Diethyl bis(2-hydroxyethyl)aminomethylphosphonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD TG 442C and in compliance with GLP without deviation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

of February 2015

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

In vitro methods are applicable for this substance and are adequate for classification and risk assessment according to REACH Regulation.

Test material

Test material information

Constituent 1

Reference substance name:

Diethyl bis(2-hydroxyethyl)aminomethylphosphonate

EC Number:

220-482-8

EC Name:

Diethyl bis(2-hydroxyethyl)aminomethylphosphonate

Cas Number:

2781-11-5

Molecular formula:

C9H22NO5P

IUPAC Name:

diethyl {[bis(2-hydroxyethyl)amino]methyl}phosphonate

Test material form:

liquid

Details on test material:

Physical state: liquid, slightly viscous
Colour: brownish-orange
Expiry date: 2019-05-13

In chemico test system

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

lysine peptide (Ac-RFAAKAACOOH)

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:
The Direct Peptide Reactivity Assay (DPRA) is an in chemico procedure proposed to address the molecular initiating event leading to skin sensitization, namely protein reactivity, by quantifying the reactivity of test chemicals towards model synthetic peptides containing either lysine or cysteine. Cysteine and lysine percent peptide depletion values are then calculated and used in a prediction model to categorize a substance in one of four classes of reactivity for supporting the discrimination between skin sensitizers and non-sensitizers.
For comparison, tests were performed with the test item, the vehicle (solvent control = negative control) and the known sensitizer Cinnamic aldehyde (positive control).

The DPRA quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 hours incubation with the test item at 25 +/-2.5ºC. Relative peptide concentration is measured by high-performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 nm. The synthetic peptides contain phenylalanine to aid in the detection.

Preparation of the cysteine or lysine containing peptides:
Stock solutions of cysteine (Ac-RFAACAA-COOH) and lysine (Ac-RFAAKAA-COOH) containing synthetic peptides of purity higher than 95% were freshly prepared just before their incubation with the test item. The final concentration of the cysteine peptide was 0.666 mM in pH 7.5 phosphate buffer, whereas the final concentration of the lysine peptide was 0.667 mM in pH 10.2 ammonium acetate buffer.

Preparation of the test item:
Solubility of the test item in an appropriate solvent was assessed before performing the assay. 92.80 mg test item were dissolved in 3 mL acetonitrile immediately before testing to prepare a 100 mM solution. A factor of 1.21 was used to correct for the purity of the test item. The test item solution was then tested as such without any further dilution by incubating at 1:10 or 1:50 ratio with the cysteine peptides and lysine peptides, respectively.

Positive control, reference controls and co-elution control:
Cinnamic aldehyde (CAS no. 14371-10-9) was used as positive control (PC) at a concentration of 100 mM in acetonitrile. In addition reference controls (i.e. samples containing only the peptide and added acetonitrile) were also included in the HPLC run sequence and these were used to verify the HPLC system suitability prior to the analysis (reference controls A) and the stability of the reference controls over time (reference control B). To verify that the solvent used to dissolve the test item does not impact the percent peptide depletion the reference control C was prepared by adding acetonitrile to the peptide solution. The reference control C was used to calculate the percent peptide depletion for the test item. In addition a co-elution control constituted by the test item alone for the test item analysed was included in the run sequence to detect possible co-elution of the test item with either the lysine or the cysteine peptide.

Incubation of the test item with the cysteine and lysine peptide solutions:
Cysteine and lysine peptide solutions were incubated in glass autosampler vials with the test item at 1:10 and 1:50 ratio, respectively. The reaction solution was left in the dark at 25 ± 2.5°C for 24 ± 2 hours before running the HPLC analysis. The test item assay was analysed in triplicate for both peptides. Samples were visually inspected prior to HPLC analysis.
If a precipitate would be observed immediately upon addition of the test item solution to the peptide solution, due to low aqueous solubility of the test item, in this case one cannot be sure how much test item remained in the solution to react with the peptide. Therefore, in such a case, a positive result could still be used, but a negative result is uncertain and would be interpreted with due care. No precipitate or phase separation was observed.

Preparation of the HPLC standard calibration curve:
A standard calibration curve was generated for both the cysteine and the lysine peptides. Peptide standards were prepared in a solution of 20% acetonitrile : buffer using 100 mM sodium phosphate buffer (pH 7.5) for the cysteine peptide and 100 mM ammonium acetate buffer (pH 10.2) for the lysine peptide. Using serial dilution standards of the peptide stock solution (nominal concentrations: 0.666 mM of cysteine peptide in sodium phosphate or 0.666 mM lysine peptide in ammonium acetate), 6 calibration standards were prepared to cover the range from 0.534 to 0.0167 mM. A blank of the dilution buffer was also included in the standard calibration curve. Suitable calibration curves should have an r2 > 0.99.
If a test item promotes the oxidation of the cysteine peptide, the peak of the dimerised cysteine peptide would have been visually monitored. If dimerisation appears to have occurred, this would have been noted as percent peptide depletion which would have been over-estimated leading to false positive predictions and/or assignment to a higher reactivity class. No dimerisation of the cysteine peptide occurred.
HPLC analysis for the cysteine and lysine peptides was performed on one day. All test item solutions were freshly prepared for both assays on one day. The analysis was timed to assure that the injection of the first sample (reference control C) started 22 to 26 hours after the test item had been mixed with the peptide solution. The HPLC run sequences were set up in order to keep the HPLC analysis time to less than 30 hours.

Data evaluation:
The concentrations of cysteine or lysine peptide were photometrically determined at 220 nm in each sample by measuring the peak area (area under the curve, AUC) of the appropriate peaks and by calculating the concentration of peptide using the linear calibration curve derived from the standards.
The percent peptide depletion was determined in each sample by measuring the peak area and dividing it by the mean peak area of the relevant reference control C.

Acceptance criteria:
The following criteria must be met for a run to be considered valid:
a) The standard calibration curve should have an r2 > 0.99.
b) The mean percent peptide depletion value of the three replicates for the positive control cinnamic aldehyde should be between 60.8% and 100% for the cysteine peptide and between 40.2% and 69.0% for the lysine peptide and the maximum standard deviation (SD) for the positive control replicates should be < 14.9% for the percent cysteine depletion and < 11.6% for the percent lysine depletion.
c) The mean peptide concentration of reference controls A should be 0.50 ± 0.05 mM and the coefficient of variation (CV) of peptide peak areas for the nine reference controls B and C in acetonitrile should be <15.0%.
If one or more of these criteria is not met, the run would have been repeated.

The following criteria must be met for a test item’s results to be considered valid:
a) The maximum standard deviation for the test item replicates should be < 14.9% for the percent cysteine depletion and < 11.6% for the percent lysine depletion.
b) The mean peptide concentration of the three reference controls C in the appropriate solvent should be 0.50 ± 0.05 mM.
If these criteria were not met, the data would have been rejected and the run have been repeated for that specific test item.

Vehicle / solvent:

acetonitrile

Positive control:

cinnamic aldehyde

Results and discussion

Positive control results:

Cinnamic aldehyde was used as positive control at a concentration of 100 mM in acetonitrile. Treatment with the positive control item revealed a cysteine and lysine peptide depletion of 68.65% for cysteine and 68.56% for lysine peptide. These values are within the required range of 60.8% and 100% for the cysteine peptide and between 40.2% and 69.0% for the lysine peptide. The maximum standard deviation (SD) for the positive control replicates were < 14.9% for the percent cysteine depletion and < 11.6% for the percent lysine depletion. Therefore, the study can be regarded as valid.

In vitro / in chemico

Results

Results 1

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

cysteine depletion

Value:

0

At concentration:

100 mM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

lysine depletion

Value:

0.84

At concentration:

100 mM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Outcome of the prediction model:

no or minimal reactivity [in chemico]

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The acceptance criteria for a DPRA test to be considered valid were met.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for vehicle control: yes
- Acceptance criteria met for positive control: yes

The test item was dissolved at a concentration of 100 mmol/L in acetonitrile. No precipitation occured before or after incubation.
Absorbance at 220 nm was not observed. Retention time similar to peptide was not observed. Co-elution of the test item with the peptides was not observed.
No relevant depletion of cysteine and lysine peptides became obvious in the DPRA for the test item (0.42%). According to the cysteine 1:10/lysine 1:50 prediction model “no or minimal reactivity” was derived for the test item in acetonitrile, leading to a DPRA prediction of “negative“. The test item is thus predicted to be a non-sensitiser (no or minimal reactivity) in the Direct Peptide Reactivity Assay (DPRA).

Any other information on results incl. tables

Results of the DPRA:

Test item	Mean % Cysteine peptide depletion	Mean % Lysine peptide depletion	Mean % Cysteine/Lysine peptide depletion	reactivity class	DPRA prediction
positive control (cinnamic aldehyde)	68.65	68.56		high	positive
test item	0.0	0.84	0.42	no or minimal	negative

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is predicted to be a non-sensitizer in the DPRA.

Executive summary:

The Direct Peptide Reactivity Assay (DPRA; OECD 442C) is an in chemico procedure proposed to address the molecular initiating event leading to skin sensitization, namely protein reactivity, by quantifying the reactivity of test chemicals towards model synthetic peptides containing either lysine or cysteine. Cysteine and lysine percent peptide depletion values are then calculated and used in a prediction model to categorize a substance in one of four classes of reactivity for supporting the discrimination between skin sensitizers and non-sensitizers.

The test item was dissolved at a concentration of 100 mmol/L in acetonitrile. No precipitaton of the test item occured before or after incubation. The cysteine 1:10/lysine 1:50 prediction model was applied to the test item. No relevant depletion of cysteine and lysine peptides became obvious in the DPRA (mean of 0.42%). According to the prediction model “no or minimal reactivity” was derived for the test item in acetonitrile, leading to a DPRA prediction of “negative“. The test item is thus predicted to be a non-sensitizer in the DPRA.