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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 November 2017 - 22 December 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:
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Name: BAL0001026
CAS number: 376653-41-7
Batch/Lot number: 012
Appearance: Off-white powder
Purity: Content by mass balance: 84.9% w/w
Expiry date: 30 June 2020
Storage conditions: Under inert gas, protected from light and humidity (tight closed container), frozen (≤-15 °C)
Safety precautions: Enhanced safety precautions were applied considering the supplied safety datasheet to assure personnel health and safety.
Specific details on test material used for the study:
Name: BAL0001026
Synonyms:
- BAL0001026-000
- (6R,7R)-7-[[(2Z)-2-amino-1,2,4-thiadiazol-3-yl)-2-[(triphenyl methoxy) imino]acetyl]amino]-3-[(E)-[(3’R)-1’-[(1,1-dimethylethoxy)carbonyl]-2-oxo-[1,3’-bipyrrolidin]-3-ylidene]methyl]8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid diphenylmethyl ester
CAS No.: 376653-41-7
Batch No.: 014
Description: Off-white powder
Storage conditions:
- In the freezer set at -20°C
- Protected from light
- Protected from humidity
- Under nitrogen atmosphere
Purity: 86.1%
Re-test date: June 2020.

In vitro test system

Details on study design:
In vitro test system:
KeratinoSens cells: the cell line KeratinoSens is stably transfected with a modified plasmid.
Supplier: this cell line was provided by Givaudan.

Control items:
Vehicle and negative control: DMSO
Positive control: Cinnamic aldehyde (CA).

Test item formulations
On the basis of solubility results, the test item was dissolved in DMSO at 200 mM.
One formulation was prepared for each run. It was then diluted in DMSO by serial dilutions, using a dilution factor of 2 to obtain a total of 12 concentrations in a 96-well plate; this 96 well plate was called "Master plate 100x". Subsequently, each formulation of the Master plate 100x was 25-fold diluted in treatment medium in another 96 well plate called "Master plate 4x" taking care to adjust all wells to the same DMSO level.
All formulations were prepared within 4 hours of use and were kept at room temperature and protected from light until use.

Study design:
Solubility assay
The test item was found soluble in DMSO at 200 mM.

Treatment:
The test item was first tested in two independent runs using cells from a different passage number. A third run was performed since non concordant results were obtained between the two first runs.
The plates were then incubated for 48 (± 2) hours at 37°C, 5% CO2, 90% humidity.

End-point measurements:
- Microscopic observation to evaluate the presence or absence of precipitate,
- Luminescence flash signal to evaluate induction signal,
- Absorbance signal to evaluate the cytotoxicity.

RESULTS ANALYSIS
Data evaluation was performed using a validated Excel sheet. The generated raw data (luminescence data for the luciferase activity and absorbance data for the MTT test) were pasted into an Excel template, and all data processing was performed automatically.

For the MTT and the luciferase data, the background value recorded in the empty well without cells (blank) was subtracted.
For the MTT data, the % viability was calculated for each well in the test plate in relation to average of the six negative control wells.
For the luciferase data, the average value of the six negative control wells was set to 1, and for each well in the plate, the fold induction was calculated in relation to this value.

For wells in which a statistically significant gene-induction (using a student test, also called T-test) over the 1.5 threshold was found, the following parameters were calculated from the processed raw data:
- Imax: maximal induction factor of luciferase activity compared to the negative control over the complete dose-response range measured,
- EC1.5: concentration at which a 1.5-fold luciferase gene induction is obtained,
- IC50 and IC30: concentrations effecting a reduction of cellular viability by 50% and 30%,
- Indication whether significant 1.5-fold gene induction occurred below the IC30.

The data were plotted in graphs and the Imax and the EC1.5 values were visually checked since uneven dose response curves or large variation may lead to wrong extrapolations.

Evaluation criteria of the test item
The results of each run are analyzed individually and if the test item is classified as positive in two runs, the final outcome is considered positive. If the test item is classified as negative in two runs, the final outcome is negative. In case, the first two runs were not concordant, a third run was performed and the final outcome was that of the two concordant runs.

The test item is considered as positive if the following four conditions are all met in two of two or in two of three runs, otherwise the KeratinoSens prediction is considered as negative:
- the Imax is > 1.5-fold and statistically significantly different as compared to the negative control (as determined by a two-tailed, unpaired Student’s T-test),
- at the lowest concentration with a gene induction > 1.5-fold (i.e. at the EC1.5 determining value), the cell viability is > 70%,
- the EC1.5 value is < 1000 µM (or < 200 µg/mL for test item without MW),
- there is an apparent overall dose-response for luciferase induction (or a reproducible biphasic response),
- In the rare cases where a statistically non significant induction > 1.5-fold was observed followed by a higher concentration with a statistically significant induction, results from this run were only considered as valid and positive if the statistically significant induction above the threshold of 1.5 is obtained for non cytotoxic concentrations.

Results and discussion

Positive control results:
All acceptance criteria were fulfilled for the positive and negative controls in each run; all runs were therefore considered to be valid.
During the two first runs, the criterion for "the average induction (Imax) in the three replicate plates for the positive control at 64 μM should be between 2 and 8" was not fulfilled (i.e. Imax of 9.5 in both runs). However, since clear dose-responses with increasing luciferase activity at increasing concentrations were obtained for the positive control in both runs, this was considered not to have any impact on the validity of the results of these runs.

In vitro / in chemico

Results
Parameter:
other: gene luciferase inductions
Run / experiment:
3
Value:
> 1.5
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Other effects / acceptance of results:
All acceptance criteria were fulfilled for the positive and negative controls in each run; all runs were therefore considered to be valid.

Applicant's summary and conclusion

Interpretation of results:
other: positive results
Conclusions:
Under the experimental conditions of this study, the test item was positive in the KeratinoSens assay and therefore was considered to activate the Nrf2 transcription factor and have the potential to be a skin sensitizer.
Executive summary:

The objective of this study was to evaluate the potential of the test item to activate the Nrf2 transcription factor which is indicative of the potential of the test item to be a skin sensitizer. This test is a part of a tiered strategy for the evaluation of skin sensitisation potential. The data generated with this test under the present Test Guideline can be used to support the discrimination between skin sensitizers and non‑sensitizers in the context of a tiered approach to testing and assessment.

 

Principle

 

This in vitro test uses the KeratinoSens cell line, an immortalized and genetically modified Human adherent HaCaT keratinocyte cell line. The KeratinoSens cell line is stably transfected with a plasmid containing a firefly luciferase gene under the transcriptional control of the SV40 origin of replication promoter. This promoter is fused with an ARE sequence. Sensitizers with electrophilic properties provoke the dissociation of Keap-1 from the transcription factor Nrf2. The free Nrf2 binds to the ARE sequence contained in the plasmid and therefore induces transcription of firefly luciferase.

 

Methods

The KeratinoSens cells were first plated on 96-well plates and grown for 24 hours at 37°C. Then the medium was removed and the cells were exposed to the vehicle control or to different concentrations of test item and of positive controls. The treated plates were then incubated for 48 hours at 37°C. At the end of the treatment, cells were washed and luciferase production was measured by flash luminescence. In parallel, the cytotoxicity was measured by a MTT reduction test and was taken into consideration in the interpretation of the sensitisation results. Three independent runs were performed as part of this study.

 

Results

All acceptance criteria were fulfilled for the positive and negative controls in each run; all runs were therefore considered to be valid.

 

First run

This first run was performed using the following test item concentrations: 0.98, 1.95, 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µM in culture medium containing 1% DMSO.

At these tested concentrations:

. slight to strong precipitate was observed in test item-treated wells at concentrations ≥ 62.5 µM at the end of the 48-hour treatment period,

. no noteworthy decrease in cell viability was noted (i.e. cell viability > 70%), thus no IC30and IC50 was calculated,

. no statistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations. Moreover, the Imaxvalue was 1.48 (i.e. < positive threshold of 1.5).

 

The evaluation criteria for a negative response were met in this first run.


Second run

During the second run, same concentrations as those used in the first run were tested.

 

At these tested concentrations:

. precipitate was observed in test item-treated wells atconcentrations ≥ 125 µM at the end of the 48‑hour treatment period,

. no noteworthy decrease in cell viability was noted (i.e. cell viability > 70%), thus no IC30and IC50 was calculated,

. statistically significant gene-fold inductions above the threshold of 1.5 were noted in comparison to the negative control at concentrations ≥ 15.63 µM (except at the highest concentration of 2000 µM) with an apparent dose response relationship,

. the Imaxvalue was 2.08 and the calculated EC1.5 was 11.39 µM.

 

The evaluation criteria for a positive response were therefore met in this second run.

However, the two runs performed were not concordant (i.e. first run negative and second run positive). Therefore, a third run was performed.

 

Third run

During the third run, same concentrations as those used in the first two runs were tested.

At these tested concentrations:

. precipitate was observed in test item-treated wells atconcentrations ≥ 125 µM at the end of the 48‑hour treatment period,

. no noteworthy decrease in cell viability was noted (i.e. cell viability > 70%), thus no IC30and IC50 was calculated,

. gene-fold inductionsabove the threshold of 1.5 were noted in comparison to the negative control at concentrations ≥ 500 µM with an apparent dose response relationship. These gene-fold inductions were found statistically significant at the highest concentrations of 2000 µM,

. the Imaxvalue was 1.78.

 

In this third run, statistically non-significant inductions above 1.5-fold (i.e. inductions of 1.7 and 1.6 at concentrations of 500 and 1000 µM, respectively) were followed by a higher concentration with a statistically significant induction (i.e. induction of 1.8 at 2000 µM). Thus, the parameters used for the EC1.5 extrapolation were corrected manually using the formula described in the § Results analysis and was 311.73 µM.

 

The evaluation criteria for a positive response were therefore met in this third run.

 

Discussion

No geometric mean IC30or IC50 was calculated since the cell viability was > 70% in all runs.

Since two of the three runs performed were considered as positive, the final outcome is therefore positive.

This positive result can be used to support the discrimination between skin sensitizers and non-sensitizers in the context of the integrated approach to testing and assessment used for skin sensitization evaluation, according to the OECD Guideline 442D.

Additionally, the Log P value of the test item is slightly > 5 (i.e. 5.66) and therefore between 5 and 7. However, this Log P value is not considered to be a limitation for the applicability of this test since the positive outcome obtained in two of the three runs performed guaranted the test system exposure to the test item.


Conclusion

Under the experimental conditions of this study, the test item was positive in the KeratinoSens assay and therefore was considered to activate the Nrf2 transcription factor and have the potential to be a skin sensitizer.