Registration Dossier

Administrative data

Description of key information

The in vitro KeratinoSens test ( ARE-Nrf2 Luciferase Test Method-OECD 442) (Aubert, CiToxLAB France 2019) with cadmium sulfate was done in 2 runs.

During the first run, the following concentrations were tested: 0.98 ; 1.95 ; 3.91 ; 7.81 ; 15.6 ; 31.3 ; 62.5 ; 125 ; 250 ; 500 ; 1000 and 2000 µM.

Due to a high cytotoxicity observed in the first run, the range of concentrations was adapted in the second run as follows: 0.10; 0.20; 0.39; 0.78; 1.56; 3.13; 6.25; 12.5; 25.0; 50.0; 100; 200 µM.

At these tested concentrations:

- A high decrease in cell viability (i.e. cell viability < 70%) was noted at concentrations ≥ 31.3 µM in the first run and 25.0 µM in the second one.

-Statistically significant gene-fold inductions above the threshold of 1.5 were noted in comparison to the negative control, from the lowest tested concentration in the first run and at concentrations ≥ 0.39 µM in the second run, and up to cytotoxic concentrations in both runs,

The evaluation criteria for a positive response are therefore met in both runs.

Therefore, and in our experimental conditions, the test itemCdSO4 is positivein the KeratinoSens assay and therefore is considered to have a potential to activate the Nrf2 transcription factor. 

The KeratinoSens test is part of a tiered testing strategy currently further being performed/discussed with the CRO.

Further investigation is currently ongoing checking if the other tests of the testing strategy are applicable for the 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:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
adopted on 25th June 2018
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- lot/batch No.of test material:X09D060
- Expiration date of the lot/batch: 04 June 2020
- Purity test date:99.99%
- Description:White solid (in the powder form)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Controlled room temperature (15-25°C, ≤70% relative humidity).
- Safety precautions: Enhanced safety precautions (half mask at least with P3 filter cartridge, nitrile gloves, lab coat) were applied considering the supplied safety datasheet to assure personnel health and safety.
Details on study design:
Skin sensitisation (In vitro test system) : KeratinoSens assay
This in vitro test uses Human adherent HaCaT keratinocytes, an immortalized cell line. The KeratinoSens is a stably transfected cell line with a plasmid containing a luciferase gene under the transcriptional control of the SV40 origin of replication promoter. This promoter is fused with an ARE sequence.
Potential skin sensitizers are applied to the cells at 12 different concentrations and for a period of 48 hours. Sensitizers with electrophilic properties will provoke the dissociation of Keap-1 from the transcription factor Nrf2. The free Nrf2 will then bind to the ARE sequence contained in the plasmid and will therefore induce transcription of firefly luciferase. The luciferase reporter gene is under control of a single copy of the ARE element of the human AKR1C2. The luciferase production will then be measured by flash luminescence.
In parallel, cytotoxicity is measured by MTT reduction and is taken into consideration in the interpretation of the sensitisation results. This evaluation is performed in at least two independent runs.


Parameter:
other: % viability
Run / experiment:
1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: A high decrease in cell viability (i.e. cell viability < 70%) was noted at concentrations ≥ 31.3 µM in the first run
Parameter:
other: % viability
Run / experiment:
2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: A high decrease in cell viability (i.e. cell viability < 70%) was noted at concentrations ≥ 25.0 µM in the second run
Parameter:
other: gene fold induction
Run / experiment:
1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Statistically significant gene-fold inductions above the threshold of 1.5 were noted in comparison to the negative control, from the lowest tested concentration (0.98µM) and up to cytotoxic concentration
Parameter:
other: gene-fold induction
Run / experiment:
1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Statistically significant gene-fold inductions above the threshold of 1.5 were noted in comparison to the negative control at concentrations ≥ 0.39 µM and up to cytotoxic concentration
Interpretation of results:
other: positive in the KeratinoSens assay and therefore is considered to have a potential to activate the Nrf2 transcription factor.
Conclusions:
Under the experimental conditions, the test item CdSO4 is positive in the KeratinoSens assay and therefore is considered to have a potential to activate the Nrf2 transcription factor.
Executive summary:

The objective of the KeratinoSens assay is to evaluate the potential of the test item toactivate the Nrf2 transcription factor. This test is part of a tiered strategy for the evaluation of skin sensitisation potential. Therefore data generated with the present Test Guideline can be used to support the discrimination between skin sensitizers and non‑sensitizers in the context of an integrated approach to testing and assessment.

In this study 2 runs were performed.

During the first run, the following concentrations were tested: 0.98 ; 1.95 ; 3.91 ; 7.81 ; 15.6 ; 31.3 ; 62.5 ; 125 ; 250 ; 500 ; 1000 and 2000 µM.

Due to a high cytotoxicity observed in the first run, the range of concentrations was adapted in the second run as follows: 0.10; 0.20; 0.39; 0.78; 1.56; 3.13; 6.25; 12.5; 25.0; 50.0; 100; 200 µM.

 

At these tested concentrations:

  • A high decrease in cell viability (i.e. cell viability < 70%) was noted at concentrations ≥ 31.3 µM in the first run and 25.0 µM in the second one.
  • Statistically significant gene-fold inductions above the threshold of 1.5 were noted in comparison to the negative control, from the lowest tested concentration in the first run and at concentrations ≥ 0.39 µM in the second run, and up to cytotoxic concentrations in both runs,

The evaluation criteria for a positive response are therefore met in both runs.

Therefore, and in our experimental conditions, the test itemCdSO4 is positive in the KeratinoSens assay and therefore is considered to have a potential to activate the Nrf2 transcription factor. 

Endpoint conclusion
Endpoint conclusion:
no study available (further information necessary)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the KeratinoSens test so far which is part of a tiered testing strategy for the evaluation of skin sensitisation potential, it cannot be used on its own to conclude on a skin sensitisation potential. Currently further investigations are ongoing. At present cadmium sulfate has not enough data (time constrains for all tests to be done) for classification which is in line with the harmonised classification in Annex I of Directive 67/548/EEC.