Phosphinylidynetrimethanol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Experimental phase: 06 July 2020 - 22 July 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

Appendix IA: In vitro Skin Sensitisation: The ARE-Nrf2 Luciferase KeratinoSens™ Test Method
(25 June 2018)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

other: ARE-Nrf2 luciferase KeratinoSens™ test method (migrated information)

Justification for non-LLNA method:

In vitro study should be performed first. An in vivo study shall be conducted only if in vitro/in chemico test methods described under point 8.3.1 of REACH are not applicable.

Test material

Test material information

Constituent 1

Reference substance name:

Phosphinylidynetrimethanol

EC Number:

213-924-6

EC Name:

Phosphinylidynetrimethanol

Cas Number:

1067-12-5

Molecular formula:

C3H9O4P

IUPAC Name:

[bis(hydroxymethyl)phosphoryl]methanol

Test material form:

liquid

Details on test material:

- Appearance: light yellow to colourless ropy liquid
- Storage conditions at test facility: at 20 +/- 5 °C, in the dark

Results and discussion

Positive control results:

The luciferase activity induction obtained with the positive control, Trans-Cinnamaldehyde, was statistically significant above the threshold of 1.5-fold in all tests.

In vitro / in chemico

Results

Results 1

Run / experiment:

run/experiment 1

Parameter:

other: IC30 [442D] (migrated information)

Remarks on result:

not determinable

Remarks:

no indication of skin sensitisation

Results 2

Run / experiment:

run/experiment 1

Parameter:

other: IC50 [442D] (migrated information)

Remarks on result:

not determinable

Remarks:

no indication of skin sensitisation

Results 3

Run / experiment:

run/experiment 2

Parameter:

other: IC30 [442D] (migrated information)

Remarks on result:

not determinable

Remarks:

no indication of skin sensitisation

Results 4

Run / experiment:

run/experiment 2

Parameter:

other: IC50 [442D] (migrated information)

Remarks on result:

not determinable

Remarks:

no indication of skin sensitisation

Any other information on results incl. tables

For each individual test, four parallel plates were used: three replicates were used for the luciferase activity induction measurements and one was needed for theMTT cell viability assay to measure the cytotoxicity induced by the test item.

1) Test item

For the test item, twelve doses ranging from 2000.00 µM to 0.98 µM were used in both tests.

Average fold induction, significance and viability (%) values for the test item concentrations in the independent experiments are appended (cf. 'Attached background material').

The summary of the results obtained in the two independent experiments are described below.

a) First test

The test item induced no cytotoxicity (meaning there was no viability value observed below 70 %) compared to the solvent/vehicle control at any concentration in KeratinoSens™ cells. Thus, IC30 and IC50 values were not calculated.

The luciferase activity induction did not exceed the threshold of 1.5 fold compared to the respective negative control. The maximal fold induction (Imax) was 1.14 fold. Therefore, EC1.5 value could not be determined. Moreover, no dose response could be observed.

No precipitation was observed at any point during the first test.

Summary of the KeratinoSens™ results for the test item in the first test:

- Statistically significant induction over 1.5-fold: no

- Viability ≥ 70 % at lowest concentration with ≥1.5-fold: no

- EC1.5 (µg/mL): -

- Clear dose response: no

- Overall result: negative

b) Second test (repeated)

The second test when conducted for the first time, did not meet all acceptance criteria concerning the positive control due to the EC1.5 value being 6 µM which was lower than the acceptable 7 µM value. Therefore the test results were not included in the Report and the test was repeated with the same conditions.

The test item induced no cytotoxicity (meaning there was no viability value observed below 70 %) compared to the solvent/vehicle control at any concentration in KeratinoSens™ cells. Thus, IC30 and IC50 values could not be calculated.

Although the luciferase activity induction exceeded the threshold of 1.5 fold compared to the respective negative control, the EC1.5 value was over the 1000 µM threshold with the value of 1455 µM, therefore not counting as a positive result. The maximal fold induction (Imax) was 1.68 fold. An overall but not clear dose response could be observed, though.  

No precipitation was observed at any point during the second test.

Summary of the KeratinoSens™ results for the test item in the second test:

- Statistically significant induction over 1.5-fold: yes

- Viability ≥ 70 % at lowest concentration with ≥1.5-fold: yes

- EC1.5: 1455 µM (KeratinoSens™ prediction is considered positive when the the EC1.5 value is less than 1000 μM)

- Clear dose response: no

- Overall result: negative

2) Negative and positive control

The coefficient of variation (CV %) of the luminescence reading for the negative control DMSO was below 20 % in both tests (13.05 % and 13.24 % respectively).

The luciferase activity induction obtained with the positive control, Trans-Cinnamaldehyde was statistically significant above the threshold of 1.5 at several concentrations in both tests. The EC1.5 values of the positive control fell between 7 µM and 30 µM (9 µM in both valid tests).

The average inductions in the parallel plates for Trans Cinnamaldehyde at 64 μM were 83.16 fold and 141.72 fold in the first and second tests, respectively.

Although the luciferase activity induction in both tests was outside of the 2 – 8-fold induction range, there was a clear dose response relationship in the luciferase activity induction with increasing concentrations and corresponding fold induction rise, therefore these results were accepted as valid.

In any of the tests, there was no cytotoxicity (cell viability lower than 70 %) induced by the positive control at any of the tested concentrations.

Each above described individual test met the acceptance criteria for the negative and positive controls therefore considered valid under the conditions described.

Applicant's summary and conclusion

Interpretation of results:

other: non-sensitiser based on the KeratinoSens™ results

Conclusions:

Results of two independent tests:
- THPO induced no cytotoxicity n KeratinoSens™ cells compared to the solvent/vehicle; thus, in none of the tests an IC30 or IC50 value could be determined.
- Both tests were concluded negative; in the first test the induction values of the test item did not exceed the 1.5-fold threshold, and in the second test EC1.5 value was over 1000 µM, thus corresponding to a negative response.
- Based on these results and the KeratinoSens™ prediction model, THPO is concluded negative for skin sensitization potential under the experimental conditions.

Executive summary:

In the course of this GLP study according to OECD 442D, the skin sensitization potential of the test item Tris(hydroxymethyl)phosphine oxide (THPO)  was studied using the KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).

In order to derive a prediction for the test item the results of two independent tests were used.

Since the results of the two tests were concordant, a third one was not needed in order to derive a conclusion. However, the second test needed to be repeated due to not fulfilling all validity criteria for the positive control.

The luciferase activity induction obtained with the positive control, Trans-Cinnamaldehyde, was statistically significant above the threshold of 1.5-fold in all tests.  

For the test item, twelve doses ranging from 2000.00 µM to 0.98 µM were used in both valid tests.

The test item induced no cytotoxicity (a substance is considered cytotoxic if the viability is below 70 %) in KeratinoSens™ cells compared to the solvent/vehicle. Thus, in none of the tests an IC30 or IC50 value could be determined.  

Both tests were concluded negative. In the first test the induction values of the test item did not exceed the 1.5-fold threshold, and in the second test EC1.5 value was over 1000 µM, thus corresponding to a negative response.  

Based on these results and the KeratinoSens™ prediction model, the test item THPO is concluded negative for skin sensitization potential under the experimental conditions of KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).