Reaction products of tetraisopropyl titanate (4+), hydroxyalkaloic acid and substituted alkanol

Endpoint summary

Description of key information

Not skin sensitising

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 May 18th to July 09th, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

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

Justification for non-LLNA method:

LLNA method was not carried out because the test substance is a complex metal

Details on the study design:

Preliminary test: Solubility test
The solubility of the test item was assessed in phosphate buffered saline (PBS), olive oil (OO), and dimethylsulfoxide (DMSO) at a concentration of 10% and 50%, at room temperature and at 37°C.
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 [3]. 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.

Vehicle / solvent control:

DMSO

Negative control:

other: DMSO

Positive control:

other: SLS, 10%= Positive control for irritation and negative control for sensitization; TNBS, 1M= Positive control for sensitization

Positive control results:

SLS at 5% was classified as irritant (number of overexpressed irritant genes > 15) and non-sensitizer, the number of overexpressed genes in both the SENS-IS and ARE groups being below 7.
TNBS at 1M was classified as sensitizer since more than 6 genes are overexpressed in at least one of the two groups of genes (SENS-IS or ARE).

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

other: Number of overexpressed SENS-IS and ARE genes

Value:

2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Vehicle: 50% DMSO

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

other: Number of overexpressed SENS-IS and ARE genes

Value:

1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Vehicle: 100% DMSO

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

other: Number of overexpressed SENS-IS and ARE genes

Value:

2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Vehicle: 10% PBS

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

other: Number of overexpressed SENS-IS and ARE genes

Value:

2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Vehicle: 1% DMSO

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

other: Number of overexpressed SENS-IS and ARE genes

Value:

2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Vehicle: 10% DMSO

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

other: Number of overexpressed SENS-IS and ARE genes

Value:

3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Vehicle: 50% DMSO

The test item was soluble at 10 and 50% (w/v) in PBS and in DMSO. It was not soluble at 10 and 50% (w/v) in olive oil.

Interpretation of results:

other: No skin sensitising according to the classification criteria described in the SENS-IS assay

Conclusions:

not 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).

The objective of this study was to evaluate the capacity of the test item to induce the expression of specific irritation and sensitization biomarkers in a 3D-reconstructed epidermis model.

The results obtained for the positive and negative controls were within acceptance criteria defined in the Study Plan.

Considering the number of over-expressed genes in the “SENS-IS” and “ARE” gene groups, the test item gave negative result (less than 7 genes induced) when it was tested at 1, 10, 50 (w/v) in DMSO and at 10% (w/v) in PBS. Moreover, negative results were obtained when the test item was incubated at 100% (w/v) in DMSO.

In conclusion, under the experimental conditions of this SENS-IS assay, the test item can be classified as a non-sensitizer.

It should be mentioned that the SENS-IS method correlate more than 92% with LLNA in potency class prediction and should demonstrated a higher performance for predicting sensitization when combined with other sensitization tests (in chemico, in vitro and in silico).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

LuSens assay

This in vitro study was performed to investigate the potential of the test item to activate the Nrf2 transcription factor, by using the LuSens cell line.

The assay was performed in two independent repetitions. 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 repetition I and II:

2.02 µg/mL, 2.42  µg/mL, 2.91  µg/mL, 3.49  µg/mL, 4.19  µg/mL, 5.02  µg/mL, 6.03  µg/mL, 7.23  µg/mL, 8.68  µg/mL, 10.42  µg/mL, 12.5  µg/mL, 15  µg/mL.

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

p-Phenylenediamine (25 µM) was used as positive control. The viability was above 70 % and a distinct increase in luciferase induction above 2.5 fold in comparison to the solvent control was detected. This luciferase induction is well within the historical data range of the positive control.

DL-lactic acid (5000 µM) was used as negative control. The viability was above 70 % and the induction of the luciferase was < 1.5 fold in comparison to the solvent control and well within the historical data range of the negative control.

The induction of the luciferase of the growth control (Medium no. 3) was < 1.5 fold.

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

In repetition I a cytotoxic effect was observed at the concentrations 8.68 µg/mL to 15 µg/mL. In repetition II, again a cytotoxic effect was observed at the concentrations 8.68 µg/mL to 15 µg/mL. Those concentrations were excluded from the evaluation of the luciferase induction.

Finally, the following test item concentrations showed a viability ≥ 70 % and could there-fore be evaluated for luciferase induction:

Repetition I and II: 2.02 µg/mL, 2.42  µg/mL, 2.91  µg/mL, 3.49  µg/mL, 4.19  µg/mL, 5.02  µg/mL, 6.03  µg/mL, 7.23  µg/mL

In all tested non-cytotoxic concentrations of the test item no increase ≥ 1.5 fold in lucifer-ase induction in comparison to the solvent control was measured.

Therefore, both repetitions are clearly negative.

In conclusion, it can be stated that under the experimental conditions of this study, the test item was negative in the LuSens assay and is therefore considered not to have the potential to activate the Nrf2 transcrip-tion factor.

 

h-CLAT assay

This in vitro study evaluates the potential of the test to change the expression levels of the cell surface markers (CD86 and CD54) associated with the process of activation of monocytes and dendritic cells (DC), in the human monocytic leukaemia cell line THP-1. The measured expression levels of CD86 and CD54 cell surface markers are then used for supporting the discrimination between skin sensitisers and non-sensitisers. This test is part of a tiered strategy for the evaluation of skin sensitization potential.   

In total a pre-test and one experiment with three runs (run I, I Wdh and II) with a treatment period of 24 hours were performed, whereby run I was invalid and had to be repeated. Therefore, in total two valid runs (run I Wdh and run II) were performed. The results and data of the invalid run are not included in this final report but will be archived with the raw data in the test facility. 

For both runs, the highest nominal applied concentration (5000 µg/mL) was chosen based on the results obtained in the pre-test. A geometric series (factor 1.2) of 7 dilutions was prepared.

As solvent control for the test item, RPMI 1640 was used in a final concentration of 1 % in culture medium.

As positive control, 2,4-dinitrochlorobenzene (DNCB, CAS n. 97-00-7, ≥ 99% purity) was used.

Precipitation of the test item was visible after 24 h incubation in all test item concentrations in the pre-test, as well as in the two runs of the experiment.

Although there were also precipitates in the pre-test, the runs were started with the highest test item concentration.The pH value of the test item stock solution was lower, than the dilutions of it in cell culture medium. The higher the pH values in the dilutions, the more precipitates were observed.

Due to no cytotoxicity, the runs were started with the highest test item concentration, with the aim of obtaining hardly any precipitates in the higher concentrations due to a lower pH value.

In both runs the RFI of CD86 was not ≥ 150 % at any tested concentration with cell viability ≥ 50 %, whereas the RFI of CD54 was ≥ 200 % in all test item concentration, but with an opposite dose response, which would indicate a false positive result. One could suspect a non-specific binding of the antibody to the precipitates. Therefore, these positive values should be viewed critically and should not be used to classify the test item.

As the result cannot be evaluated of the above reasons, the test item cannot be classified.

 

SENS-IS test

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).

Considering the number of over-expressed genes in the “SENS-IS” and “ARE” gene groups, the test item gave negative result (less than 7 genes induced).

In conclusion, under the experimental conditions of this SENS-IS assay, the test item can be classified as a non-sensitizer.

Justification for classification or non-classification

According to the CLP Regulation (EC) no. 1272/2008, a skin sensitiser is an agent that will lead to an allergic response in susceptible individuals following skin contact. As a consequence of a secondary - usually organ-specific - subsequent re-exposure, adverse health effects occur on the skin (allergic contact dermatitis). Skin sensitisers are classified in Category 1 - H317. Where data is sufficient, skin sensitisers can be divided into sub-categories. If data are not sufficient for sub-categorisation, Category 1 must be chosen. The CLP (and UN GHS) criteria for classifying sensitisers are based on standard animal data and human data; data obtained from non-standard methods such as read-across or in vitro/in chemico test methods may be used in combination in a Weight of Evidence approach.

Indicators of potency of a substance can be obtained from in chemico/in vitro testing; specifically, the following tests may be accepted to fulfill the requirements of Annex VII:

(i) Direct Peptide Reactivity Assay (DPRA) addresses AOP Key Event 1: Peptide/protein binding

(ii) ARE-Nrf2 Luciferase Test Method (KeratinoSensTM) addresses AOP Key Event 2: Keratinocyte response

(iii) the Human Cell Line Activation Test (h-CLAT) addresses AOP Key Event 3: Monocytic /Dendritic cell response.

These test methods were developed to address specific events of the skin sensitisation AOP (OECD, 2012). The AOP for skin sensitisation describes the current understanding of key events linked to skin sensitisation. As each of the test methods only addresses a specific key event of skin sensitisation, currently they should not be used in isolation to identify a potential skin sensitiser but rather in combination in a Weight of Evidence approach.

The DPRA could not be performed due to structural incompatibility.

 

For this reason at the beginning a LuSens assay (inflammatory response in keratinocytes) and a h-CLAT (dendritic cell activataion) were performed. The first one gave negative result while in the second one, the RFI of CD54 was ≥ 200 % in all test item concentration, but with an opposite dose response, which would indicate a false positive result, probably due to a non-specific binding of the antibody to the precipitates. As the result cannot be evaluated for the above reasons, the test item cannot be classified in this assay.

Being the substance an organometallic a LLNA assay was not considered due to structural limitations.

However as a new in vitro study has been developped in order to assess all biological biomarkers leading to a phenomenon of sensitisation, the test substance was assessed following the method described by 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.

 

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.

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

 

Classification criteria:

A test item is classified as irritant if at least 16/23 genes of the “IRRITATION” group are significantly overexpressed.

A test item is classified as sensitizer if at least 7/17 genes of the “ARE” group, and/or 7/21 genes in the “SENS-IS” group are significantly overexpressed.

Moreover, the results obtained with the different concentrations allow the classification of the test item according to the lowest concentration that gives a positive result (DE> 1.25). Thus, a test item is classified in:

-      category 1A: strong to extreme skin sensitizer, when a positive result is obtained at concentrations of 0.1 and/or 1%,

-      category 1B: weak to moderate sensitizer, whena positive result is obtained at concentrationsof 10 and/or 50%.

A test item is classified as a non-sensitizer when negative results are observed at 100% and at all other analysed concentrations.

 

According to the classification criteria reported in the SENS-IS assay, the test item is considered to be a non sensitizer, as less than 7 genes of the SENS-IS groups have been induced.