Reaction mass of 2-hydroxyethyl laurate and ethylene dilaurate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

13 Dec 2016 - 11 Jan 2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

Limited documentation

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:

adopted in 2015

Deviations:

yes

Remarks:

Limited data on materials and methods available (such as information on general materials, suppliers etc. are documented solely in the study data and not in the report);

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of hydroxyethyl laurate and ethylenedilaurate

EC Number:

908-917-6

Molecular formula:

C14H28O3 C26H50O4

IUPAC Name:

Reaction mass of hydroxyethyl laurate and ethylenedilaurate

In chemico test system

Details on the study design:

The test item was incubated for 24 h (± 2 h) at 25 ± 2.5 °C in solution at 100 mM in combination with either cysteine (96% purity) or lysine (98% purity) containing peptides and then run on an HPLC system (20-minute run-time) using gradient elution and UV detection at 220 nm to measure peptide concentration. The test item was compared to reference controls containing the test item solvent in combination with either cysteine or lysine peptide in order to determine the relative percent peptide depletion. Relative percent peptide depletion values were used in a prediction model to assign the test item to one of four reactivity classes.

Statistical method: Data analysis for this study was performed using the validated EURL-ECVAM analysis template (DPRA validated study template) available from the EURL-ECVAM website.
The template is a Microsoft Excel workbook containing formulae to process the raw data as per OECD TG 442C, it has been validated initially for internal use at XCellR8 during the DPRA proficiency study 16XC014. The final data output is a percentage peptide depletion value for the cysteine and lysine peptides after exposure to the test items. The validated template also assesses adherence to the acceptance criteria.

Results and discussion

Positive control results:

Cinnamic aldehyde, CAS 104-55-2, 100 mM in HPLC Grade acetonitrile (CAS 75-05-8), freshly prepared on Day 1 of main testing

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: 1/2

Parameter:

other: Mean % peptide depletion (cysteine + lysine)

Value:

5.286 %

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Run / experiment:

other: 1/2

Parameter:

other: % cysteine peptide depletion

Value:

9.732 %

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 3

Key result

Run / experiment:

other: 1/2

Parameter:

other: % lysine peptide depletion

Value:

0.84 %

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 4

Key result

Run / experiment:

other: 3

Parameter:

other: % cysteine peptide depletion

Value:

12.541 %

Vehicle controls validity:

not valid

Negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: not specified

DEMONSTRATION OF TECHNICAL PROFICIENCY: due to limited reporting details not specifiable

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for vehicle control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: due to limited reporting details not specifiable
- Range of historical values if different from the ones specified in the test guideline: due to limited reporting details not specifiable

Any other information on results incl. tables

ACCEPTANCE CRITERIA

Acceptance criteria for all controls and the test item were met in both runs with the exception of the RefC controls highlighted in bold. The cysteine peptide in the RefC controls was deemed to have been partially depleted by the acetone solvent used. However, as the test items were also solubilised in acetone the control remained valid for use as a reference for the test item depletion values. RefA and RefB controls in acetonitrile met all the acceptance criteria and therefore the cysteine runs were deemed valid. As acetone partially depleted the cysteine peptide, the positive control was compared to RefB controls in order to calculate percent depletion, as both the positive control and RefB were prepared in acetonitrile.

Criterion	Run 1 (Cysteine)	Run 2 (Lysine)	Run 3 (Cysteine Additional)	Outcome
Std Curve r^2>0.99	0.993	0.997	0.991	PASSED
PC 60.8% to 100% depletion Cysteine	66.249	-	68.930	PASS
PC 40.2% to 69.0% depletion Lysine	-	68.210	-	PASS
SD Cysteine Depletion PC < 14.9 %	4.949	-	9.731	PASS
SD Lysine Depletion PC < 11.6 %	-	1.477	-	PASS
RefA Mean Conc 0.50 ±0.05 mM	0.536	0.508	0.504	PASS
Peak Area CV RefB < 15.0 %	8.615	3.008	12.934	PASS
Peak Area CV RefC < 15.0 %	3.520	0.773	19.876	PASS/PASS/FAILED
SD Cysteine Depletion Test ltem < 14.9 %	9.046	-	10.861	PASS
SD Lysine Depletion Test ltem < 11.6 %	-	0.734	-	PASS
RefC Mean Conc 0.50 ±0.05 mM	0.326	0.499	0.333	FAIL/PASS/FAIL
PC = positive control, SD = standard deviation, CV = coefficient of variation

RESULTS SUMMARY

The test item produced 5.286 % mean cysteine and lysine peptide depletion, therefore, using the cysteine 1:10 / lysine 1:50 prediction model, the test item was classified as a Non­ Sensitizer with No or Minimal Activity. A single HPLC analysis for the lysine peptide was considered sufficient for the test item as the result was unequivocal with this peptide. However, for the cysteine peptide an additional run was carried out as the first result generated was borderline. The second result was assessed using the cysteine 1:10 prediction model. This also produced a borderline result with the same outcome.

Run	Test item ID	% Cysteine Peptide Depletion	% Lysine Peptide Depletion	Mean % Peptide Depletion (Cys + Lys)	DPRA Prediction	DPRA Reactivity Class
1/2	TEN0001	9.732	0.840	5.286	Non-Sensitizer	No or Minimal Reactivity
3	TEN0001	12.541	N/A	N/A	Non-Sensitizer	No or Minimal Reactivity

 N/A = not applicable

Applicant's summary and conclusion

Interpretation of results:

other: no skin sensitising potential based on the key event “protein reactivity”

Conclusions:

There is regulatory acceptance in the EU for the application of the Direct peptide reactivity assay to address key event 1: peptide/protein binding in the skin sensitisation Adverse Outcome Pathway. Under the conditions of the test, the test substance did not show reactivity towards selected proteins. The result is not conclusive with respect to the non-classification or classification as skin sensitiser of the test substance and therefore further evaluation and/or data generation is required.