Registration Dossier

Administrative data

Description of key information

Due to the complexity of the skin sensitisation endpoint no single non-animal test method is able to provide information sufficient to classify or not a substance as sensitisier. No in-vitro tests have been developed for use as stand-alone methods and therefore, a combination of these non-animal methods (e.g. in silico, in chemico and in vitro) should be used within a Weight of Evidence approach. According to OECD guides line on skin sensitisation some in-vitro tests were not applicable to the test item, Acid Brown 396:1.

The selected tests on the basis of the UVCB and metal-complex nature of the test items have been h-Clat (OECD 442E) and LuSens (OECD 442D). Only one of them was feasable so that a QSAR estimation was used to support the lacking information. Acid Brown 396:1 is resulted to be a sensitising substance.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 07th August 2018 to 20th September 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
OECD 442D
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
Adopted 25. June 2018
Deviations:
yes
Remarks:
Not critical.
Qualifier:
according to
Guideline:
other: EU-Method B.60 “In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method”
Version / remarks:
Commission Regulation (EU) No. 2017/735 adopted 14. Feb. 2017.
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes
Specific details on test material used for the study:
Name: ACID BROWN 396:1
EINECS-No. 947-779-0
Batch no.: 77171
Appearance: Brown powder
Purity: 100% (UVCB)
Homogeneity: homogeneous
Production date 18. Sep. 2015
Expiry date: 18. Sep. 2020
Storage: Room temperature (20 ± 5 °C)
Details on study design:
The LuSens cell line was specially designed for this test system by the BASF SE. It employs the use of a luciferase reporter gene placed under the control of the antioxidant response element (ARE) and hence monitors Nrf2 transcription factor activity. For designing this cell line, a human keratinocyte cell line was transfected with the pGL4.20 [luc2/Puro] vector carrying the regulatory antioxidant response element (ARE) upstream of the luciferase gene at the Institute of Anatomy and Cell Biology of the RWTH, Aachen (laboratory of PD Dr. Wruck).
The LuSens cell line was obtained from the BASF SE. For mycoplasma contamination screened stocks of LuSens cells are stored in liquid nitrogen in the cell bank of LAUS GmbH to allow a continuous stock of cells (mycoplasma contamination free), which guarantees similar parameters of the experiment and reproducible characteristics of the cells. For the Cytotoxicity Range Finder Assay cells of passage 10 were used. After thawing, the cells were cultivated in DMEM in cell culture flasks at 37 ± 1 °C in a humidified atmosphere.Three experiments were performed.
Key result
Parameter:
other: Luciferase induction
Remarks:
(fold)
Value:
>= 1.5
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation

VALIDITY OF THE TEST

In the following table the criteria for acceptability as well as the corresponding results in experiment I, II and III are given. 

Criteria

Found in

experiment I

Found in

experiment II

Found in

experiment III

The average induction for the positive control should be ≥ 2.5 fold and it should have a relative viability of at least 70 %.

Positive control

Fold induction:

4.5

Relative viability: 90.2 %

Positive control

Fold induction:

4.6

Relative viability: 89.8 %

Positive control

Fold induction:

6.2

Relative viability: 93.3 %

The induction triggered by the negative control and growth control should be < 1.5 fold as compared to the induction of the solvent control and the viability should be above 70 %.

Negative control:

Fold induction:

0.9

Relative viability:

106.7 %

Growth control:

Fold induction:

1.2

Relative viability:

149.9 %

Negative control:

Fold induction:

1.0

Relative viability:

104.5 %

Growth control:

Fold induction:

1.45

Relative viability:

154.4 %

Negative control:

Fold induction:

1.1

Relative viability:

110.9 %

Growth control:

Fold induction:

0.9

Relative viability:

130.0 %

The average percentage standard deviation (luciferase induction) of the variability in at least 21 solvent control wells should be below 20 %.

5.54 %

11.73 %

8.25 %

At least 3 test concentrations must be within viability limits, i.e. have relative viability of at least 70 %.

7 concentrations are analysable

8 concentrations are analysable

7 concentrations are analysable

All validity criteria were met. Therefore, the study is valid.


Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
Each valid experiment (i.e. meeting all acceptance criteria, according to the procedure described above) is interpreted as follows:
A test compound is considered to have the potential to activate the Nrf2 transcription factor if the luciferase induction is ≥ 1.5 fold and statistically significant compared to the vehicle control in 2 (or more than) consecutive non-cytotoxic (relative viability ≥ 70 %) tested concentrations whereby at least three tested concentrations must be non-cytotoxic in two independent valid experiments.
A test compound is considered not to have the potential to activate the Nrf2 transcription factor if the effects mentioned above are not observed.
The skin sensitizing potential (corresponding to the potential to activate the Nrf2 transcription factor) of a test substance is determined by the result of the majority of the repetitions of an experiment. If two of two or two of three experiments are nega-tive/positive, the substance is considered as negative/positive: in this case, the luciferase induction was ≥ 1.5 fold and statistically significant compared to the solvent control in more than 2 consecutive non-cytotoxic tested concentrations in experiment II and III.
Therefore, the test item ACID BROWN 396:1 is considered to have the potential to activate the Nrf2 transcription factor (sensitizing potential) under the conditions of the LuSens test.
Executive summary:

This in vitro study was performed to investigate the potential of ACID BROWN 396:1 to activate the Nrf2 transcription factor (sensitizing potential), by using the LuSens cell line.

The assay was performed in three independent experiments: 12 concentrations of the test item were evaluated. The exposure time was 48 h.

The following nominal concentrations of the test item were investigated in experiment I, II and III:

6.7 µg/mL, 8.1 µg/mL, 9.7 µg/mL, 11.6 µg/mL, 14.0 µg/mL, 16.7 µg/mL, 20.1 µg/mL, 24.1 µg/mL, 28.9 µg/mL, 34.7 µg/mL, 41.7 µg/mL, 50.0 µg/mL.

None of the real treatment concentrations in both experiments deviated more than 10 % from the nominal concentration. Precipitation of the test item was not visible up to the highest concentration.

Since all acceptability criteria of the assay were met the study is valid.

In experiment I cytotoxic effects were observed in the concentrations 24.1 µg/mL to 50.0 µg/mL.

In experiment II, again cytotoxic effects were observed in the concentrations 28.9 µg/mL to 50.0 µg/mL.

In experiment III at the highest test item concentration (50.0 mg/ml) there was one outlier in the three replicates in the plate for evaluation of the relative viability and one outlier in the three replicates in the plate for evaluation of Luciferase induction.

The cytotoxic concentrations and the highest concentration in experiment III were excluded from the evaluation of the luciferase induction.

Finally, the following test item concentrations showed a viability ≥ 70 % and are analysable and could therefore be evaluated for luciferase induction:

Experiment I: 6.7 µg/mL, 8.1 µg/mL, 9.7 µg/mL, 11.6 µg/mL, 14.0 µg/mL, 16.7 µg/mL, 20.1 µg/mL

Experiment II: 6.7 µg/mL, 8.1 µg/mL, 9.7 µg/mL, 11.6 µg/mL, 14.0 µg/mL, 16.7 µg/mL, 20.1 µg/mL, 24.1 µg/mL

Experiment III: 6.7 µg/mL, 8.1 µg/mL, 9.7 µg/mL, 11.6 µg/mL, 14.0 µg/mL, 16.7 µg/mL, 20.1 µg/mL, 24.1 µg/mL, 28.9 µg/mL, 34.7 µg/mL, 41.7 µg/mL

 

A statistically significant increase≥ 1.5 foldin luciferase induction was measured in the following concentrations in experiment I:20.1 µg/mL

A statistically significant increase≥ 1.5 foldin luciferase induction was measured in the following concentrations in experiment II:20.1 µg/mL, 24.1 µg/mL

A statistically significant increase≥ 1.5 foldin luciferase induction was measured in the following concentrations in experiment II:24.1 µg/mL, 28.9 µg/mL, 34.7 µg/mL, 41.7 µg/mL

 

Therefore, experiment I is clearly negative and experiment II and III are clearly positive.

 

In conclusion, it can be stated that under the experimental conditions of this study, the test item ACID BROWN 396:1 was positive in the LuSens assay and is therefore considered to have the potential to activate the Nrf2 transcription factor (sensitising potential).


Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Two in vitro studies have been planned but the study according to OECD 442E was not feasible for a not sufficient solubility.

Under the experimental conditions of the LuSens assay , the test item ACID BROWN 396:1 was positive and is therefore considered to have the potential to activate the Nrf2 transcription factor (sensitising potential).

QSAR estimation has been performed as supporting study and it has confirmed a sensitising potential for the tested substance.

Acid Brown 396:1 is therefore considered as sensitising.