Reaction mass of diammonium (2R,3R)-2,3-dihydroxybutanedioate and diammonium bis[(2R,3R)-2,3-dioxidobutanedioato]diantimonate(2-)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

May - Aug 2018

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:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

04 February 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(from the competent authority) Landesamt für Umwelt Rheinland-Pfalz

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

in vitro turnkey testing strategy

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of diammonium (2R,3R)-2,3-dihydroxybutanedioate and diammonium bis[(2R,3R)-2,3-dioxidobutanedioato]diantimonate(2-)

EC Number:

943-693-3

IUPAC Name:

Reaction mass of diammonium (2R,3R)-2,3-dihydroxybutanedioate and diammonium bis[(2R,3R)-2,3-dioxidobutanedioato]diantimonate(2-)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 170492F01
- Purity test date: 100.0 area-% (HPLC fingerprint)
- Content:
21.4 g/100 g Test item
23.5 g/100 g (NH4)2C2H4O6
30 g/100 g Tartrate
6.7 g/100 g Ammonium
9.1 g/100 g Antimony
53.5 g/100 g Water
- Physical state / color: liquid / colorless to yellowish, clear

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Solubility and stability of the test substance in the solvent/vehicle: the test substance was soluble in deionized water

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was prepared as a 100 mM preparation in deionized water (considering a molecular weight of 575.74 g/mol and a purity / contents of 46.5 %). After short stirring the test substance was soluble in the vehicle.

FORM AS APPLIED IN THE TEST (if different from that of starting material): solution

In vitro test system

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:
The test substance was dissolved in a suitable vehicle. Three samples of the test substance were incubated with each peptide. Additionally, triplicates of the concurrent vehicle control (= NC) were incubated with the peptides. The remaining non-depleted peptide concentration was determined thereafter by HPLC with gradient elution and UV-detection at 220 nm. In addition, calibration samples of known peptide concentration, prepared from the respective peptide stock solution used for test substance incubation, were measured in parallel with the same analytical method.

Controls:
Negative control (NC): vehicle control = deionized water
Positive control (PC): Ethylene glycol dimethacrylate (EGDMA), prepared as 50 mM emulsion in deionized water
Co-elution control (SK): Sample prepared of the respective peptide buffer and the test substance but without peptide

Test substance solubility:
Prior to the assay the solubility of the test substance at a concentration of 100 mM was tested. A suitable non-reactive, water-miscible solvent which dissolves the test substance completely (no visible precipitation or cloudiness of the test substance preparation) should be used. The preferred solvent was acetonitrile. When not soluble in acetonitrile solutions in water, isopropanol, acetone, propanol, methanol or mixtures of these solvents were tried.

Preparation of peptide stock solutions:
Peptide stock solutions in a concentration of 0.667 mM were prepared in pH 7.5 phosphate buffer (C-containing peptide) or pH 10.2 ammonium acetate buffer (K-containing peptide). The peptide stock solution was used for preparing the calibration samples and the test substances and control samples.

Preparation of the test substance samples:
The samples were prepared in triplicates for each peptide according to the following pipetting scheme.
C-peptide: 750 µL C-peptide stock solution, 200 µL solvent (vehicle), 50 µL test substance preparation (or PC-preparation or solvent (VC)).
K-peptide: 750 µL K-peptide stock solution, 250 µL test substance preparation (or PC-preparation or solvent (VC)).
The samples were prepared in suitable tubes, capped tightly and incubated at 25 °C +/- 2.5 °C in the dark for 24 +/- 2 hours. Visual inspection for solubility was performed directly after sample preparation and prior to HPLC analysis. Unsolved samples were centrifuged and / or filtrated prior to injection into the HPLC in order to remove any unsolved particles. The HPLC analysis of the batch of samples started about 24 hours after sample preparation and the analysis time itself did not exceed 30 hours.

Preparation of the vehicle controls:
Several vehicle controls were prepared in triplicates in the same way as the test substance samples described above but with the vehicle (deionized water) instead of the test substance: One set (set A) was analyzed together with the calibration samples without incubation and serves as a performance control. Another three sets (two sets B and set C) were prepared and incubated with the samples. Sets B were placed at the very start and ending of the sample list and serve as stability control of the peptide over the analysis time. Set C was analyzed with the samples and serves for calculation of the peptide depletion of any chemical formulated in the vehicle.

Preparation of the co-elution control:
One sample per peptide was prepared in the same wa as the test substance samples described above but without the peptides. Instead the respective peptide buffer was used. The samples were analyzed together with the calibration samples. Samples which were visually turbid or display precipitates were centrifuged and / or filtrated prior to injection into the HPLC in order to remove any unsolved particles.

Results and discussion

In vitro / in chemico

Results

Results 1

Run / experiment:

other: cysteine-peptide

Parameter:

other: mean peptide depletion [%]

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: true value is negative (- 11.21)

Results 2

Run / experiment:

other: lysine-peptide

Parameter:

other: mean peptide depletion [%]

Value:

0.77

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: precipitation after 24 h incubation time

Other effects / acceptance of results:

Solubility of the test substance samples with the peptides:
The test substance was dissolved in deionized water at a concentration of 100 mM. The samples of the test substance with the C-containing peptide were solutions at the time of preparation and after the 24-hour incubation time. The samples of the test substance with the K-containing peptide were emulsions at the time of preparation and precipitates were noted after the 24-hour incubation time.

Co-elution:
No co-elution of the test substance and peptides occurred as demonstrated by consistent values of the area ratios 220 nm / 258 nm and chromatograms of the co-elution control.

Mean peptide depletion:
Due to the insolubility of the test substance in the K-peptide samples calculation of mean peptide depletion is not applicable for the test substance.

Any other information on results incl. tables

Table 1: Peak area, peptide concentration and peptide depletion of NC, PC and the test substance for cysteine-peptide

Reaction with cysteine-peptide	Peak area [mAU*s] at 220 nm			Peptide concnetration [mM]					Peptide depletion [%]
	Sample 1	Sample 2	Sample 3	Sample 1	Sample 2	Sample 3	Mean	SD	Sample 1	Sample 2	Sample 3	Mean	SD
NC: H2O	400.2	392.6	398.4	0.496	0.486	0.493	0.492	0.005	- 0.79	1.14	- 0.35	0.00	1.01
Test substance	443.8	438.5	441.2	0.550	0.543	0.543	0.547	0.003	- 11.88	- 10.54	- 11.21	- 11.21	0.67
PC: EGDMA	70.2	117.9	67.8	0.083	0.142	0.080	0.102	0.035	83.18	71.03	83.79	79.33	7.20

Table 2: Area ratio 220 nm / 258 nm of NC, PC and the test substance for cysteine-peptide

Reaction with cysteine-peptide	Peak area [mAU*s] at 258 nm			Area ratio 220 nm / 258 nm
	Sample 1	Sample 2	Sample 3	Sample 1	Sample 2	Sample 3
NC: H2O	12.7	13.6	12.6	31.6	28.8	31.7
Test substance	15.1	15.0	15.1	29.4	29.3	29.3
PC: EGDMA	n.a.	n.a.	n.a.	-	-	-

Table 3: Peak area, peptide concentration and peptide depletion of NC, PC and the test substance for lysine-peptide

Reaction with lysine-peptide	Peak area [mAU*s] at 220 nm			Peptide concnetration [mM]					Peptide depletion [%]
	Sample 1	Sample 2	Sample 3	Sample 1	Sample 2	Sample 3	Mean	SD	Sample 1	Sample 2	Sample 3	Mean	SD
NC: H2O	408.7	407.9	407.4	0.516	0.515	0.514	0.515	0.001	- 0.18	0.01	0.16	0.00	0.17
Test substance	402.0	405.5	407.3	0.507	0.512	0.514	0.511	0.004	1.51	0.63	0.17	0.77	0.68
PC: EGDMA	362.3	368.0	366.7	0.456	0.463	0.462	0.460	0.004	11.47	10.03	10.35	10.61	0.76

Table 4: Area ratio 220 nm / 258 nm of NC, PC and the test substance for lysine-peptide

Reaction with lysine-peptide	Peak area [mAU*s] at 258 nm			Area ratio 220 nm / 258 nm
	Sample 1	Sample 2	Sample 3	Sample 1	Sample 2	Sample 3
NC: H2O	14.2	14.3	14.3	28.8	28.5	28.5
Test substance	14.0	14.3	14.1	28.7	28.4	29.0
PC: EGDMA	12.6	12.9	12.8	28.9	28.4	28.8

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

No prediction can be made for skin sensitisation according to GHS criteria based on the results of this in vitro study alone.

Executive summary:

The reactivity of the test substance towards synthetic cysteine (C)- or lysine (K)-containing peptides was evaluated in the Direct Peptide Reactivity Assay (DPRA). For this purpose, the test substance was incubated with synthetic peptides for ca. 24 hours at ca. 25 °C and the remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and UV-detection at 220 nm.

The test substance was dissolved at 100 mM in deionized water. Three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-containing peptide) or 1:50 (for K-containing peptide). Additionally, triplicates of the concurrent vehicle control (= VC) were incubated with the peptides. Further, in order to detect possible interference of the test substance with the peptides, a co-elution control was performed and the samples were analyzed by measuring UV absorbance at 258 nm in order to calculate the area ratio 220 nm / 258 nm.

The following results were obtained in the DPRA:

The test substance was dissolved in deionized water at a concentration of 100 mM. The samples of the test substance with the C-containing peptide were solutions at the time of preparation and after the 24 -hour incubation time. The samples of the test substance with the K-containing peptide were emulsions at the time of preparation and precipitates were noted after the 24 -hour incubation time.

The mean C-peptide depletion, caused by the test substance was determined to be - 11.21 %.

The mean K-peptide depletion, caused by the test substance was determined to be 0.77 %.

No co-elution of test substance and peptides was present. Although a distinct negative C-peptide depletion values is noticed, the absence of interference is clearly demonstrated by the co-elution control and the consistent area ratio 220 nm / 258 nm.

Due to the insolubility of the test substance in the K-peptide samples calculation of mean peptide depletion is not applicable and the cysteine 1:10 prediction model is used for evaluation.

Based on the observed results and applying the cysteine 1:10 prediction model it was concluded that the test substance shows minimal or no chemical reactivity in the DPRA under the test conditions chosen.