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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 January 2018 - January 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
read-across: supporting information
Reference
Endpoint:
skin sensitisation: in chemico
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
COMPLETEREPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Comparing the structures of the main constituents of the target substance and the source substance, we note that both main constituents are composed of the same chemical groups (Acetyl and Ester groups). The only difference between the source and the target main constituents is the presence of the C=C double bonds, one in each chain, in the source substance.
Consequently, the overall molecular weight of the source substance is 1059.5403 which is 6.0476 units (6 H) less than the target substance which is 1065.5879.
The overall elemental composition of the 2 substances are also very similar, with the percent Carbon between 71.0-71.4%, the percent Hydrogen between 10.5-11.0% and the percent Oxygen between 18.0-18.1%.
Neither the source or target substance main constituents contain any hydrogen donor or hydrogen acceptor sites.
Predicted dermal absorption of both the source and target substance is of the 0.1 mg/kg day magnitude (appendix 1).
These similarities mean that the physical chemical properties are also very similar, in particular:
• Both are water insoluble (<1.5 µg/mL)
• Both are liquids at room temperature with complete glass transition points <-50°C and decompose before boiling (>250 °C).
• Both have a relative density of 0.955 – 0.97 g/cm3
• Both have high partition coefficients (Log KoW) of >7.2 at 35 °C pH 7
The molecular weight, number of hydrogen bond donors, number of hydrogen bond acceptors, water solubility, and partition coefficient values for both substances indicate they will have similar bioavailability.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

Source Target
Common name Dermol GTR Hetester HCA
IUPAC name propane-1,2,3-triyl tris(12-acetoxyoctadec-9-enoate) propane-1,2,3-triyl tris(12-acetoxyoctadecanoate)
Physical state Liquid Liquid
Molecular weight 1059.5403 1065.5879
Molecular formula C63H110O12 C63H116O12


3. ANALOGUE APPROACH JUSTIFICATION
The source substance does not fulfil Lipinski’s rule of five and as such is not expected to be bioavailable (see section 2.2). The source substance also has very low dermal absorption (0.1 mg/kg day). As such the source substance is not expected to be react with the peptides, proteins, and surface markers in the in chemico and in vitro tests.
The hypothesis is proven by the results of the source substance, which failed to induce all but the slightest of reactions, and therefore could not be considered a positive skin sensitiser under the conditions of the three in chemico and in vitro tests.
The target substance does not fulfil Lipinski’s rule of five and as such is not expected to be bioavailable. The target substance also has very low dermal absorption (0.1 mg/kg day).
The target substance is therefore predicted to fail to induce sufficient reaction in the OECD 442 C, OECD 442 E, and OECD 442 D tests to be considered a positive skin sensitiser, and by extension, be considered to fulfil the criteria for skin sensitisation under the Classification, Labelling, and Packaging (CLP) regulation (1272/2008).


4. DATA MATRIX
Please see appended document for full data matrix.
Reason / purpose:
read-across source
Related information:
Composition 1
Reason / purpose:
read-across source
Related information:
Composition 1
Reason / purpose:
read-across source
Related information:
Composition 1
Test material information:
Composition 1
Parameter:
other: ARE-NRF2
Remarks on result:
no indication of skin sensitisation
Remarks:
Based upon read across substance
Parameter:
other: h-CLAT
Remarks on result:
no indication of skin sensitisation
Remarks:
Based upon read across substance
Parameter:
other: DPRA
Remarks on result:
no indication of skin sensitisation
Remarks:
Based upon read across substance
Interpretation of results:
GHS criteria not met

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
February 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
This is an in chemico test and the test material is used in cosmetic ingredients. Regulation 1223/2009 Article 18 restricts the use of in vivo studies on these types of raw materials.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Test material form:
liquid
Specific details on test material used for the study:
The test article, a clear yellow liquid, was identified as Dermol GTR and was received at Covance on 31 October 2017 as follows:
- CAS Number: 101-34-8
- Storage:15 to 25°C, protected from light
- Purity: 100%

In chemico test system

Details on study design:
The study was conducted to investigate the potential of the test material 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.

Specifications
KeratinoSens™ cell line supplied by Givaudan Schweiz, Zurich, Switzerland. Identification
The test system was appropriately labelled with the study number, assay type, experiment number and test/positive/negative control.
Preparation of Cultures
A fresh vial of cells was used for each experimental occasion and cultured using Dulbecco’s modified Eagle medium (DMEM) containing serum and Geneticin.
Treatment Plate Preparation
The cells were 80-90% confluent (see Section 9 for details of protocol deviations). On the day prior to treatment, cells were harvested and distributed into 96-well plates (10000 cells/well) and incubated at 37±1°C, 5% (v/v) CO2, for 24±1 hours.
For each repetition, three replicates were used for the luciferase activity measurements and one parallel replicate used for the cell viability assay.
Treatment
At the end of the 24-hour incubation period, the medium was removed and replaced with fresh culture medium (containing serum but without Geneticin) to which test article and control formulations were added.
One well per plate was left empty (no cells and no treatment) to assess background values.
Each plate was sealed and incubated at 37±1°C, 5% (v/v) CO2 in air, in a humidified environment for 48±1 hours.
For each test article and positive control, one experiment was needed to derive a prediction (positive or negative), consisting of at two independent repetitions each containing three replicates of each concentration.
The data for repetition 1 was obtained from a repeat experiment as the initial experiment did not meet the acceptance criteria for the positive or negative controls.
The data from the initial experiment has not been reported.
Discordant results were obtained between the two repetitions, therefore a third repetition containing three replicates was performed.
Each independent repetition was performed on a different day with fresh stock solutions of chemicals and independently harvested cells. The cells came from different passages.
Cytotoxicity Assessment
After the 48-hour exposure period, the medium was replaced with fresh medium containing MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide).The plate was sealed and incubated for 4 hours at 37±1°C, 5% (v/v) CO2.
The MTT medium was 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.

Luciferase Activity Measurements
After the 48-hour exposure period, the cells were washed with phosphate buffered saline and lysis buffer for luminescence readings was added to each well. The plates were then incubated for 20 minutes at 25±2°C, loaded into the luminescence plate
reader and read using the following parameters: 100 µL injection (Luciferase assay substrate), 15 second delay, 7 second luminescence integration time.

Results and discussion

Positive control results:
Luciferase activity induction obtained with the positive control was statistically significant above the threshold of 1.5 at concentrations of 16 to 64 µM in Experiment 1 and at concentrations of 32 to 64 µM in Experiment 2. The EC1.5 value for the positive control were 11.02 and 18.11 µM in Experiments 1 and 2, respectively. The average induction in the two replicates for the positive control at 64 µM were 10.56 and 3.08 in Experiments 1 and 2, respectively.

In vitro / in chemico

Resultsopen allclose all
Parameter:
other: Mamimal fold increase
Remarks:
Imax
Run / experiment:
Experiment 1
Value:
0.78
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Parameter:
other: Maximal fold increase
Remarks:
Imax
Run / experiment:
Experiment 2
Value:
1.05
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
- Viability:
The cell viability measurement was not applicable as there were no EC1.5 determining concentrations in Experiments 1 or 2.
The IC50 value could not be calculated for Experiment 1 as all viability results were below 50% or in Experiment 2 as the minimum viability did not drop below 50%.
The IC30 value was 958.72 µM in Experiment 2. An IC30 value could not be calculated for Experiment 1 as all viability results were below 70%.

- Assay Acceptance:
Luciferase activity induction obtained with the positive control was statistically significant above the threshold of 1.5 at concentrations of 16 to 64 µM in Experiment 1 and at concentrations of 32 to 64 µM in Experiment 2.
The EC1.5 value for the positive control were 11.02 and 18.11 µM in Experiments 1 and 2, respectively. The average induction in the two replicates for the positive control at 64 µM were 10.56 and 3.08 in Experiments 1 and 2, respectively.
The average coefficient of variation of the luminescence reading for the negative control (DMSO) was 9.87% and 6.63% in Experiments 1 and 2, respectively.
All acceptance criteria were met, with the exception that in Experiment 1, the EC1.5 for the positive control was above the range specified in the protocol at 10.56. However, as luciferase induction values showed a clear dose response, it was considered that this was a valid experiment.
The assay was considered valid as the results in Experiment 1 were replicated in Experiment 2.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The Imax in both experiments was less than 1.5 and not statistically significant, therefore the test article, Dermol GTR, was considered to be negative in the ARE-Nrf2 Luciferase Test.
Executive summary:

The study was conducted to investigate the potential of Dermol GTR 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 dimethylformamide (DMF) to the final stock concentration (200 mM). Serial dilutions were then made using DMF 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 concentrationswere 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 environmentfor 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 minutes at 25±2°C, loaded into the luminescence plate reader and read.

The results are summarised as follows:

Criteria

Experiment 1

Experiment 2

Imax

0.78

1.05

Cell Viability

N/A

N/A

EC1.5

>2000 µM

>2000 µM

Dose Response

No

No

 

All acceptance criteria were met, with the exception that in Experiment 1, the EC1.5 for the positive control was above the range specified in the protocol at 10.56. However, luciferase induction values showed a clear dose response, therefore it was considered that this was a valid experiment.

The test article, Dermol GTR, was considered to be negative in the ARE-Nrf2 Luciferase Test.