Sodium (2-butoxyethoxy)acetate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

16 January 2019 - 21 March 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015

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:

Sodium (2-butoxyethoxy)acetate

EC Number:

268-040-3

EC Name:

Sodium (2-butoxyethoxy)acetate

Cas Number:

67990-17-4

Molecular formula:

C8H16O4.Na

IUPAC Name:

sodium (2-butoxyethoxy)acetate

Test material form:

liquid: viscous

Details on test material:

Batch Number: P59564
Appearance: white-yellow, low melting point paste
Purity: 100 %
Expiration date: 31.12.2018
Storage: room temperature (ca. 20°C)

In chemico test system

Details on the study design:

Synthetic peptides:
Sequence Cys-Peptide (Cysteine): Ac-RFAACAA-COOH (MW = 750.9 g/mol), Batch No.:150219HS-MHe
Sequence Lys-Peptide (Lysine): Ac-RFAAKAA-COOH (MW = 775.9 g/mol), Batch No.: 020517HS_MHEW0119-2

Instruments and Devices:
Analytical and precision scales
Volumetric measurement tools
pH-meter
Standard laboratory glassware
Incubation chamber

Chemicals:
Water for chromatography
H2O, HPLC grade
Demineralised water
Acetonitrile for chromatography
CH3CN, ACN, HPLC grade
Trifluoroacetic acid
TFA, for spectroscopy, Merck
Isopropanol
CH3CHOHCH3, p.a.

Buffers:
Phosphate buffer pH 7.5 ± 0.05
18 (v/v) % 0.1 M sodium phosphate monobasic (of Sodium Phosphate Monobasic Monohydrate (NaH2PO4 • H2O) in purified water)
82 (v/v) % 0.1 M sodium phosphate dibasic (of Sodium Phosphate Dibasic Heptahydrate (Na2HPO4 • 7H2O) in purified water) pH was adjusted with either the monobasic or dibasic solution
Ammonium acetate buffer pH 10.2 ± 0.05
0.1 M Ammonium Acetate (CH3CO2NH4) in purified water
pH was adjusted with Ammonium Hydroxide (NH4OH) drop by drop

Positive control:
Cinnamaldehyde (CAS 104-55-2, 99.5 %,100 mM solution in acetonitrile for the cysteine peptide
100 mM solutions of the positive control chemical in acetonitrile were prepared just before use.

Peptide stock solutions
Cysteine peptide stock solution, 0.667 mM, 0.501 mg/mL:
The needed amount of peptide was calculated based on the equation below (0.01085 g ± 10%). The previously calculated amount of the peptide (0.01153 g) was pre-weighted and 21.25 mL of pH 7.5 phosphate buffer was added right before beginning the assay in the valid run.
Lysine peptide stock solution, 0.667 mM, 0.518 mg/mL:
The needed amount of peptide was calculated based on the equation below (0.01065 g ± 10%). The previously calculated amount of the peptide (0.01003 g) was pre-weighted and 18.83 mL of pH 10.2 acetate buffer was added right before beginning of the assay in the valid run.

Test item stock solution:
100 mM solutions of the test chemical in the appropriate solvent were prepared just before use. The needed amount of test chemical was calculated (0.0991 g ± 10 %) and weighted based on the single average molecular weight. 0.0993 g test chemical was weighted for the stock solution used for the cysteine peptide depletion determination and 0.0991 g test chemical was weighted for the stock solution used for lysine peptide depletion determination in the valid runs.

Test item samples:
Samples are prepared in triplicate for each peptide. The Cys-peptide samples are prepared in 1:10 molar ratio (0.5 mM peptide: 5 mM test item), the Lys-peptide samples in 1:50 molar ratio (0.5 mM peptide and 25 mM test item) using the stock solutions described. A final volume of 1 mL per sample is prepared for each sample

Incubation:
The positive control, solvent control and test item samples are incubated in closed amber glass HPLC vials in an incubation chamber at 25.0 ± 2.5 °C for 24 ± 2 h. When the test item stock solution was combined with the cysteine and lysine stock solutions (reaction samples) or the phosphate and ammonium acetate buffer (co-elution controls), the samples appeared to be clear and homogenous.

Measurements
HPLC system with UV/VIS-Detector

Evaluation of results:
Evaluation criteria of results according to the cysteine 1:10 / lysine 1:50 prediction model.
Mean peptide depletion [%] Reactivity Evaluation
> 42.47 high reactivity positive
> 22.62 ≤ 42.47 moderate reactivity positive
> 6.38 ≤ 22.62 low reactivity positive
0- ≤ 6.385 minimal or no reactivity negative

Acceptance criteria
-the standard calibration curve should have an r2 > 0.99
-the mean peptide concentration of reference controls A should be 0.50 ± 0.05 mM and the coefficient of variation (CV) of peptide peak areas for the nine reference controls B and C in acetonitrile should be ≤ 15.0%.
-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 69.0% 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

The following criteria should be met for a test chemical’s results to be considered valid:
-the maximum standard deviation for the test chemical replicates should be less than 14.9 % for the percent cysteine depletion and less than 11.6% for the percent lysine depletion
-the mean peptide concentration of the three reference controls C in the appropriate solvent should be 0.50 ± 0.05 mM.

Results and discussion

Positive control results:

The acceptance criteria were met for the positive control with a cysteine peptide depletion value of 70.60 % and a mean lysine peptide depletion value of 53.00 %. The standard deviation of the percent peptide depletions of the positive control was 1.05 % and 0.49 % for the cysteine and lysine depletion, respectively. Thus, this validity criterion was also met.

In vitro / in chemico

Results

Key result

Run / experiment:

other: 1st experiment

Parameter:

other: mean peptide depletion of both peptides (%)

Value:

2.44

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
Co-elution:
The test chemical did not absorb at 220 nm significantly (<10 % compared to the respective reference control) when tested with the cysteine and lysine peptides. The range of retention time for cysteine peptide was between 8.383 and 8.480 and the range of retention time for lysine peptide was between 6.118 and 6.317.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Prior to routine use of the method, TOXI-COOP ZRT. demonstrated technical proficiency in a separate study by correctly obtaining the expected DPRA prediction for 10 proficiency substances as recommended in the OECD TG 442C guideline.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Any other information on results incl. tables

Table 1: Cysteine peptide depletion values for the positive control and the test item

Sample	Peptide conc. calculated (mM)	Mean peptide conc. (mM)	Peptide depletion (%)	Standard deviation (SD) for peptide depletion (%)
ref C upw, rep I	0.50	0.49	-	-
ref C upw, rep II	0.49		-
ref C upw, rep III	0.49		-
CINNAMALDEHYDE, rep I	0.14	0.14	71.81%	1.05%
CINNAMALDEHYDE, rep II	0.15		70.09%
CINNAMALDEHYDE, rep III	0.15		69.91%
sodium (2-butoxyethoxy) acetate,  rep I	0.48	0.47	4.41%	1.72%
sodium (2-butoxyethoxy) acetate, rep II	0.48		1.57%
sodium (2-butoxyethoxy) acetate, rep III	0.47		4.69%

(upw = ultrapure water)

Table 2: Lysine peptide depletion values for the positive control and the test item

Sample	Peptide conc. calculated (mM)	Mean peptide conc. (mM)	Peptide depletion (%)	Standard deviation (SD) for peptide depletion (%)
ref C upw, rep I	0.49	0.49	-	-
ref C upw, rep II	0.49		-
ref C upw, rep III	0.49		-
CINNAMALDEHYDE, rep I	0.23	0.23	53.35%	0.49%
CINNAMALDEHYDE, rep II	0.24		52.44%
CINNAMALDEHYDE, rep III	0.23		53.22%
sodium (2-butoxyethoxy) acetate,  rep I	0.49	0.49	1.17%	0.19%
sodium (2-butoxyethoxy) acetate, rep II	0.49		1.53%
sodium (2-butoxyethoxy) acetate, rep III	0.49		1.27%

Table 3: Mean peptide depletion values for the positive control and the test chemical

Name, replicate number	Obtained mean % cysteine peptide depletion	Obtained mean % lysine peptide depletion	Mean % obtained peptide depletion
sodium (2-butoxyethoxy) acetate	3.56	1.32	2.44
CINNAMALDEHYDE	70.60	53.00	61.80

Applicant's summary and conclusion

Interpretation of results:

other: no peptide depletion

Remarks:

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.

Conclusions:

Based on these results and the cysteine 1:10 / lysine 1:50 prediction model, the test item was concluded to have no or minimal reactivity towards the synthetic peptides and thus is not a potential skin sensitizer under the experimental conditions of the in chemico Direct Peptide Reactivity Assay (DPRA) method.

Executive summary:

A study according OECD TG 442 C was conducted for detection of the sensitising potential of the test item by quantifying its reactivity towards synthetic peptides containing either lysine or cysteine.

For the test chemical and positive control substance, in order to derive a prediction five independent tests were conducted, since the first run with cysteine peptide and the first two runs with lysine peptide did not meet acceptance criteria those results were rejected. All in all, the results of the two valid runs were used for the classification of the test item.

Peptide depletion resulted from the positive control cinnamaldehyde was 70.60 % with cysteine peptide and 53.00 % with the lysine peptide while the standard deviation (SD) of the percent peptide depletions were 1.05 % and 0.49 % for the cysteine and lysine peptides respectively in the valid runs. The back-calculated values of the reference control replicates were within the expected molarity concentration range for the cysteine (0.50 – 0.49 mM) and lysine peptides (0.50 – 0.49 mM) and the CV % for the nine reference controls B and C in acetonitrile were 2.6 % and 0.3 % percentages for the cysteine and lysine peptides. In the last runs for each peptide all validity criteria were also met, confirming the validity of the assay.

The percent cysteine peptide depletion value of the test item was 3.56 % while the percent lysine peptide depletion was 1.32 %. The mean depletion value of the peptides was used to categorize the test chemical in one of the four classes of reactivity. No co-elution was observed with either cysteine or lysine peptides; therefore the cysteine 1:10 / lysine 1:50 prediction model was used for the discrimination between sensitisers and non-sensitisers. The mean peptide depletion of the test item was 2.44 %, which did not exceed the 6.38 % threshold of the applicable prediction model.