Sophorolipids: fermentation products of glucose and fatty acids, C18 (unsaturated), glycerol esters with yeast Candida Bombicola

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-03-07 to 2016-04-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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:

adopted: February 04, 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

January 12, 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

The Direct Peptide Reactivity Assay (DPRA) is accepted as a reliable and relevant replacement test for in vivo skin sensitisation testing.

Test material

Test material information

Constituent 1

Reference substance name:

Sophorolipids: fermentation products of glucose and fatty acids, C18-unsatd., esters with glycerol with yeast Candida Bombicola, partially hydrolysed

EC Number:

941-809-7

IUPAC Name:

Sophorolipids: fermentation products of glucose and fatty acids, C18-unsatd., esters with glycerol with yeast Candida Bombicola, partially hydrolysed

Test material form:

liquid

Specific details on test material used for the study:

Preparation of the Test Item
The test item was freshly prepared immediately prior to use, unless stability data demonstrate the acceptability of storage. The test item was pre-weighted into a glass vial and was dissolved in an appropriate solvent previously determined in a pre-experiment. A stock solution with a concentration of 100 mM was prepared.

In chemico test system

Details on the study design:

The DPRA is supposed to address the molecular initiating event of the adverse outcome pathway (AOP), namely protein reactivity, by quantifying the reactivity of test chemicals towards synthetic model peptides containing either lysine or cysteine. The percentage depletion value of the cysteine and lysine peptide is used to categorize a substance in one of four reactivity classes to support discrimination between skin sensitisers and non-sensitisers.
The correlation of protein reactivity with skin sensitisation potential of a chemical is well established and represents the first and initial key event in the skin sensitisation process as defined by the AOP. It is therefore a crucial step for the sensitising potential of a chemical.
This test may be used for the hazard identification of sensitising chemicals in accordance with UN GHS “Category 1”. It does not allow the classification of chemicals to the subcategories 1A and 1B as defined by UN GHS nor predict potency for safety assessment decisions. Therefore, all substances giving a positive result in the DPRA will be classified into UN GHS “Category 1”.
For detailed information on experimental procedure, materials, methods, controls, prediction model and acceptance criteria please see section “any other details on materials and methods incl. tables”.

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 62.90 %.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Both Cysteine and Lysine

Parameter:

other: mean peptide depletion [%]

Value:

2.03

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Run / experiment:

other: Cysteine

Parameter:

other: mean peptide depletion [%]

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

Values were set to zero due to negative depletion

Results 3

Run / experiment:

other: Lysine

Parameter:

other: mean peptide depletion [%]

Value:

4.07

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: not reported
ACCEPTANCE OF RESULTS: all criteria were fulfilled. For details, please see Tables 10 and 11 in section “any other details on results incl. tables”.

Any other information on results incl. tables

Pre-Experiments

Solubility of the test item was determined prior to the main experiment. All test item solutions were freshly prepared immediately prior to use. The test item was not soluble in acetonitrile but completely soluble in water. No turbidity, precipitation and phase separation were observed for the test item solutions. All test item preparations of the main experiment were prepared using water.

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 turbidity or precipitation was observed when diluted with the cysteine peptide solution.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution.

After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples of the cysteine peptide run were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the test item samples, positive control, RC A, RC B, RC C and for STD 1 and 2. Samples were not centrifuged prior to the HPLC analysis.

After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples of the lysine peptide run were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the any test item samples. Slight phase separation was observed for the positive control and the respective co-elution control.

After the HPLC run samples of the cysteine peptide run were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the test item samples, positive control, RC A, RC B, RC C and for STD 1, 2 and 3.

After the HPLC run samples of the lysine peptide run were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the any test item samples. Slight phase separation was observed for the positive control and the respective co-elution control.

Co-elution with the peptide peak

No co-elution of the test item with any of the peptide peaks was observed.

Results Calibration Curve

Table 6: 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	3495.8242	0.5340	3675.5410	0.5340
STD2	1805.4337	0.2670	1867.7395	0.2670
STD3	944.5201	0.1335	985.2802	0.1335
STD4	466.7887	0.0667	516.4465	0.0667
STD5	237.0339	0.0334	282.1445	0.0334
STD6	116.4341	0.0167	179.8809	0.0167
STD7	0.0000	0.0000	0.0000	0.0000

Based on these results, linear regression was performed and the following calibration curves were determined:

Cysteine Peptide Calibration Curve : y = 6546.17x + 26.29 ; R²= 0.9995

Lysine Peptide Calibration Curve : y = 6805.79x + 50.30 ; R²= 0.9996

 

Results of the Cysteine Peptide Depletion

Table 7: Depletion of the Cysteine Peptide. * Values were set to zero due to negative depletion.

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	906.7542	0.1345	71.54	72.27	0.90	0.01
	892.3105	0.1323	71.99
	851.7282	0.1261	73.27
Test Item	3217.3611	0.4875	0.00*	0.00	0.00	-
	3224.2549	0.4885	0.00*
	3206.4368	0.4858	0.00*

 

 Results of the Lysine Peptide Depletion

Table 8: 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	1608.7321	0.2290	53.57	53.53	0.22	0.00
	1618.3588	0.2304	53.29
	1603.4867	0.2282	53.72
Test Item	3266.0764	0.4725	4.10	4.07	0.10	0.02
	3271.2881	0.4733	3.95
	3264.7769	0.4723	4.14

Detailed results about the reference controls can be found in Table 15.

Categorization of the Test Item

Based on the results of the peptide depletion, categorization according to the prediction model was performed. In case that no co-elution was detected, the prediction model based on the combination of cysteine and lysine peptide should be used. Since no co-elution was observed the prediction model of cysteine and lysine was used.

Table 9:     Categorization of the Test Item

Predicition 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	2.03	Minimal reactivity	No sensitiser	0.00	Minimal reactivity	No sensitiser
Positive Control	62.90	High reactivity	sensitizer	72.27	Moderate reactivity	sensitizer

 

Acceptance Criteria

Table 10: Acceptance Criteria for Cysteine Peptide

Cysteine Peptide Run
Acceptance Criterion	Range	Value	pass/fail
coefficient of determination	R^2> 0.99	0.9995	pass
mean peptide concentration of RC A	0.45 ≤ x ≤ 0.55 mM	0.4969	pass
mean peptide concentration of RC C (PC)	0.45 ≤ x ≤ 0.55 mM	0.4827	pass
mean peptide concentration of RC C (TI)	0.45 ≤ x ≤ 0.55 mM	0.4584	pass
CV of the peak area of RC B	< 15 %	2.38	pass
CV of the peak area of RC C (PC)	< 15 %	0.85	pass
CV of the peak area of RC C (TI)	< 15 %	9.07	pass
mean peptide depletion of the PC	60.8 % < x < 100 %	72.27	pass
SD of peptide depletion of the PC replicates	< 14.9 %	0.90	pass
SD of peptide depletion of the TI replicates	< 14.9 %	0.00	pass

Table 11: Acceptance Criteria for Lysine Peptide

Lysine Peptide Run
Acceptance Criterion	Range	Value	pass/fail
coefficient of determination	R² > 0.99	0.9996	pass
mean peptide concentration of RC A	0.45 ≤ x ≤ 0.55 mM	0.5067	pass
mean peptide concentration of RC C (PC)	0.45 ≤ x ≤ 0.55 mM	0.5017	pass
mean peptide concentration of RC C (TI)	0.45 ≤ x ≤ 0.55 mM	0.4930	pass
CV of the peak area of RC B	< 15 %	0.54	pass
CV of the peak area of RC C (PC)	< 15 %	0.61	pass
CV of the peak area of RC C (TI)	< 15 %	0.54	pass
mean peptide depletion of the PC	40.2 % < x < 69.0 %	53.53	pass
SD of peptide depletion of the PC replicates	< 11.6 %	0.22	pass
SD of peptide depletion of the TI replicates	< 11.6 %	0.00	pass

Table 12: Historical Data Cysteine Peptide

Cysteine Peptide
	mean	SD	N
linearity of the calibration curve	0.9991	0.0006	8
mean peptide concentration of reference A [mM]	0.52	0.00	8
mean peptide concentration of reference C [mM]	0.50	0.00	10
CV of the peak area of control B [%]	2.10	0.34	8
CV of the peak area of control C [%]	1.60	0.85	10
mean peptide depletion of the PC [%]	74.67	2.32	8
SD of peptide depletion of the PC replicates [%]	0.84	0.72	8
SD of peptide depletion of the test items [%]	4.60	14.80	20

Table 13:   Historical Data Lysine Peptide

Lysine Peptide
	mean	SD	N
linearity of the calibration curve	0.9998	0.0001	7
mean peptide concentration of reference A [mM]	0.49	0.02	7
mean peptide concentration of reference C [mM]	0.49	0.24	9
CV of the peak area of control B [%]	1.26	0.24	7
CV of the peak area of control C [%]	0.81	0.89	9
mean peptide depletion of the PC [%]	59.54	6.09	7
SD of peptide depletion of the PC replicates [%]	2.58	1.90	7
SD of peptide depletion of the test items [%]	1.02	1.08	21

Table 14:   Exemplary Analysis Sequence

Run 1 Run 2 Run 3 Run 4 Run 5 Run 6 Run 7 Run 8 Run 9 Run 10 Run 11	STD1 STD2 STD3 STD4 STD5 STD6 SDT7 (DB) Reference Control A, replicate 1 Reference Control A, replicate 2 Reference Control A, replicate 3
Run 12 Run 13	Co-Elution Control Positive Control Co-Elution Test Item 1
Run 14 Run 15 Run 16	Reference Control B, replicate 1 Reference Control B, replicate 2 Reference Control B, replicate 3
Run 17 Run 18 Run 19	Reference Control C, replicate 1 Positive Control, replicate 1 Test Item 1, replicate 1
Run 20 Run 21 Run 22	Reference Control C, replicate 2 Positive Control, replicate 2 Test Item 1, replicate 2
Run 23 Run 24 Run 25	Reference Control C, replicate 3 Positive Control, replicate 3 Test Item 1, replicate 3
Run 26 Run 27 Run 28	Reference Control B, replicate 4 Reference Control B, replicate 5 Reference Control B, replicate 6

Table 15: Results of the Reference Controls for the Cysteine Peptide

Cysteine Peptide Run
Sample	Peptide Peak Area				Peptide Concentration [mM]
	PA	Mean	SD	CV [%]	Peptide Concentration	Mean	SD	CV [%]
Reference A 1	3295.67	3278.9512	26.0108	0.79	0.4994	0.4969	0.0040	0.80
Reference A 2	3292.20				0.4989
Reference A 3	3248.98				0.4923
Reference B 1	3211.87	3162.2388	74.5299	2.36	0.4866	0.4791	0.0114	2.38
Reference B 2	3253.24				0.4930
Reference B 3	3222.13				0.4882
Reference B 4	3097.92				0.4692
Reference B 5	3090.32				0.4681
Reference B 6	3097.95				0.4692
Reference C 1 (PC solvent)	3208.51	3186.06	26.9566	0.85	0.4861	0.4827	0.0041	0.85
Reference C 2 (PC solvent)	3193.52				0.4838
Reference C 3 (PC solvent)	3156.16				0.4781
Reference C 1 (TI solvent)	3205.63	3027.02	272.2805	8.99	0.4857	0.4584	0.0416	9.07
Reference C 2 (TI solvent)	2713.64				0.4105
Reference C 3 (TI solvent)	3161.80				0.4790

Table 16:   Results of the Reference Controls for the Lysine Peptide

Lysine Peptide Run
Sample	Peptide Peak Area				Peptide Concentration [mM]
	PA	Mean	SD	CV [%]	Peptide Concentration	Mean	SD	CV [%]
Reference A 1	3484.17	3498.5079	12.6544	0.36	0.5046	0.5067	0.0019	0.37
Reference A 2	3508.10				0.5081
Reference A 3	3503.26				0.5074
Reference B 1	3472.14	3482.0649	18.3933	0.53	0.5028	0.5042	0.0027	0.54
Reference B 2	3506.05				0.5078
Reference B 3	3497.27				0.5065
Reference B 4	3473.01				0.5029
Reference B 5	3487.77				0.5051
Reference B 6	3456.15				0.5004
Reference C 1 (PC solvent)	3445.89	3465.01	20.6789	0.60	0.4989	0.5017	0.0030	0.61
Reference C 2 (PC solvent)	3462.18				0.5013
Reference C 3 (PC solvent)	3486.96				0.5050
Reference C 1 (TI solvent)	3426.42	3405.85	18.0704	0.53	0.4961	0.4930	0.0027	0.54
Reference C 2 (TI solvent)	3392.54				0.4911
Reference C 3 (TI solvent)	3398.60				0.4920

 

Applicant's summary and conclusion

Interpretation of results:

other: Expert judgement: no indication of sensitisation

Conclusions:

In this study under the given conditions the test item showed minimal reactivity towards the peptides. The test item can be considered as “non-sensitizer”. The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and will be considered in the context of integrated approach.

Executive summary:

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential

of a test item by addressing the molecular initiating event of the adverse outcome pathway (AOP),

namely protein reactivity, by quantifying the reactivity of test chemicals towards synthetic peptides

containing either lysine or cysteine. The percentage depletion value of the cysteine and lysine

peptide is used to categorize a substance in one of four reactivity classes to support discrimination

between skin sensitiser and non-sensitisers.

In the present study, the test item was dissolved in water and 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 analysis.

After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples of the cysteine and lysine peptide run were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the test item samples, RC A, RC B, RC C, positive control and for STD 1 and 2 of the cysteine run. No centrifugation was necessary to perform the HPLC analysis. Slight phase separation was observed for the positive control and the respective co-elution control of the lysine run.

After the HPLC run samples of the cysteine peptide run were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the test item samples, RC A, RC B, RC C, positive control and for STD 1, 2 and 3 of the cysteine runs. Slight phase separation was observed for the positive control and the respective co-elution control of the lysine run.

Since the turbidity noted for the test item samples was also observed for reference controls, positive controls and standard solutions it can be considered that it is related to the peptide and that it is not a precipitation of the test substance. Additionally, the turbidity did not change during the HPLC analysis period. Since stability of the cysteine peptide in the used acetonitrile batch was demonstrated successfully, the reactivity of the positive control towards the cysteine peptide and peptide depletion were identified correctly and the validity of the cysteine run was acceptable the precipitation was considered as not relevant.

No co-elution of test item with the peptide peaks was observed. Sensitizing potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC C).

The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was ≤6.38 % (2.03 %). Based on the prediction model 1 the test item can be considered as non-sensitiser.

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 62.90 %.

The controls confirmed the validity of the study for both, the cysteine and lysine run. For the cysteine run the coefficient of determination for the calibration curve was >0.99 (0.9995). The mean peptide depletion of the cysteine peptide by the positive control was between 60.8 % and 100 % (72.27 %).

The mean peptide concentration of reference control A and reference control C (acetonitrile and water) was between >0.45 and <0.55 mM (RC A: 0.4969 mM, RC C (acetonitrile): 0.4827 mM, RC C (water): 0.4584 mM). The coefficient of variation (CV) of the peak area (PA) of reference control B and reference control C (acetonitrile and water) was <15 %. (RC B: 2.38 %, RC C (acetonitrile): 0.85 %, RC C (water): 9.07 %). The SD of the peptide depletion for the replicates of the positive control as well as for the tested test item samples was <14.9 % (PC: 0.90 %; test item 0.00 %).

For the lysine run the coefficient of determination for the calibration curve was >0.99 (0.9996). The mean peptide depletion of the lysine peptide by the positive control was between 40.2 % and 69.0 % (53.53 %). The mean peptide concentration of reference control A and reference control C (acetonitrile and water) was between >0.45 and < 0.55 mM (RC A 0.5067 mM, RC C (acetonitrile): 0.5017 mM, RC C water): 0.4930 mM). The coefficient of variation (CV) of the peak area (PA) of reference control B and reference control C (acetonitrile and water) was <15 %. (RC B: 0.54 %, RC C (acetonitrile: 0.61 %, RC C water): 0.54 %). The SD of the peptide depletion for the replicates of the positive control as well as for the tested test item samples was <11.6 % (PC: 0.22 %; test item: 0.10 %).

The test item can be considered as non-sensitiser. The data generated with this test will be considered in the context of an integrated approached such as IATA, combining this result with other complementary information.