methyl-bis(2-arylpropyl)dihydro-heteropolycycle

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-01-14 to 2019-01-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

February 04, 2015

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154, January 12, 2013

Version / remarks:

January 12, 2013

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

In order to replace in vivo experiments validation studies on alternative, mechanistically based in chemico and in vitro test methods on skin sensitisation were conducted under the auspices of ECVAM and have been considered scientifically valid for the evaluation of the skin sensitisation hazard of chemicals. It was concluded that the direct peptide reactivity assay (DPRA) showed evidence of being a reliable and relevant method to test for skin sensitisation testing. However, only combinations of several non-animal testing methods within an Integrated Approach to Testing and Assessment (IATA) will be able to fully substitute for the animal test currently in use.

Test material

Test material information

Constituent 1

Test material form:

liquid

In chemico test system

Details on the study design:

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.
In the present study methyl-bis(2-arylpropyl)dihydro-heteropolycycle was dissolved in acetonitrile, based on the results of the pre-experiments. Based on a molecular weight of 365.51 g/mol a 100 mM stock solution was prepared.
The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.

Results and discussion

Positive control results:

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 67.86%.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: cysteine run

Parameter:

other: mean peptide depletion

Value:

9.14

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: value in %; no predicxtion can be made due to phase separation

Results 2

Key result

Run / experiment:

other: lysine run

Parameter:

other: mean peptide depletion

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: value in %; no predicxtion can be made due to phase separation

Other effects / acceptance of results:

Acceptance Criteria for Cysteine Peptide
- coefficient of determination R² > 0.99 0.9999 pass
- mean peptide concentration of RC A 0.45 ≤ x ≤ 0.55 mM 0.5097 pass
- mean peptide concentration of RC C (PC) 0.45 ≤ x ≤ 0.55 mM 0.5049 pass
- mean peptide concentration of RC C (TI) 0.45 ≤ x ≤ 0.55 mM 0.5049 pass
- CV of the peak area of RC B < 15% 0.81 pass
- CV of the peak area of RC C (PC) < 15% 0.34 pass
- CV of the peak area of RC C (TI) < 15% 0.34 pass
- mean peptide depletion of the PC 60.8% < x < 100% 69.38 pass
- SD of peptide depletion of the PC replicates < 14.9% 0.58 pass
- SD of peptide depletion of the TI replicates < 14.9% 4.27 pass

Acceptance Criteria for Lysine Peptide
- coefficient of determination R² > 0.99 1.0000 pass
- mean peptide concentration of RC A 0.45 ≤ x ≤ 0.55 mM 0.5014 pass
- mean peptide concentration of RC C (PC) 0.45 ≤ x ≤ 0.55 mM 0.5001 pass
- mean peptide concentration of RC C (TI) 0.45 ≤ x ≤ 0.55 mM 0.5001 pass
- CV of the peak area of RC B < 15% 0.84 pass
- CV of the peak area of RC C (PC) < 15% 0.26 pass
- CV of the peak area of RC C (TI) < 15% 0.26 pass
- mean peptide depletion of the PC 40.2% < x < 69.0% 66.34 pass
- SD of peptide depletion of the PC replicates < 11.6% 0.83 pass
- SD of peptide depletion of the TI replicates < 11.6% 0.00 pass

Any other information on results incl. tables

Precipitation and Phase Separation

All test item solutions were freshly prepared immediately prior to use.

For the 100 mM stock solution of the test item no phase separation or precipitation was observed however turbidity was noted when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Slight phase separation was observed for all the samples of the test item. Samples were not centrifuged prior to the HPLC analysis.

For the 100 mM stock solution of the test item no phase separation or precipitation was observed, however turbidity was noted when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Phase separation was observed for the samples of the positive control including the co-elution control. Slight phase separation was also observed for all the samples of the test item. Samples were not centrifuged prior to the HPLC analysis.

Since the acceptance criteria for the depletion range of the positive control was fulfilled, the observed phase separation was regarded as not relevant.

Co-elution with the Peptide Peaks

No co-elution of the test item with any of the peptide peaks was observed. Representative HPLC chromatograms are presented in Appendix 3: Representative HPLC Chromatograms Figure 6 and Figure 12.

To detect a co-elution of the test item with the peptide peak, a ratio of the 220 nm peak area and the 258 nm peak area was calculated too (peak purity indication). If the ratio of the control samples and the test item samples do not differ more than 10% from each other, no sign for a co-elution is given.

For the cysteine measurement the peak purity indication was < 10% (0.3 % test item replicate 1; 0.0 % test item replicate 2; 0.2% test item replicate 3) and for the lysine measurement the peak purity indication was < 10% (1.2 % test item replicate 1; 0.8 % test item replicate 2; 1.1 % test item replicate 3)

Categorization of the Test Item

Based on the results of the peptide depletion, categorization according to the prediction model might be performed.

Since no co-elution was observed, prediction model 1 based on the combination of cysteine and lysine peptide depletion should be considered.

Due to the observed phase separation after the incubation period in the cysteine and the lysine peptide samples containing also test item, no prediction can be made.

Prediction Model	Prediction Model 1 (Cysteine Peptide and Lysine Peptide / Ratio: 1:10 and 1:50)			Prediction Model 2 (Cysteine Peptide / Test Item Ratio: 1:10)
Test Substance	Mean Peptide Depletion [%]	Reactivity Category	Prediction	Mean Peptide Depletion [%]	Reactivity Category	Prediction
Test Item	4.57	Minimal Reactivity	(negative)	9.14	Minimal Reactivity	(negative)
Positive Control	67.86	High Reactivity	positive	69.38	Moderate Reactivity	positive

Cysteine and Lysine Values of the Calibration Curve

Sample	Cysteine Peptide		Lysine Peptide
	Peak Area at 220 nm	Peptide Concentration [mM]	Peak Area at 220 nm	Peptide Concentration [mM]
STD1	16.4390	0.5340	14.6340	0.5340
STD2	8.2610	0.2670	7.3130	0.2670
STD3	4.0740	0.1335	3.6450	0.1335
STD4	1.9450	0.0667	1.8080	0.0667
STD5	0.9340	0.0334	0.9000	0.0334
STD6	0.4240	0.0167	0.4440	0.0167
STD7	0.0000	0.0000	0.0000	0.0000

Depletion of the Cysteine Peptide

Cysteine Peptide
Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	4.7360	0.1551	69.56	69.38	0.58	0.84
	4.8660	0.1593	68.73
	4.6910	0.1537	69.85
Test Item	13.3730	0.4342	14.06	9.14	4.27	46.69
	14.5480	0.4721	6.51
	14.4960	0.4705	6.84

Depletion of the Lysine Peptide

Lysine Peptide
Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	4.4950	0.1644	67.20	66.34	0.83	1.25
	4.6220	0.1690	66.27
	4.7210	0.1726	65.55
Test Item	13.8210	0.5044	0.00	0.00	0.00	0.00
	13.7520	0.5019	0.00
	13.7710	0.5026	0.00

Prediction Model 1

Cysteine 1:10/ Lysine 1:50 Prediction Model ¹

Mean Cysteine andLysine PPD	Reactivity Class	DPRA Prediction²
0.00% ≤ PPD ≤ 6.38%	No or Minimal Reactivity	Negative
6.38% < PPD ≤ 22.62%	Low Reactivity	Positive
22.62% < PPD ≤ 42.47%	Moderate Reactivity
42.47% < PPD ≤ 100%	High Reactivity

1 The numbers refer to statistically generated threshold values and are not related to the precision of the measurement.

2 DPRA predictions should be considered in the framework of an IATA.

Prediction Model 2

Cysteine 1:10 Prediction Model

Cysteine PPD	ReactivityClass	DPRA Prediction²
0.00% ≤ PPD ≤ 13.89%	No or Minimal Reactivity	Negative
13.89% < PPD ≤ 23.09%	Low Reactivity	Positive
23.09% < PPD ≤ 98.24%	Moderate Reactivity
98.24% < PPD ≤ 100%	High Reactivity

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Remarks:

No prediction can be made

Conclusions:

In this study under the given conditions the test item could not be classified due to the observed phase separation of the test item with both peptide peaks.
The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.

Executive summary:

In the present study methyl-bis(2-arylpropyl)dihydro-heteropolycycle was dissolved in acetonitrile, based on the results of the pre-experiments. Based on a molecular weight of 365.51 g/mol a 100 mM stock solution was prepared.

The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.

For the 100 mM stock solution of the test item no phase separation or precipitation was observed however turbidity was noted when diluted with the cysteine peptide solution. After the 24 h±2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Slight phase separation was observed for all the samples of the test item. Samples were not centrifuged prior to the HPLC analysis.

For the 100 mM stock solution of the test item no phase separation or precipitation was observed, however turbidity was noted when diluted with the lysine peptide solution. After the 24 h±2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Phase separation was observed for the samples of the positive control including the co-elution control. Slight phase separation was also observed for all the samples of the test item. Samples were not centrifuged prior to the HPLC analysis.

Since the acceptance criteria for the depletion range of the positive control was fulfilled, the observed phase separation was regarded as not relevant.

The stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was <= 6.38% (4.57%). Since a phase separation with both peptides was observed, no firm conclusion on the lack of reactivity should be drawn from a negative result. Therefore, no prediction can be made.

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 67.86%.

In this study under the given conditions the test item could not be classified due to the observed phase separation of the test item with both peptide peaks.

The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.