Aluminum magnesium sodium oxide (Al10.33Mg0.67Na1.67O17)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Remarks:

Direct Peptide Reactivity Assay (DPRA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Feb 2017

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)

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

Aluminum magnesium sodium oxide (Al10.33Mg0.67Na1.67O17)

EC Number:

807-888-6

Cas Number:

110621-40-4

Molecular formula:

Al10.33Mg0.67Na1.67O17

IUPAC Name:

Aluminum magnesium sodium oxide (Al10.33Mg0.67Na1.67O17)

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: BASF, 10978-52 H_K
- Purity: 82%
- Homogenity: homogeneous by visual inspection

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: protect against humidity (store dry), ambient
- Stability under test conditions: guranteed by the sponsor
- Solubility and stability of the test substance in the solvent/vehicle: good homogenity was achieved with DMSO


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: stirring and ultra-sonic preparation
- Preliminary purification step: 4x stock concentration of highest concentration and further serial dilutions in DMSO
- Final dilution of a dissolved solid: 1:1.2 serial dilution of maximum concentration

FORM AS APPLIED IN THE TEST: suspension, unsolved but homogenous

In chemico test system

Details on the study design:

TEST SYSTEM
- Synthetic peptides: Cysteine- (C-) containing peptide: Ac-RFAACAA-COOH (MW=751.9 g/mol); Lysine-(K-)containing peptide: Ac-RFAAKAA-COOH (MW=776.2 g/mol)
- Source: The peptides are custom material (Supplier: GenScript, Piscataway, NJ, USA and RS Synthesis, Louisville KY, USA) containing phenylalanine to aid in detection and either cysteine or lysine as the reactive center.
- 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 (K­containing peptide). The peptide stock solution was used for preparing the calibration samples, the test-substance and control samples.

CONTROLS
- vehicle control: de-ionized water: Set A) performance control (analyzed together with the calibration samples without incubation); Set B) Stability control (placed at the very start and ending of the sample list for HPLC analysis); Set C) for calculation of the peptide depletion (analyzed with the samples)
- Positive control: ethylene glycol dimethacrylate (EGDMA; CAS no. 97-90-5) (prepared as a 50 mM emulsion in de-ioinzed water)
- Co-elution control: Sample prepared of the respective peptide buffer and the test substance but without peptide; visually turbid or precipitating samples were centrifuged and/or filtrated prior to injection into the HPLC

VEHICLE
- Vehicle: de-ionized water
- Reason for choice of the vehicle: The test substance was not soluble in one of the vehicles used for the assay (acetonitrile solutions in water, isopropanol, acetone, propanol, methanol, ethanol, N,N-dimethylformamide and tetrahydrofuran were tried). In de-ionized water a homogeneous suspension was achieved.

SAMPLE PREPARATION
- Peptide stock solutions were mixed with the test substance or positive control or vehicle control at a ration of 1:10 (C-peptide, cysteine) or 1:50 (K-peptide, lysine)

EXPERIMENTAL PROCEDURE
- No. of replicates: 3 (for each peptide)
- The test substance was prepared at a ca. 100 mM concentration. The C-containing peptide was incubated with the test substance in a ratio of 1:10 (0.5 mM peptide, 5 mM test substance) and the K-containg peptide in a ratio of 1:50 (0.5 mM peptide, 25 mM test substance). Due to solubility issues, the maximum soluble test-substance concentration was tested additionally. The test substance was dissolved in de-ionized water at a concentration of 0.0108% (0.108 mg/mL) and was incubated as such without any further dilution in a 1:10 and 1:50 ratio with the cysteine and lysine peptide.
- Visual inspection for solubility was performed directly after sample preparation and prior to HPLC analysis
- Samples were incubated at 25°C ± 2.5°C in the dark for 24 +/- 2 hours.
- The remaining non-depleted peptide concentration was determined by HPLC with gradient elution and UV-detection at 220 nm about 24 hours after sample preparation (for details on HPLC conditions see table 3). The analysis time itself did not exceed 30 hours.
- Calibration samples of known peptide concentration (dissolved in 20% de-ionized water in the respective buffer), prepared from the respective peptide stock solution used for test-substance incubation were measured before analysis of the test-substance samples with the same analytical method (for details see table 1)
- Possible interference of the test substance with the peptides was detected by performing a co-elution control for each tested concentration. The samples were analyzed by measuring UV absorbance at 258 nm in order to calculate the area ratio 220 nm / 258 nm.

DATA EVALUATION (for detailed formulas see "Any other information on material and methods")
Calculation of the peptide concentrations:
- For each peptide a calibration curve is generated from the measured peak areas of the calibration samples of known peptide concentration. The peptide concentration of the samples is calculated with the respective calibration curve using linear regression (b = axis intercept; m = slope).
Calculation of the peptide depletion:
- The mean peptide depletion for each of the two peptides is calculated as the mean value of the three samples conducted for each peptide and test substance. When a negative value for C- or K-containing peptide depletion is obtained the value is considered zero for calculation of the mean peptide depletion. The mean peptide depletion of a test substance is calculated as the mean value of C-containing peptide depletion and K-containing peptide depletion.

ACCEPTANCE CRITERIA
- The standard calibration curve should have an r² >0.99.
- The negative control (vehicle control) samples of sets A and C should be 0.50 mM +/- 0.05 mM.
- The CV of the nine vehicle controls B and C should be < 15%.
- Since the mean peptide depletion for each peptide is determined from the mean of three single samples, the variability between these samples should be acceptably low (SD < 14.9% for % cysteine depletion and < 11.6% for % lysine depletion).
- The positive control should cause depletion of both peptides comparable to historic data.

Results and discussion

In vitro / in chemico

Results

Results 1

Run / experiment:

other: test substance with a 100 mM stock concentration

Parameter:

other: mean peptide depletion (cysteine and lysine peptides)

Value:

4.46

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: due to limited solubility: inconclusive

Results 2

Run / experiment:

other: test substance with a maximum soluble stock concentration

Parameter:

other: mean peptide depletion (cysteine and lysine peptides)

Value:

0.82

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: due to limited solubility: inconclusive

Other effects / acceptance of results:

OTHER EFFECTS:
Due to the limited solubility of the test substance at 100 mM with both peptides the result could be underpredictive. Following OECD TG 442C a “negative” result should be considered “inconclusive” in this case.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

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
- Range of historical values: see tab. 9a+b

The samples of the 100 mM stock concentration with the peptides were suspensions at the time of preparation. Visual observation after the 24-hour incubation time revealed precipitates in the samples of the test substance with the C-containing peptide. Samples with the K-containing peptide were suspensions.

No co-elution of test substance and peptides was present.

Any other information on results incl. tables

Table 6: Peptide depletion for C-peptide

Reaction with cysteine-peptide	peptide depletion [%]
	sample 1	sample 2	sample 3	mean	SD
NC: H2O	-0.69	0.25	0.43	0.00	0.60
Test substance 100mM stock concentration	3.01	3.67	7.38	4.69	2.36
Test substance maximum soluble stock concentration	0.31	1.07	1.34	0.91	0.53
PC: EGDMA in H2O	63.36	81.15	78.01	74.17	9.50

Table 7: Peptide depletion for K-peptide

Reaction with lysine-peptide	peptide depletion [%]
	sample 1	sample 2	sample 3	mean	SD
NC: H2O	-1.50	-1.62	3.12	0.00	2.70
Test substance 100mMstockconcentration	-0.81	3.13	10.37	4.23	5.67
Test substance maximum soluble stock concentration	-0.61	1.51	1.33	0.74	1.18
PC: EGDMA in H2O	6.84	5.93	11.25	8.01	2.84

Table 8: Mean peptide depletions

	Cysteine-Peptide  mean depletion [%] SD [%]		Lysine-Peptide  mean depletion [%] SD [%]		mean of both depletions [%]
Test substance 100mM stock concentration	4.69	2.36	4.23	5.67	4.46
Test substance maximum soluble stock concentration	0.91	0.53	0.74	1.18	0.82
PC: EGDMA in H2O	74.17	9.50	8.01	2.84	41.09

Table 9a: Historic control data of vehicle control (de-ionized water) (not including present study)

	C-peptide concentration	K-peptide concentration
	[mM]	[mM]
Min	0.441	0.459
Max	0.510	0.528
Mean	0.479	0.504
SD	0.014	0.013
n	22	21

Table 9b: Historic control data of positive control (EGDMA, 50 mM in de-ionized water) (not including present study)

	C-peptide concentration [mM]	C-peptide depletion [%]	K-peptide concentration [mM]	K-peptide depletion [%]
Min	0.032	44.32	0.403	5.76
Max	0.323	93.44	0.481	16.01
Mean	0.167	65.87	0.456	9.35
SD	0.083	13.54	0.018	2.39
n	18		18

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model it was concluded that the test substance shows minimal or no chemical reactivity in the DPRA under the test conditions chosen. However, it should be noted that due to the limited solubility of the test substance, the samples with both peptides were suspensions at the 100 mM stock concentration and that the result could therefore be under-predictive. Following OECD TG 442C a “negative” result should be considered “inconclusive” in this case.

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 formulated at 100 mM in de-ionized water. Further, the maximum soluble concentration of the test substance in de-ionized water (0.0108%) was tested. Three samples of each test-substance concentration 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 for each tested concentration and the samples were analyzed by measuring UV absorbance at 258 nm in order to calculate the area ratio 220 nm / 258 nm.

The test substance was not soluble in one of the vehicles used for the assay at a concentration of 100 mM: The test substance was a suspension in de-ionized water at 100 mM and dissolved at a concentration of 0.0108% (maximum soluble concentration). The samples of the 100 mM stock concentration with the peptides were suspensions at the time of preparation. Visual observation after the 24-hour incubation time revealed precipitates in the samples of the test substance with the C-containing peptide. Samples with the K-containing peptide were suspensions. The samples of the 0.0108% concentration were solutions with both peptides at the time of preparation and after the 24-hour incubation time. No co-elution of test substance and peptides was present.

Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model it was concluded that the test substance shows minimal or no chemical reactivity in the DPRA under the test conditions chosen.

However, it should be noted that due to the limited solubility of the test substance, the samples with both peptides were suspensions at the 100 mM stock concentration and that the result could therefore be under-predictive. Following OECD TG 442C a “negative” result should be considered “inconclusive” in this case.