Phosphoric acid, 2-ethylhexyl ester, ammonium salt

Endpoint summary

Description of key information

OECD 442C DPRA (2017): Negative

OECD 442D KSA (2017): Negative

In accordance with the integrated testing strategy for skin sensitisation, no further studies require being conducted and the endpoint can be considered negative (not sensitising).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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.

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)

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

Positive control results:

Mean PPD = 55.84 %

High reactivity (Positive result)

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

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 %).

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

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.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 June 2017 to 16 April 2018

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.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

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

Treatment plate preparation

The cells were 80-90 % confluent. On the day prior to treatment, cells were harvested and distributed into 96-well plates (10000 cells/well) and incubated at 37 ± 1 °C, 5 % (v/v) CO2, for 24 ± 1 hours.
For each repetition, three replicates were used for the luciferase activity measurements and one parallel replicate used for the cell viability assay.

Treatment

At the end of the 24-hour incubation period, the medium was removed and replaced with fresh culture medium (containing serum but without Geneticin) to which test article and control formulations were added.
One well per plate was left empty (no cells and no treatment) to assess background values.
Each plate was sealed and incubated at 37 ± 1 °C, 5 % (v/v) CO2 in air, in a humidified environment for 48 ± 1 hours.
For each test article and positive control, one experiment was needed to derive a prediction (positive or negative), consisting of at two independent repetitions each containing three replicates of each concentration.
Each independent repetition was performed on a different day with fresh stock solutions of chemicals and independently harvested cells. The cells came from the same passage.

Cytotoxicity assessment

After the 48-hour exposure period, the medium was replaced with fresh medium containing MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide).The plate was sealed and incubated for 4 hours at 37 ± 1 °C, 5 % (v/v) CO2.
The MTT medium was removed and SDS (at 10% w/v) added per well. The plate was sealed and placed into an incubator at 37 ± 1 °C, 5 % (v/v) CO2 in air and left overnight.
After the overnight incubation, the plate was shaken to ensure homogeneity of the solution in the wells and then absorption read at 600 nm using a SpectraMax M2e.

Luciferase activity measurements

After the 48-hour exposure period, the cells were washed with phosphate buffered saline and lysis buffer for luminescence readings was added to each well. The plates were then incubated for 20 minutes at 25 ± 2 °C, loaded into the luminescence plate reader and read using the following parameters: 100 µL injection (Luciferase assay substrate), 15 second delay, 7 second luminescence integration time.

Positive control results:

Luciferase activity induction obtained with the positive control was statistically significant above the threshold of 1.5 at concentrations of 16, 32 and 64 µM in Experiments 1 and 2.

The EC1.5 values for the positive control were 15.42 and 10.35 µM in Experiments 1 and 2, respectively.

The average induction in the three replicates for the positive control at 64 µM was 4.32 in both experiments.

Key result

Run / experiment:

other: other: Two experimental runs with Imax (fold increase) of 4.27 (statistically signifiant) and 1.52 (not statistically significant)

Parameter:

other:

Remarks:

ARE-Nrf2 Luciferase induction

Value:

1.52

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

Luciferase activity induction obtained with the positive control was statistically significant above the threshold of 1.5 at concentrations of 16, 32 and 64 µM in Experiments 1 and 2.

The EC1.5 values for the positive control were 15.42 and 10.35 µM in Experiments 1 and 2, respectively.

The average induction in the three replicates for the positive control at 64 µM was 4.32 in both experiments.

The average coefficient of variation of the luminescence reading for the negative control (DMSO) was 10.02 % and 16.20 % in Experiments 1, and 2, respectively.

Table 1       Luminescence Readings for Experiment 1

Substance		Concentration (µg/mL)
		0.20	0.39	0.78	1.56	3.13	6.25	12.5	25	50	100	200	400
Test Article	Plate 1	1595543	1949671	2150748	1977587	1939608	1503695	1466992	1411049	7414562	-4	-1334	-891
	Plate 2	1425323	2405398	2444141	2252869	1967323	1588885	1382291	1761253	8290327	1672	-913	-615
	Plate 3	1875925	1876982	2098413	2115341	2253219	1562467	1647061	1685263	8939677	506	-1112	-773
	Mean Fold Induction	0.85	1.08	1.16	1.10	1.07	0.81	0.78	0.84	4.27	0.00	0.00	0.00

 

Substance		Individual Values
Negative Control	Plate 1	1964652	2133206	1808828	1981876	2009225	1725812
	Plate 2	1854369	1817851	2077112	1941072	2001819	1929047
	Plate 3	1564894	1808335	1860888	2489171	1886345	1816473

  

Substance		Concentration (µM)
		4	8	16	32	64
Positive Control	Plate 1	2214660	2247164	2805564	3624814	9044607
	Plate 2	2171338	2723033	3029193	3895171	7591194
	Plate 3	2571740	2779511	2904452	4639515	8310327
	Mean Fold Induction	1.20	1.34	1.51	2.11	4.32

Table 2       Luminescence Readings for Experiment 2

Substance		Concentration (µg/mL)
		0.20	0.39	0.78	1.56	3.13	6.25	12.5	25	50	100	200	400
Test Article	Plate 1	681637	813061	836969	814404	760686	693972	611022	671329	3249179	186	-432	-310
	Plate 2	916614	977708	927934	999602	940968	885310	852727	1137502	105122	-633	-644	-366
	Plate 3	1126957	1091203	1013303	932404	895338	795742	807759	823055	608566	-380	-436	-302
	Mean Fold Induction	0.92	0.98	0.95	0.94	0.89	0.81	0.77	0.89	1.52	0.00	0.00	0.00

 

Substance		Individual Values
Negative Control	Plate 1	745443	820675	821712	1047885	813494	770470
	Plate 2	938363	983923	1365594	1092872	862394	946182
	Plate 3	1126957	1091203	1013303	932404	895338	795742

 

Substance		Concentration (µM)
		4	8	16	32	64
Positive Control	Plate 1	929885	1092452	1512082	2284728	3861519
	Plate 2	1272749	1734957	1807804	2041535	4905376
	Plate 3	1207810	1344946	1610995	2376396	3804812
	Mean Fold Induction	1.16	1.42	1.69	2.32	4.32

 

Table 3       MTT-Absorbance Readings

Substance		Concentration (µg/mL)
		0.20	0.39	0.78	1.56	3.13	6.25	12.5	25	50	100	200	400
Test Article	Experiment 1	0.963	1.254	1.149	1.145	1.096	0.991	0.744	0.612	0.149	0.001	0.001	0.003
	Experiment 2	0.665	0.828	0.824	0.771	0.736	0.657	0.607	0.661	0.006	0.005	0.002	0.001
Mean Viability (%)		82.33	104.94	100.14	96.71	92.44	83.12	69.32	66.89	6.78	0.35	0.16	0.20

 

Substance		Individual Values
Negative Control	Experiment 1	1.023	1.140	1.218	1.189	1.173	1.186
	Experiment 2	0.726	0.884	0.824	0.811	0.803	0.857

 

Substance		Concentration (µM)
		4	8	16	32	64
Positive Control	Experiment 1	1.187	1.362	1.266	1.295	1.019
	Experiment 2	0.828	1.045	1.024	1.045	1.048
Mean Viability (%)		102.01	122.87	117.44	120.01	108.20

Table 4. Result Summaries

Condition	Repetition 1	Repetition 2
Imax(fold increase)	4.27 (at 50 μg/mL) and statistically significant	1.52 (at 50 μg/mL), but not statistically significant
Cell Viability at 50 μg/mL	12.9%	Not applicable
Cell viability at 25 μg/mL	53%	Not applicable
EC1.5(μg/mL)	29.81	Not applicable
Dose Response for Luciferase Induction	No	No

 

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the condition of this study, the test article was considered to be negative in the ARE-Nrf2 Luciferase Test according to EU CLP and UN GHS.

Executive summary:

In an OECD 442D 2017: the test item potential was investigated at concentration range of 0.2 to 400 μg/mL. Aliquots of 50 μL of each of the final concentrations were transferred to each of three luciferase plates and a single viability plate. Each plate was sealed using a plate sealer and then incubated at 37±1°C, 5% (v/v) CO2 in air, in a humidified environment for 48±1 hours.

After the 48-hour exposure period, the medium in the viability plate was replaced with fresh medium containing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The plate was sealed and incubated for 4 hours at 37±1°C, 5% (v/v) CO2. The MTT medium was then removed and SDS (at 10% w/v) added per well. The plate was sealed and placed into an incubator at 37±1°C, 5% (v/v) CO2 in air and left overnight. After the overnight incubation, the plate was shaken to ensure homogeneity of the solution in the wells and then absorption read at 600 nm using a SpectraMax M2e. After the 48-hour exposure period, the cells in the luciferase plates were washed with phosphate buffered saline and lysis buffer for luminescence readings was added to each well. The plates were then incubated for 20 minutes at 25±2°C, loaded into the luminescence plate reader and read.

The maximal fold increase (Imax) was 4.27 in Experiment 1 and was statistically significant. However, the viability at the lowest concentration with > 1.5-fold induction (50 mg/mL) was only 12.9%. Therefore this was considered to be a false positive result.

In Experiment 2, the Imax was 1.52, but was not statistically significant, therefore this was considered a negative result.

It was concluded that under the condition of the study, the test item was considered to be negative in the ARE-Nrf2 Luciferase Test according to EU CLP and UN GHS.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

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 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.

OECD 442D (2017): the test item potential was investigated at concentration range of 0.2 to 400 μg/mL. Aliquots of 50 μL of each of the final concentrations were transferred to each of three luciferase plates and a single viability plate then incubated at 37±1°C, 5% (v/v) CO2 in air, in a humidified environment for 48±1 hours.

After the 48-hour exposure period, the medium in the viability plate was replaced with fresh medium containing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and incubated overnight. This is followed by washing cells in luciferase plates using phosphate buffered saline and lysis buffer for luminescence readings.

The maximal fold increase (Imax) was 4.27 in Experiment 1 and was statistically significant. However, the viability at the lowest concentration with > 1.5-fold induction (50 mg/mL) was only 12.9%. Therefore this was considered to be a false positive result. 

In Experiment 2, the Imax was 1.52, but was not statistically significant, therefore this was considered a negative result. 

It was concluded that under the condition of the study, the test item was considered to be negative in the ARE-Nrf2 Luciferase Test according to EU CLP and UN GHS. 

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The substance did not meet the classification criteria in accordance with Regulation (EC) No 1272/2008 (CLP).