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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:
November 2018 - january 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
The objective of the KeratinoSens assay is to evaluate the potential of the test item to activate the Nrf2 transcription factor. This test is part of a tiered strategy for the evaluation of skin sensitisation potential. Thus, data generated with the present Test Guideline should be used to support the discrimination between skin sensitizers and non sensitizers in the context of an integrated approach to testing and assessment. It is an alternative method in order to avoid animal study

Test material

Constituent 1
Chemical structure
Reference substance name:
2-[2,4-bis(tert-pentyl)phenoxy]-N-(3,5-dichloro-4-ethyl-2-hydroxyphenyl)butyramide
EC Number:
300-634-0
EC Name:
2-[2,4-bis(tert-pentyl)phenoxy]-N-(3,5-dichloro-4-ethyl-2-hydroxyphenyl)butyramide
Cas Number:
93951-12-3
Molecular formula:
C28H39Cl2NO3
IUPAC Name:
2-[2,4-bis(tert-pentyl)phenoxy]-N-(3,5-dichloro-4-ethyl-2-hydroxyphenyl)butyramide
Test material form:
solid
Details on test material:
Batch 44029
colour : White to off-white
retest date : 01/06/2019
Specific details on test material used for the study:
At room temperature (on 29 October 2018, the test item was found at +4°C, whereas it should have been at room temperature. After investigations, it was deducted that the test item was mistakenly stored at +4°C for up to 12 days. This was considered not to have any impact on test item integrity, as stated by the Sponsor in an email dated 29 October 2019

. Protected from light

. Protected from humidity

In vitro test system

Details on the study design:
The study design was based on the OECD Guideline 442D: In Vitro Skin Sensitisation assays addressing the AOP key event on keratinocyte activation, adopted on June 2018.
With the following exception:
. the OECD guideline 442D mentions that test items prepared in sterile water or culture medium have to be filtered after preparation for sterilization prior treatment. Filtration of such preparations can be possible when a test item formulation is a solution. However, in case the test item formulation is a suspension, filtration process can retain test item particles in the filter and considerably decrease test item concentration in the filtered formulation. Therefore, in view of the above and considering that pre-treatment filtration procedure is not of common practice in other in vitro cell-based assays, and that water and culture media used in the assay are already sterile, test item formulations were not filtered prior treatment.
The test item was tested in three 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.

Results and discussion

Positive control results:
Gene induction values, Imax and EC1.5 values obtained with the positive control (cinnamic aldehyde)
Imax mean = 4.24
EC1.5 geometric mean = 13.08 µM

Evaluation of the viability (%) of cultures treated with the positive control for each run:
cinnamic aldehyde Concentrations (μM) : 4 ; 8 ; 16 ; 32 ; 64
Viability (%) in Run 1 106 ; 113 ; 116 ; 117 ; 97
Viability (%) in Run 2 111 ; 109 ; 128 ; 126 ; 129
Viability (%) in Run 3 107 ; 108 ; 112 ; 116 ; 106
Mean viability (%) 108 ; 110 ; 119 ; 120 ; 111
Geometric Mean (%) 108 ; 110 ; 119 ; 120 ; 110
SD 3 ; 3 ; 8 ; 5 ; 17

In vitro / in chemico

Resultsopen allclose all
Key result
Run / experiment:
other: 1
Parameter:
other: IC30
Remarks:
(µM)
Value:
3.69
Vehicle controls validity:
other: same as negative control
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 1
Parameter:
other: IC50
Remarks:
(µM)
Value:
18.91
Vehicle controls validity:
other: same as negative control
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 1
Parameter:
other: Imax
Value:
1.5
Vehicle controls validity:
other: same as negative control
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 2
Parameter:
other: IC30
Remarks:
(µM)
Value:
25.37
Vehicle controls validity:
other: same as negative control
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 2
Parameter:
other: IC50
Value:
29.65
Vehicle controls validity:
other: same as negative control
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 2
Parameter:
other: Imax
Value:
1.67
Vehicle controls validity:
other: same as negative control
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 2
Parameter:
other: EC1.5
Remarks:
calculated (µM)
Value:
1.87
Vehicle controls validity:
other: same as negative control
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 3
Parameter:
other: IC30
Value:
15.23
Vehicle controls validity:
other: same as negative control
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 3
Parameter:
other: IC50
Value:
19.29
Vehicle controls validity:
other: same as negative control
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 3
Parameter:
other: Imax
Value:
1.5
Vehicle controls validity:
other: same as negative control
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
First run
All acceptance criteria were fulfilled for the positive and negative controls. The run was therefore considered to be valid.
Second run
All acceptance criteria were met for the positive and negative controls, this run was therefore considered to be valid.
Third run
Since non-concordant results were obtained throughout the two first runs, a third run was performed. In the third run, all acceptance criteria were met for the positive and negative controls, with one exception regarding positive control EC1.5 slightly higher than historical data (i.e. 15.6 μM instead of ranging from 2.8 to 13.9 μM) which was considered not to have any impact on the validity of the results, the run was therefore considered to be valid.

Any other information on results incl. tables

IC30 and IC50: Concentrations effecting a reduction of cellular viability by 50% and 30%

Imax : Maximal induction factor of luciferase activity compared to the negative control over the complete dose-response range measured

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions of this study, the test item, 80ACQ, was negative in the KeratinoSens assay and therefore was considered to have no potential to activate the Nrf2 transcription factor.
Executive summary:

test item was found soluble in DMSO at 200 mM and 100 mM. Precipitates were observed once these formulations were 100-fold diluted in culture medium.

The three runs were performed using the following concentrations in culture medium containing 1% DMSO:

. first run: 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 μM,

. second run: 0.71, 1.01, 1.42, 2, 2.82, 3.98, 5.61, 7.91, 11.15, 15.72, 22.16 and 31.25 μM,

. third run: 0.02, 0.03, 0.06, 0.12, 0.24, 0.49, 0.98, 1.95, 3.91, 7.81, 15.63 and 31.25 μM.

At these tested concentrations:

. slight to strong precipitates were observed in treated wells at concentrations ≥ 500 μM,

. a decrease in cell viability (i.e. cell viability < 70%) was noted at concentrations ≥ 3.91 μM in the first run, ≥ 31.25 μM in the second run and ≥ 0.98 μM in the third run,

. the corresponding geometric means IC30 and IC50 of the three validated runs were calculated to be 11.26 and 22.11 μM, respectively,

. no statistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to

the negative control in non-cytotoxic and non-precipitating concentrations for both the first and third runs. In the second run, statistically gene-fold inductions above the threshold of 1.5 were noted at

2 μM and at concentrations between 3.98 to 11.15 μM with an apparent dose-response until 11.15 μM.

The evaluation criteria for a negative response are met in two out of three runs (i.e. first and third runs), the final outcome is therefore negative. This negative result can be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment. It cannot be used on its own to conclude on a skin sensitisation potential.