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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From September 27 to November 30, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

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

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
Method described in the following papers:
1. Genes specifically modulated in sensitized skins allow the detection of sensitizers in a reconstructed human skin model. Development of the SENS-IS assay. Cottrez F., Boitel E., Auriault C., Aeby P., Groux H. Toxicology in vitro 29: 787-802, 2015.
2. SENS-IS, a 3D reconstituted epidermis based model for quantifying chemical sensitization potency: reproductibility and predictivity results from an inter-laboratory study. Cottrez F., Boitel E., Ourlin J.C., Peiffer J.L., Fabre I., Henaoui I.S., Mari B., Vallauri A., Paquet A., Barbry P., Auriault C., Aeby P., Groux H. . Toxicology in Vitro 32: 248-260, 2016.
GLP compliance:
no
Remarks:
Not fully validated method
Type of study:
other: activation of ARE and SENS-IS gene subset

Test material

Constituent 1
Reference substance name:
Chromium(3+) neodecanoate
IUPAC Name:
Chromium(3+) neodecanoate

In vitro test system

Details on the study design:
Skin sensitisation (In vitro test system) - Details on study design

Test systems
The test system used in this study was the EpiSkin™ model, an in vitro reconstructed human epidermis from normal human keratinocytes cultured on a collagen matrix at the air-liquid interface. This model is histologically similar to the in vivo human epidermis, with a functional horny layer.
Some quality controls are performed by the provider of the model and must fulfill the following criteria: IC50 value of SLS by the MTT test ≥ 1.2 mg/mL.

Preliminary test: Solubility test
The test item was solubilized in olive oil (OO) at a concentration of 10% and 50% at room temperature and at 60°C, respectively.
The solubility of the test item was assessed by visual inspection of each preparation.

Main test: SENS-IS assay
The test item (30 µL) was deposited on the epidermis surface and gently spread on the entire surface. After 15 minutes of exposure, the Episkin™ was rinsed with PBS and then incubated at 37°C for 6 hours.
After incubation, reconstructed epidermis was removed from the inserts with forceps and placed in a cryotube for freezing in liquid nitrogen. The epidermis was then transferred in a tube containing 1 mL of Qiazol reagent and 2 steel beads. Epidermis was homogenized using the TissueLyser II. After centrifugation, the supernatant was collected and stored at -20°C until RNA extraction.
After addition of bromochloropropane, total RNA were purified using the miRNeasy extraction Kit according to the manufacturer’s instructions (Qiagen, Courtaboeuf, France). RNA quality was assessed by measuring 260/280 absorbance ratio. The mRNA was then reversed as cDNA using SuperScript III Reverse Transcriptase kit and RNase inhibitor.
After reverse transcription, quantitative gene expression was measured by qRT-PCR using a SYBRGreen® buffer and specific primers defined for the SENS-IS test. The housekeeping genes (Glucuronidase ß, ß2 microglobuline, and Nono « non-POU domain containing octamer-binding ») were analyzed in parallel.

Results and discussion

In vitro / in chemico

Resultsopen allclose all
Run / experiment:
other: Experiment 1
Parameter:
other: Number of SENS-IS genes
Value:
3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: highest value found at 50 % of test item in olive oil
Run / experiment:
other: Experiment 2
Parameter:
other: Number of SENS-IS genes
Value:
9
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: highest value found at 50 % in olive oil
Run / experiment:
other: Experiment 3
Parameter:
other: Number of SENS-IS genes
Value:
8
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: highest value found at 10 % and 50 % in olive oil

Applicant's summary and conclusion

Interpretation of results:
other: Skin Sensitizer 1B
Remarks:
according to the classification criteria described in the SENS-IS assay
Conclusions:
Skin Sensitising

Executive summary:

Method

The substance has been tested for its potential to induce skin sensitisation according to the SENS-IS assay (Cottrez F. et al, Toxicology in vitro 29 (2015) 787 -802).

As reported in Annex XI of the REACH Regulation N. 1907 -2006, the results of non-validated in vitro methods are accepted with no further confirmation, if the following conditions are met:

(1) results are derived from anin vitro method whose scientific validity has been established by a validation study, according to internationally agreed validation principles;

(2) results are adequate for the purpose of classification and labelling and/or risk assessment; and

(3) adequate and reliable documentation of the applied method is provided.

All three conditions are fullfilled because:

1) the SENS-IS method has been internally validated (Cottrez F. et al 2016, Toxicology in vitro V.32, 248 -260). Validation study has been submitted to ECVAM and it is under evaluation as declared in the TSAR website (https://tsar.jrc.ec.europa.eu/test-method/tm2011 -11). The only limitation to the formal validation derives from the status of the patented method.

2)The results of the assay lead to a classification according to the CLP. The confirmation is requested only in case of negative results.

3) The study is well documented and performed in the spirit of GLP.

The objective of this study was to evaluate the in vitro sensitization potential of the test item applied onto 3D human reconstructed epidermis (EpiSkin™) using the SENS-IS assay, based on the quantitative analysis of specific gene biomarkers.

The expression profile of 61 genes divided in three sets were analysed: one set of 23 genes related to the irritation process and the two other sets of genes, named “SENS-IS” and “ARE”, with 21 and 17 biomarkers respectively, involved in skin sensitization.

The expression level of these separate sets of genes was determined by qRT-PCR (quantitative real-time polymerase chain reaction) after a 15 min incubation period with the test item and a 6 hours post-incubation period at 37 °C.

The fold expression of each gene were calculated as a ratio between the mRNA level of test item-treated epidermis versus control (vehicle treated) epidermis.

Results

The results obtained for the positive and negative controls were within acceptance criteria and validated the study, with the exception of DMSO in the experiments 2SI18007.

Considering the number of over-expressed gene in the “SENS-IS” and “ARE” gene groups, the test item gave positive result (more than 7 genes induced) when it was tested diluted at 50% (v/v). Moreover, positive results were also obtained when the test item was tested at 10 % in one experiment out of two and at 100% (not diluted).

Conclusion

Under the experimental conditions of this SENS-IS assay, the test itemcan be classified as a sensitizer in class 1B.