Reaction mass of oxybispropanediol and tetraglycerol and triglycerol, esterification products with lactic acid and lauric acid

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

January 22nd 2021 to February 27th 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

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:

Reaction mass of oxybispropanediol and tetraglycerol and triglycerol, esterification products with lactic acid and lauric acid

Molecular formula:

not applicable - UVCB substance

IUPAC Name:

Reaction mass of oxybispropanediol and tetraglycerol and triglycerol, esterification products with lactic acid and lauric acid

Specific details on test material used for the study:

Test item name: Finestar 2009
CAS Number: 2225876-48-0
Water solubility: soluble in water
Molecular formula: C12 H22 O8
Molecular weight: 294.30
Batch/Lot number: 3253082031
analyzed purity: 100% active
Saponification value: 45.87
Date of manufacture: August 27, 2020
Date of expiry: August 26, 2021
Appearance: Pale yellow viscous liquid
Storage temperature: Room temperature (15 to 30°C)
Storage condition: Keep away from light, moisture, and oxidizing - reducing chemicals
Storage container: Keep in original container

In chemico test system

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

lysine peptide (Ac-RFAAKAACOOH)

Details on the study design:

Principle
The DPRA is an in chemico method, which quantifies the covalent binding potential of the test item by determining the remaining concentration of Cysteine or Lysine containing peptide, following 24 ± 2 hours incubation with the test item at 25°C ± 2.5 oC. The synthetic peptides contain phenylalanine to aid in the detection. Relative peptide concentration is measured by high-performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 nm. Cysteine and Lysine peptide percent depletion values are calculated and used in a prediction model which allow assigning the test item to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

Test System and Source
Synthetic heptapeptides containing either Lysine (Ac-RFAAKAA-COOH) or Cysteine (Ac-
RFAACAA-COOH) are used as the test system for the direct peptide reactivity assay. Synthetic
heptapeptides used in this study were obtained from RS Synthesis, Louisville, KY 40270, USA.

Preparation of Cysteine and Lysine Containing Peptides
Stock solutions of Cysteine (Ac-RFAACAA-COOH) and Lysine (Ac-RFAAKAA-COOH) were
freshly prepared just before their incubation with the test item. The final concentration of the Cysteine peptide was 0.667 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. The HPLC run sequence was set up in order, to keep the HPLC analysis time less than 30 hours. This run sequence size permitted the first HPLC injection to occur 24 hours after mixing of test item and the peptide and the last HPLC injection to occur no more than 30 hours later.

Preparation of Cysteine Peptide
An appropriate amount of pH 7.5 phosphate buffer was added to make a 0.667mM solution of Cysteine peptide.

Preparation of Lysine Peptide
An appropriate amount of pH 10.2 ammonium acetate buffer was added to make a 0.667 mM solution of Lysine peptide.

Preparation of the HPLC Standard Calibration Curve
Standards were prepared in a solution of 20% Acetonitrile: phosphate buffer (Cysteine peptide) and/or ammonium acetate buffer (Lysine peptide). Standards of the Cysteine and Lysine peptide stock solution covering the range from 0.534 mM - 0.0167 mM were prepared by serial dilution. A volume of 10 mL of dilution buffer was prepared by mixing 8 mL of buffer (pH 7.5 phosphate buffer for Cysteine peptide, 0.667 mM; pH 10.2 ammonium acetate buffer for Lysine peptide, 0.667 mM) with 2 mL of acetonitrile. 500 μL of standard STD1 was diluted with an equal volume of dilution buffer and continued in a serial manner to prepare standards with nominal concentrations noted below. A blank of dilution buffer as STD 7 was included.
Before starting the analysis HPLC system was equilibrated for around 2 h with mobile phase A (0.1% v/v Trifluoroacetic acid in Milli Q water) and Mobile phase B (0.085% v/v Trifluoroacetic acid in Acetonitrile).

Incubation of the Test Item with the Cysteine and Lysine Peptide Solutions
Cysteine and Lysine peptide solutions were incubated in glass auto sampler vial with the test item at 1:10 and 1:50 ratio, respectively. The reaction solution and standards prepared were left in the dark at 25°C ± 2.5 ºC for 24 ± 2 hours before the HPLC analysis. Test item was tested in triplicate for both peptides. Samples were visually inspected for precipitation prior to HPLC analysis. Precipitation was not observed with the test item and positive control. Depletion of the peptide in the reaction mixture was measured by high pressure liquid chromatography (HPLC) using UV detection.

Reference Controls
Three types of "Reference Control” were included on each day of analysis. Reference Control is a
peptide solution where the test item is replaced by the solvent used to dissolve test item.
Reference Control A was prepared with acetonitrile and used to verify the accuracy of the calibration curve for peptide quantification.

Preparation of Reference Control A (0.5 mM): 750 μL Cysteine/Lysine peptide (0.667 mM) + 250 μL of acetonitrile Reference Control B was prepared with acetonitrile and its replicates were injected at the beginning and end of the experimental run to verify the stability of the peptide over the analysis time.

Preparation of Reference Control B: Same as preparation for Reference Control A (but run before and after 24 ± 2 hours incubation at 25°C ± 2.5 ºC in the dark).
Reference Control C was prepared with the solvent used to solubilize the test items i.e., Milli-Q-water and was included in every assay run together with the samples. They were used to verify that the solvent does not impact the Percent Peptide Depletion. Reference Control C was used to calculate Percent Peptide Depletion.

Preparation of Reference Control C:
i. 750 μL of Cysteine peptide stock + 200 μL of acetonitrile + 50 μL Milli-Q-water (for Cysteine).
ii. 750 μL of Lysine peptide stock + 250 μL of Milli-Q-water (for Lysine).

Sample Preparation and Co-elution Control
Samples were prepared in triplicate for both peptides. Each assay (Cysteine and Lysine) was
performed on separate days. Samples were prepared in 1 mL auto sampler vials. The time of
addition of the test item to the peptide solution was recorded. Vials were caped, vortexed and
were placed in the HPLC auto sampler at 25°C ± 2.5 oC in dark for 24 ± 2 hours. HPLC analysis
of the batch of samples was started 24 ± 2 hours after the test item was added to the peptide
solution.

Sample preparation:
750 μL of Cysteine peptide stock + 200 μL of acetonitrile + 50 μL test item (Cysteine).
750 μL of Lysine peptide stock + 250 μL of test item (Lysine).
Co-elution control was prepared without peptide, to verify whether the test item absorbs at 220 nm and has a similar retention time as a peptide and/or interfere with the data analysis.

Co-elution Control preparation:
750 μL of phosphate buffer (pH 7.5) + 200 μL of acetonitrile + 50 μL test item (Cysteine).
750 μL of ammonium acetate buffer (pH 10.2) + 250 μL of test item (Lysine)

Assay Acceptance and Evaluation Criteria
Acceptance Criteria
Once acceptance criteria for a valid assay had been met, responses observed in the assay were
evaluated.
a) For determining the system suitability, the standard calibration curve should have r² > 0.99.
b) The mean Percent Peptide Depletion value of the three replicates for the positive control
Cinnamaldehyde should be between 60.8% and 100% for the Cysteine peptide and between
40.2% and 69% 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 Control A, B and C should be 0.50 ± 0.05 mM
and the coefficient of variability (CV) of peptide peak areas for the Reference Control A, B and
C in acetonitrile should be < 15.0%.
d) The maximum standard deviation for the test item replicates should be <14.9 for the percentage depletion of Cysteine and <11.6 for the percent depletion of Lysine.
e) For solvent used, the mean of the peptide concentrations of the Reference Control C should be 0.50 ± 0.05 mM.

Evaluation Criteria
The concentration of Cysteine or Lysine peptide was determined in each sample at 220 nm, by
measuring the peak area obtained by appropriate peak integration and by calculating the concentration of peptide using the linear calibration curve derived from the standard.

Analysis
Data from individual test item replicates and Reference Control replicates, e.g., mean peak area, mean percent peptide depletion, mean peptide concentration of Reference Controls, standard deviation and relative coefficient of variability for test item were determined in this study.

Vehicle / solvent:

water

Positive control:

cinnamic aldehyde

Results and discussion

Positive control results:

Positive Control
Cinnamaldehyde (at a concentration of 100 mM in acetonitrile) was used as the positive control in
each assay with Cysteine and Lysine peptides. The mean percent peptide depletion value of the positive control was 74% for Cysteine peptide and 60% for Lysine peptide (TABLE 6) and the Maximum Standard Deviation (SD) for the positive control replicate was 0.58% for percent Cysteine depletion and 0.40% for percent Lysine depletion. These values met the acceptance criteria for the positive control.

In vitro / in chemico

Results

Results 1

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

lysine depletion

Value:

1 %

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

cysteine depletion

Value:

0 %

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Outcome of the prediction model:

negative [in vitro/in chemico]

Any other information on results incl. tables

Solubility and Precipitation Results
The test item, Finester 2009, was checked for solubility in acetonitrile. To achieve a concentration of 100 mM, 88.29 mg of the test item was weighed (considering molecular weight 294.30 g/mol and purity: 100%) and volume was made up to 3 mL with acetonitrile. Since the test item was insoluble in acetonitrile, solubility of the test item was further checked with other recommended solvents, as specified in the OECD Test Guideline No. 442C, i.e., Milli-Q-water.
Finester 2009 was found to be soluble in Milli-Q-water at 100 mM. Precipitation was not observed in Milli-Q-water. Hence, Milli-Q-water was selected as the vehicle for the experiment.

Reference Controls
The relative coefficient of variability (RCV) of peptide peak areas for the Reference Control A was
0.57 % for Cysteine peptide and 0.13 % for Lysine peptide. For Reference Control B relative
coefficient of variability (RCV) of peptide peak areas was 0.41% for Cysteine peptide and 0.32 % for Lysine peptide Lysine, respectively.

The relative coefficient of variability (RCV) of peptide peak areas for the Reference Control C in
acetonitrile was 0.41% for Cysteine peptide and 0.33% for Lysine peptide.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

From the results of this study, under the specified experimental conditions, Finester 2009 is predicted by DPRA to be a non-sensitiser (Negative).

Executive summary:

Study Guidelines
The present study was conducted according to:
OECD, 2020: The Organization for Economic Co-operation and Development (OECD) Guidelines for the Testing of Chemicals, OECD 442C, In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), adopted by the Council on June 26, 2020.

RESULTS
Solubility and Precipitation Results
The test item, Finester 2009, was checked for solubility in acetonitrile. To achieve a concentration of 100 mM, 88.29 mg of the test item was weighed (considering molecular weight 294.30 g/mol and purity: 100%) and volume was made up to 3 mL with acetonitrile. Since the test item was insoluble in acetonitrile, solubility of the test item was further checked with other recommended solvents, as specified in the OECD Test Guideline No. 442C, i.e., Milli-Q-water.
Finester 2009 was found to be soluble in Milli-Q-water at 100 mM. Precipitation was not observed in Milli-Q-water. Hence, Milli-Q-water was selected as the vehicle for the experiment.

Reference Controls
The relative coefficient of variability (RCV) of peptide peak areas for the Reference Control A was
0.57 % for Cysteine peptide and 0.13 % for Lysine peptide. For Reference Control B relative
coefficient of variability (RCV) of peptide peak areas was 0.41% for Cysteine peptide and 0.32 % for Lysine peptide Lysine, respectively.

The relative coefficient of variability (RCV) of peptide peak areas for the Reference Control C in
acetonitrile was 0.41% for Cysteine peptide and 0.33% for Lysine peptide.