reaction mass of disodium;3-[2-(2-carboxylatoethylamino)-ethylamino]propanoate and sodium 3-[(2-aminoethyl)amino]propanoate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

15 September 2016 - 8 March 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

04 February 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

reaction mass of disodium;3-[2-(2-carboxylatoethylamino)-ethylamino]propanoate and sodium 3-[(2-aminoethyl)amino]propanoate

Molecular formula:

C5H11N2O2Na and C8H14N2O4Na2

IUPAC Name:

reaction mass of disodium;3-[2-(2-carboxylatoethylamino)-ethylamino]propanoate and sodium 3-[(2-aminoethyl)amino]propanoate

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: Do_0164

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient, under nitrogen
- Stability under test conditions: guaranteed

In chemico test system

Details on the study design:

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)
The peptides are custom material (Supplier: GenScript, Piscataway, NJ, USA and/or RS Synthesis, Louisville KY, USA) containing phenylalanine to aid in detection and either cysteine or lysine as the reactive center.

Material and technical equipment:
HPLC: Liquid chromatograph Thermo Scientific, Dionex Ultimate 3000 consisting of the following modules:
Pump: HPG-3400RS
Autosampler: WPS-3000TSL
Column oven: TCC-3000
UV-Detector: DAD-3000
Column: ZORBAX SB-C18 2.1 x 100 mm, 3.5 μm with guard column SecurityGuard Ultra Cartridges, UHPLC C18 for 4.6 mm ID(Phenomenex)
HPLC mobile phase A: H2O/ACN/TFA 950/50/1 V/V/V
HPLC mobile phase B: ACN/H2O/TFA 950/50/0.85 V/V/V
Reagents for preparing the buffers for pH 7.5 phosphate buffer (used for solving C-containing peptide): Sodium phosphate, monobasic monohydrate, CAS no. 10049-21-5
Sodium phosphate, dibasic heptahydrate, CAS no. 7782-85-6 for pH 10.2 ammonium acetate buffer (used for solving K containing peptide): Ammonium acetate, CAS no. 631-61-8, Ammonium hydroxide, 28% – 30%, CAS no. 1336-21-6

Controls:
Negative control (NC): vehicle control = de-ionized water (The test substance was soluble de-ionized water.)
Positive control (PC): Ethylene glycol dimethacrylate (EGDMA; CAS-no. 97-90-5), prepared as 50 mM emulsion in de-ionized water
Co-elution control: Sample prepared of the respective peptide buffer and the test substance but without peptide.

Test substance preparation:
The test substance was prepared as a 100 mM (considering a molecular weight of 154.144 g/mol and a purity/contents of 93.6%) preparation in de-ionized water. After short stirring the test substance was soluble in the vehicle.

Experimental procedure:
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.

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-substance and control samples.

Preparation of calibration samples:
The calibration samples were prepared from the peptide stock solutions in 20% deionized water in the respective buffer (= dilution buffer) using serial dilutions.
(0.534, 0.267, 0.134, 0.067, 0.033, 0.017, 0.000 mM peptide)

Selection of concentrations
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-containing peptide in a ratio of 1:50 (0.5 mM peptide, 25 mM test substance).

The samples were 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 or filtrated prior to injection into the HPLC in order to remove any unsolved particles. The HLPC 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 co-elution control
One sample per peptide was prepared in the same way as the test-substance samples 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 or filtrated prior to injection into the HPLC in order to remove any unsolved particles.

Data evaluation:
For evaluation of peptide depletions peak areas at 220 nm are used. When samples were additionally analyzed by measuring UV absorbance at 258 nm, the area ratio 220 nm/ 258 nm may be calculated and serve as a measure of peak purity. The ratio of a pure peptide peak should be consistent over all samples (100% ± 10% of the mean of the vehicle controls). However, due to small peak areas calculation of the area ratio may not be possible for all samples.

Calculation of the peptide concentrations:
For each peptide and test run 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 concentration (C-containing and K-containing peptide depletion; example calculation for C-containing peptide). 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.

Acceptance criteria of the DPRA:
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). In addition the positive control should cause depletion of both peptides comparable to historic data.

Evaluation of results:
Evaluation criteria of DPRA; 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
> 8.11 ≤ 22.62 low reactivity positive
> 4.65 ≤ 8.11 no to low reactivity borderline1
≤ 4.65 minimal or no reactivity negative
In the case mean peptide depletion [%] cannot be determined due to invalid K-peptide depletion (e.g. insolubility of the K-peptide samples or interference in the samples of the K-peptide) but valid C-peptidedepletion is available, evaluation is performed as follows:

Evaluation criteria of DPRA; cysteine 1:10 prediction model.
C-peptide depletion Reactivity Evaluation
[%]
> 98.24 high reactivity positive
> 23.09 ≤ 98.24 moderate reactivity positive
> 17.28 ≤ 23.09 low reactivity positive
> 10.50 ≤ 17.28 no to low reactivity borderline2
≤ 10.50 minimal or no reactivity negative
1 The „borderline“-evaluation was determined statistically using historic BASF data and hence considers the variance of the test method. This evaluation is an amendment to the evaluation given in OECD TG442C. OECD TG442C defines mean depletions ≤ 6.38 as “negative” (minimal or no reactivity) and mean depletions > 6.38 ≤ 22.62 as “positive” (low reactivity).
2 The „borderline“-evaluation was determined statistically using historic BASF data and hence considers the variance of the test method. This evaluation is an amendment to the evaluation given in OECD TG442C. OECD TG442C defines mean depletions ≤ 13.89 as “negative” (minimal or no reactivity) and mean depletions > 13.89 ≤ 23.09 as “positive” (low reactivity).

Limitations of the evaluation by insolubility and gravimetric procedure:
For test substances that are not completely soluble by visual observation in the sample preparations containing the peptides immediately after preparation or after 24 hours, or when a gravimetric procedure is applied (with the exception of application of the undiluted test substance (liquids) or the maximal soluble test-substance concentration (solids)), the result may be under-predictive due to limited availablity of the test substance. In this case mean peptide reactivity < 8.11% (cysteine 1:10 / lysine 1:50 prediction model) or < 17.28% (cysteine 1:10 prediction model) is interpreted as “inconclusive”. However, a mean peptide depletion > 8.11% or > 17.28% is considered as “positive”.

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: 1st run

Parameter:

other: mean peptide depletion of both peptides (%)

Value:

9.5

Vehicle controls validity:

valid

Remarks:

0%

Positive controls validity:

valid

Remarks:

35.26%

Remarks on result:

other: low reactivity positive

Results 2

Run / experiment:

other: 1st run

Parameter:

other: peptide depletion (%) cysteine peptide

Value:

19.01

Vehicle controls validity:

valid

Remarks:

0%

Positive controls validity:

valid

Remarks:

61.86%

Results 3

Run / experiment:

other: 1st run

Parameter:

other: peptide depletion (%) lysine peptide

Value:

0

Vehicle controls validity:

valid

Remarks:

0%

Positive controls validity:

valid

Remarks:

8.67%

Other effects / acceptance of results:

OTHER EFFECTS:
Solubility of the test-substance samples with the peptides: Visual observation after the 24-hour incubation time did not reveal precipitates in any samples of the test substance with the peptides.

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

ACCEPTANCE OF RESULTS:
The acceptance criteria were met. The positive control caused depletion of both peptides comparable to historic data as can be
seen in the following table:

Historic control data of negative control / vehicle control (not including present study).
De-ionized water
Historic period: Feb 2014 - Mar 2016
C-peptide concentration [mM] K-peptide concentration [mM]
Min 0.441 0.488
Max 0.510 0.528
Mean 0.479 0.506
SD 0.015 0.009
n 19 19
Historic control data of positive control (not including present study):
EGDMA, 50 mM in de-ionized water
Historic period: Feb 2014 - Mar 2016
C-peptide concentration [mM] C-peptide depletion [%] K-peptide concentration [mM] K-peptide depletion [%]
Min 0.032 44.32 0.427 5.76
Max 0.323 93.44 0.481 16.01
Mean 0.164 66.47 0.459 9.27
SD 0.072 14.31 0.014 2.45
n 16 16

Any other information on results incl. tables

Table 1: DPRA. Mean peptide depletions of Cysteine, Lysine and both peptides.

	Cysteine-Peptide Mean depletion       SD      [%]                     [%]	Lysine-Peptide Mean depletion        SD       [%]                     [%]	mean of both depletions                   [%]
Test item	19.01                 2.35	-0.18                   1.25	9.50
PC: EGDMA in H20	61.86                 2.84	8.67                   1.72	35.26

 

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria