Reaction mass of N-(2-hydroxyethyl)alkyl] alkylamide and glycerol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12th September 2016 - completed 18th January 2017 - reported 23rd February 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154,
January 12, 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

ECHA Guidelines 7a July 2017, require the use of this test prior to the LLNA as animal testing is a last resort. The correlation of protein reactivity with skin sensitisation potential of a chemical is well established and represents the first and initial key event in the skin sensitisation process as defined by the AOP .
[4], [12]. It is therefore a crucial step for the sensitising potential of a chemical.

Test material

Test material information

Constituent 1

Test material form:

liquid

Details on test material:

Chemical Name: N-[2-(2-Hydroxyethoxy)ethyl]acetamide
CAS No.: 118974-46-2
Batch: 27191705
Purity: 82% (dose calculation was adjusted to puriy)
Appearance: Pale to yellow liquid
Expiry Date: 16 June 2017
Storage Conditions: At room temperature
Stability in Solvent: Stable in water (not quantified)
Purpose of Use: Industrial chemical

In chemico test system

Details on the study design:

Preparation of the Test Item
The test item was freshly prepared immediately prior to use, unless stability data demonstrate the acceptability of storage. The test item was pre-weighed into a glass vial and was dissolved in an appropriate solvent previously determined in a pre-experiment. A stock solution with a concentration of 100 mM was prepared.

Controls
Reference controls, co-elution controls and a positive control (PC) were set up in parallel to the test item in order to confirm the validity of the test.
Positive Control
Cinnamic aldehyde ((2E)-3-phenylprop-2-enal) was solved in acetonitrile and was used as positive control. A stock concentration of 100 mM was prepared and was included in every assay run for both peptides.

Co-elution Control
Co-elution controls were set up in parallel to sample preparation but without the respective peptide solution. The controls were used to verify whether a test chemical absorbs at 220 nm and co-elutes with the cysteine or lysine peptide. The co-elution controls were prepared for every test item preparation and the positive control and were included in every assay run for both peptides.

Reference Control
Reference controls (RCs) were set up in parallel to sample preparation in order to verify the validity of the test run.

Reference control A was prepared using acetonitrile in order to verify the accuracy of the calibration curve for peptide quantification. Its replicates were injected in the beginning of each HPLC run .

Reference control B was prepared using acetonitrile in order to verify the stability of the respective peptide over the analysis time. Its replicates were injected in the beginning and in the end of each HPLC run
.
Reference control C was set up for the test item and the positive control. RC C for the test item was prepared using the respective solvent used to solubilize the test item. RC C for the positive control was prepared using acetonitrile. The RC C was used to verify that the solvent does not impact the percent peptide depletion (PPD). Additionally reference control C was used to calculate PPD. The RC C was included in every assay run for both peptides and was injected together with the samples.

Peptides
20.36 mg cysteine peptide with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (39.532 mL) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM.

21.37 mg lysine peptide with an amino acid sequence of Ac-RFAAKAA were pre-weighed in a vial and dissolved in a defined volume of ammonium acetate buffer with pH 10.2 (37.732 mL) to reach a concentration of 0.667 mM.

All peptides used for this study were stored at -80 °C and protected from light. Peptides were thawed only immediately prior to use.

Dose Groups
Reference Control C (solvent control) undiluted
Test Item 100 mM stock solution
Positive Control 100 mM stock solution

Pre-Experiments
Solubility of the test item was determined prior to the main experiment and was tested at the highest final concentration applied in the study (100 mM). The test item was dissolved in the following solvents suitable for the test:
- acetonitrile
- water
The test item was soluble in water, which was used as solvent for the test.

Experimental Procedure
Incubation of the Test Item with the Cysteine and Lysine Peptide The test item solutions were incubated with the cysteine and lysine peptide solutions in glass vials using defined ratios of peptide to test item (1:10 cysteine peptide, 1:50 lysine peptide). The reaction solutions were left in the dark at 25 ± 2.5 °C for 24 ± 2 h before running the HPLC analysis. Reference controls, co-elution controls as well as the positive control were set up in parallel. Samples were prepared according to the scheme described in Table 2.

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Parameter:

other: Peptide depletion %

Remarks:

Prediction model 1

Value:

0.46

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Parameter:

other: Preptide depletion %

Remarks:

Prediction Model 2

Value:

0.59

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Any other information on results incl. tables

Pre-Experiments

Solubility of the test item was determined prior to the main experiment. All test item solutions were freshly prepared immediately prior to use. The test item was soluble in water. No turbidity,

precipitation and phase separation was observed for the test item solutions. All test item preparations of the main experiment were prepared using water.  

Precipitation and Phase Separation

All test item solutions were freshly prepared immediately prior to use.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution.

After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples of the cysteine peptide run were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for test item samples.  

After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples of the lysine peptide run were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for test item samples. A phase separation was observed for the samples of the positive control and the respective co-elution control. No centrifugation was necessary to perform the HPLC analysis.

Since the positive control fulfilled all quality criteria the observed precipitations were considered as irrelevant.

Co-elution with the Peptide Peaks

No co-elution of the test item with any of the peptide peaks was observed.

Table 6 Cysteine and Lysine Values of the Calibration Curve

Sample	Cysteine Peptide		Lysine Peptide
	Peak Area  at 220 nm	Peptide Concentration [mM]	Peak Area  at 220 nm	Peptide Concentration [mM]
STD1	4655.6279	0.5340	4493.0674	0.5340
STD2	2355.5945	0.2670	2277.1133	0.2670
STD3	1163.3136	0.1335	1160.3005	0.1335
STD4	583.4137	0.0667	577.1267	0.0667
STD5	288.7250	0.0334	292.0436	0.0334
STD6	140.0172	0.0167	147.2783	0.0167
STD7	0.0000	0.0000	0.0000	0.0000

Based on these results, linear regression was performed and the calibration curves were determined.

Results of the Cysteine Peptide Depletion

Table 7: Results of the Cysteine Peptide Depletion

Cysteine Peptide
Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	1333.1064	0.1526	69.69	70.06	0.48	0.68
	1323.4985	0.1515	69.91
	1292.8408	0.1479	70.60
Test Item	4333.3467	0.4960	0.00	0.59	0.64	109.09
	4306.2905	0.4929	0.49
	4272.4668	0.4890	1.27

 

Table 8: Results of the Lysine Peptide Depletion

Lysine Peptide
Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	1802.2493	0.2125	57.49	57.48	1.25	2.18
	1749.5433	0.2062	58.73
	1855.6707	0.2188	56.23
Test Item	4122.5361	0.4883	0.18	0.33	0.14	43.44
	4110.8330	0.4870	0.46
	4115.9360	0.4876	0.34

 

Table 9: Categorization of the Test Item

Based on the results of the peptide depletion, categorization according to the prediction model might be performed. Since precipitation of the lysine peptide was observed, prediction model 2 should be considered.

Predicition Model	Prediction Model 1 (Cysteine Peptide and Lysine Peptide / Ratio: 1:10 and 1:50)			Prediction Model 2  (Cysteine Peptide / TestItemRatio: 1:10)
Test Substance	Mean Peptide Depletion [%]	Reactivity Category	Prediction	Mean Peptide Depletion [%]	Reactivity Category	Prediction
Test Item	0.46	Minimal Reactivity	no sensitizer	0.59	Minimal Reactivity	no sensitizer
Positive Control	63.77	High Reactivity	sensitizer	70.06	High Reactivity	sensitizer

 

Table 10 Acceptance Criteria for Cysteine Peptide

Cysteine Peptide Run
Acceptance Criterion	Range	Value	pass/fail
coefficient of determination	R² > 0.99	1.0000	pass
mean peptide concentration of RC A	0.45≤x≤0.55 mM	0.5149	pass
mean peptide concentration of RC C (PC)	0.45≤x≤0.55 mM	0.5033	pass
mean peptide concentration of RC C (TI)	0.45≤x≤0.55 mM	0.4953	pass
CV of the peak area of RC B	< 15%	1.54	pass
CV of the peak area of RC C (PC)	< 15%	0.77	pass
CV of the peak area of RC C (TI)	< 15%	1.43	pass
mean peptide depletion of the PC	60.8% < x < 100%	70.06	pass
SD of peptide depletion of the PC replicates	< 14.9%	0.48	pass
SD of peptide depletion of the TI replicates	< 14.9%	0.64	pass

 

Table 11 Acceptance Criteria for Lysine Peptide

Lysine Peptide Run
Acceptance Criterion	Range	Value	pass/fail
coefficient of determination	R² > 0.99	0.9999	pass
mean peptide concentration of RC A	0.45≤x≤0.55 mM	0.5024	pass
mean peptide concentration of RC C (PC)	0.45≤x≤0.55 mM	0.5023	pass
mean peptide concentration of RC C (TI)	0.45≤x≤0.55 mM	0.4892	pass
CV of the peak area of RC B	< 15%	0.28	pass
CV of the peak area of RC C (PC)	< 15%	0.15	pass
CV of the peak area of RC C (TI)	< 15%	0.18	pass
mean peptide depletion of the PC	40.2% < x < 69.0%	57.48	pass
SD of peptide depletion of the PC replicates	< 11.6%	1.25	pass
SD of peptide depletion of the TI replicates	< 11.6%	0.14	pass

 

Historical Data

Table 12 Historical Data Cysteine Peptide

Cysteine Peptide
	mean	SD	N
linearity of the calibration curve	0.9995	0.0005	22
mean peptide concentration of reference A [mM]	0.5019	0.0000	22
mean peptide concentration of reference C [mM]	0.4873	0.0000	36
CV of the peak area of control B [%]	2.43	1.39	22
CV of the peak area of control C [%]	1.72	1.45	36
mean peptide depletion of the PC [%]	73.11	1.54	22
SD of peptide depletion of the PC replicates [%]	0.69	0.45	22
SD of peptide depletion of the test items [%]	13.02	26.77	57

 

Table 13 Historical Data Lysine Peptide

Lysine Peptide
	Mean	SD	N
linearity of the calibration curve	0.9999	0.0001	21
mean peptide concentration of reference A [mM]	0.4868	0.0157	21
mean peptide concentration of reference C [mM]	0.4868	0.1938	34
CV of the peak area of control B [%]	0.64	0.19	21
CV of the peak area of control C [%]	0.50	0.62	34
mean peptide depletion of the PC [%]	59.28	6.86	21
SD of peptide depletion of the PC replicates [%]	1.67	1.48	21
SD of peptide depletion of the test items [%]	1.62	2.33	57

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item showed minimal reactivity towards the cysteine and lysine peptide. The test item might be considered as “non-sensitizer”.
The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach
such as IATA.

Executive summary:

Results

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing

either lysine or cysteine.  

In the present study N-[2-(2-Hydroxyethoxy)ethyl]acetamide was given into water, based on the results of the pre-experiments. Based on the weighted molecular weight of 137.26 g/mol a 100 mM

stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.

The test item was completely soluble and the resulting solution was used for further testing.

After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples of the cysteine and lysine peptide run were inspected for precipitation, turbidity or phase separation.  

For the cysteine peptide experiment no precipitation, turbidity or phase separation was observed for any test sample.

For the lysine peptide experiment no precipitation, turbidity or phase separation was observed for the test item samples. A phase separation was observed for the samples of the positive control and

the respective co-elution control. No centrifugation was necessary to perform the HPLC analysis.

No co-elution of test item with the peptide peaks was observed. Sensitizing potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by

comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC C).

The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was < 6.38% (0.46%). Based on prediction model 1 the test

item can be considered as non-sensitiser.

Since the positive control fulfilled all quality criteria the observed precipitations were considered as irrelevant. The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high

reactivity towards the synthetic peptides. The mean depletion of both peptides was 63.77%.

Conclusion

In this study under the given conditions the test item showed minimal reactivity towards the cysteine and the lysine peptide. The test item might be considered as “non-sensitizer”.

The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach

such as IATA.