt

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study Initiation Date: 05 September 2017 Exerimental Completion Date: 06 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

The study was conducted to meet the known requirements of OECD Guidelines for Testing of Chemicals Method 442D (adopted February 2015).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Analysis for stability, achieved concentration and homogeneity of test article formulations was not conducted as part of this study as it was not a requirement of the Test Guideline.

Type of study:

other: In Vitro Skin Sensitisation (ARE-Nrf2 Luciferase Test Method)

Justification for non-LLNA method:

The data will be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.

Test material

Test material information

Constituent 1

Reference substance name:

Neodecanoic acid, iron salt

EC Number:

257-446-6

EC Name:

Neodecanoic acid, iron salt

Cas Number:

51818-55-4

Molecular formula:

C10H20O2.xFe

IUPAC Name:

λ²-iron(2+) bis(2-ethyl-2,5-dimethylhexanoate)

Test material form:

solid

Details on test material:

CAS Number: 51818-55-4
EC Number 257-446-6
Molecular formula: C30H57FeO6
Molecular weight: 569.6
Purity: 100%
Storage conditions: Refrigerated under Nitrogen ( 2-8 °C )

Specific details on test material used for the study:

Storage: 2 to 8°C, protected from light, under nitrogen
Batch Number: Ln11013894

In vitro test system

Details on the study design:

The study was conducted to investigate the potential of the test item to induce genes that are regulated by the antioxidant response element (ARE). The data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.
The ARE-Nrf2 luciferase test method utilises an immortalised adherent cell line derived from HaCaT human keratinocytes. The cell line is stably transfected with a plasmid containing a luciferase gene under the transcriptional control of the SV40 promoter fused with the ARE from a gene known to be up-regulated 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 substances.

Results and discussion

Positive control results:

The EC1.5 values for the positive control were 20.81 and 9.54 µM in Repetitions 1 and 2, respectively. The average induction in the two replicates for the positive control at 64 µM was 3.09 and 6.40 in Repetitions 1 and 2, respectivley.

In vitro / in chemico

Results

Key result

Parameter:

other: The four conditions specified in the prediction model were not met in either repetition. The test article was therefore considered to be negative in the ARE-Nrf2 Luciferase Test.

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

The four conditions specified in the prediction model were not met in either repetition. The test article was therefore considered to be negative in the ARE-Nrf2 Luciferase Test.

Any other information on results incl. tables

Calculation of Imax and EC1.5 Values

Luminescence readings and fold increases are given in the attached Table 10.1 and Table 10.2.

The maximal fold increases (Imax) were 0.94 and 3.79 for Repetitions 1 and 2, respectively.

There was no EC1.5 value for Repetition 1 as there were no statistically significant increases in induction.

The EC1.5 value for Repetition 2 was 0.78 mM.

Viability

MTT-absorbance readings are given in the attached Table 10.3 and Table 10.4.

This measurement was not applicable for Repetition 1 as there was no EC1.5 determining concentration.

Cell viability was 21.51% at the EC1.5 determining concentration in Repetition 2.

In Repetition 1, there was no apparent overall dose response for lucipherase and the dose response curve was not biphasic.

In Repetition 2 the dose response curve for luciferase induction was biphasic.

Assay Acceptance

Luciferase activity induction obtained with the positive control was statistically significant above the threshold of 1.5 at concentrations of 32 and 64 µM in Repetition 1 and at concentrations of 16 to 64 µM in Repetition 2.

The EC1.5 values for the positive control were 20.81 and 9.54 µM in Repetitions 1 and 2, respectively. The average induction in the two replicates for the positive control at 64 µM was 3.09 and 6.40 in Repetitions 1 and 2, respectivley.

The average coefficient of variation of the luminescence reading for the negative control (DMSO) was 6.56% and 11.62% in repetitions 1 and 2, respectively.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The four conditions specified in the prediction model were not met in either repetition. The test article, was therefore considered to be negative in the ARE-Nrf2 Luciferase Test.

Executive summary:

The study was conducted to investigate the potential of the test item to induce genes that are regulated by the antioxidant response element (ARE). The data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.

The test article was dissolved in Isopropanol to the final concentration (200 mM). Serial dilutions were then made using Isopropanol to obtain 12 master concentrations of the test article (0.098, 0.196, 0.391, 0.781, 1.563, 3.125, 6.25, 12.5, 25, 50, 100 and 200 mM).

The master concentrations were then further diluted 25 fold into culture medium containing serum and finally used for treatment with a further 4 fold dilution factor so that the final concentrations ranged from 0.98 to 2000 µM.

Aliquots of 50 µL of each of the final concentrations were transferred to each of three luciferase plates and a single viability plate. Each plate was sealed using a plate sealer and then incubated at 37±1¿C, 5% (v/v) CO2 in air, in a humidified environment for 48±1 hours.

After the 48-hour exposure period, the medium in the luciferase plates was replaced with fresh medium containing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The plate was sealed and incubated for 4 hours at 37±1°C, 5% (v/v) CO2. The MTT medium was then removed and SDS (at 10% w/v) added per well. The plate was sealed and placed into an incubator at 37±1°C, 5% (v/v) CO2 in air and left overnight. After the overnight incubation, the plate was shaken to ensure homogeneity of the solution in the wells and then absorption read at 600 nm using a SpectraMax M2e.

After the 48-hour exposure period, the cells in the viability plate were washed with phosphate buffered saline and lysis buffer for luminescence readings was added to each well. The plates were then incubated for 20 to 22 minutes at 25±2°C, loaded into the luminescence plate reader and read.

The results are summarised as follows:

Condition	Rep 1	Rep 2
IMAX (fold increase)	0.94	3.79
Cell Viability at the EC1.5 (%)	N/A	21.51
EC1.5 (mg/mL)	N/A	0.78
Dose response for luciferase Induction	No	Yes

The four conditions specified in the prediction model were not met in either repetition. The test article, was therefore considered to be negative in the ARE-Nrf2 Luciferase Test.