Phosphoric acid, 2-ethylhexyl ester, ammonium salt

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 May 2017 to 06 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

Ammonium mono(2- ethylhexyl)phosphate

Molecular formula:

C8H22NO4P

IUPAC Name:

Ammonium mono(2- ethylhexyl)phosphate

Constituent 2

Reference substance name:

Ammonium bis(2-ethylhexyl) phosphate

EC Number:

225-615-3

EC Name:

Ammonium bis(2-ethylhexyl) phosphate

Cas Number:

4971-47-5

Molecular formula:

C16H38NO4P

IUPAC Name:

Ammonium bis(2- ethylhexyl)phosphate

Constituent 3

Reference substance name:

2-ethylhexyl diphosphate ammonium salts / esters of 2-ethylhexan-1-ol with diphosphoric acid and their corresponding ammonium salts

Molecular formula:

C8H23NO7P2

IUPAC Name:

2-ethylhexyl diphosphate ammonium salts / esters of 2-ethylhexan-1-ol with diphosphoric acid and their corresponding ammonium salts

Test material form:

liquid

Details on test material:

Storage Conditions: Room temperature.

In chemico test system

Details on the study design:

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

The study was conducted to quantify the reactivity of the test article (Reaction mass of Ammonium mono(2-ethylhexyl)phosphate, Ammonium bis(2-ethylhexyl)phosphate and 2-ethylhexyl diphosphate ammonium salts) towards model synthetic peptides containing either lysine or cysteine.

The test article and positive control was dissolved in purified water and acetonitrile, respectively at a concentration of 100 mM.

The test solutions were incubated at 1:10 and 1:50 ratios with the cysteine and lysine peptides, respectively, for 24±2 hours in glass autosampler vials, protected from light and set at 25 °C.

The remaining concentration of cysteine- or lysine-containing peptides following the 24 hour incubation period was measured by high performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 nm.

The mean percent cysteine and percent lysine depletion value was calculated.

Negative depletion was considered as “0” when calculating the mean. By using the cysteine 1:10/lysine 1:50 prediction model below, the threshold of 6.38 % average peptide depletion was used to support the discrimination between skin sensitisers and non-sensitisers in the framework of an IATA.


0% ≤ mean % depletion ≤6.38% = No or minimal reactivity/ Negative for sensitisation
6.38% < mean % depletion ≤22.62% Low reactivity/ Positive for sensitisation
22.62% < mean % depletion ≤42.47% = Moderate reactivity/ Positive for sensitisation
42.47% < mean % depletion ≤100% = High reactivity/ Positive for sensitisation

Results and discussion

Positive control results:

Mean PPD = 55.84 %

High reactivity (Positive result)

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: other: Lysine mean test item PPD

Parameter:

other: Peptide Percent Depletion

Value:

0.55

Vehicle controls validity:

not examined

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Run / experiment:

other: other: Cysteine mean test item PPD

Parameter:

other: Peptide Percent Depletion

Value:

11.27

Vehicle controls validity:

not examined

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: n/a

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

The following criteria should be met for a run to be considered valid: a) the standard calibration curve should have an r2 >0.99 (actual >0.9935), 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 (actual = 73.11 %) and between 40.2 % and 69.0 % for the lysine peptide (actual = 55.84 %) and the maximum standard deviation (SD) for the positive control replicates should be <14.9 % for the percent cysteine depletion (actual = 0.4 %) and <11.6 % for the percent lysine depletion (actual = 2.5 %) and c) the mean peptide concentration of reference controls A should be 0.50 ± 0.05 mM (actual = 0.53 and 0.46 mM for cysteine and lyseien standards, respectively)and the coefficient of variation (CV) of peptide peak areas for the nine reference controls B and C in acetonitrile should be <15.0 % (actual = 1.92 %).

Any other information on results incl. tables

Table 1       Lysine Depletion Summary

 

Substance	Replicate Peptide Peak Areas	Reference Control C Mean Peptide Peak Area	PPD	Mean PPD	SD
Test Article	38.717	38.38	-0.88	0.55	0.9
	38.588		-0.54
	37.749		1.64
Positive Control	16.192	35.78	54.75	55.84	2.5
	16.414		54.13
	14.792		58.66

 

Table 2       Cysteine Depletion Summary

 

Substance	Replicate Peptide Peak Areas	Reference Control C Mean Peptide Peak Area	PPD	Mean PPD	SD
Test Article	23.579	26.115	9.71	11.27	1.5
	23.126		11.45
	22.809		12.66
Positive Control	7.243	26.748	72.92	73.11	0.4
	7.059		73.61
	7.277		72.79

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the condition of the study, the test article was considered to be negative in the Direct Peptide Reactivity Assay according to EU CLP and UN GHS.

Executive summary:

OECD 442C (2017) - The in chemico study was conducted to quantify the reactivity of the test item towards model synthetic peptides containing either lysine or cysteine.  The test item and positive control were dissolved in purified water and acetonitrile, respectively at a concentration of 100 mM.  The test solutions were incubated at 1:10 and 1:50 ratios with the cysteine and lysine peptides, respectively, for 24 ± 2 hours in glass autosampler vials, protected from light and set at 25 °C.  The remaining concentration of cysteine- or lysine-containing peptides following the 24 hour incubation period was measured by high performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 nm.

 

The mean percent cysteine and percent lysine depletion values were calculated and determined to be 11.27 and 0.55 %, respectively.  Based on the condition of the study, the test article was considered to be negative in the Direct Peptide Reactivity Assay, according to EU CLP and UN GHS.