complex mixture from biological origin

Endpoint summary

Description of key information

No substance-specific data on skin sensitization potential is available. Data from a suitable read-across partner was used to assess the skin sensitization potential. For details and justification of read-across please refer to the report attached in section 13 of IUCLID.

The potential of the substance to induce skin sensitisation was evaluated in two in vitro tests conducted according or similar to OECD 442D and OECD 442E and in an in chemico test according to OECD 442C. Based on the results (2 of 3 tests are negative), the substance can be considered as a non-sensitiser. The result is supported by an in vivo study (GPMT) conducted with a structural related compound.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

For details and justification of read-across please refer to the report attached in section 13 of IUCLID.

Reason / purpose for cross-reference:

read-across source

Positive control results:

The positive control (DCNB) led to an upregulation of CD54 and CD86 in both experiments. The threshold of 150 % for CD86 (252 % in experiment 1 and 351 % in experiment 2) and 200 % for CD54 (431 % in experiment 1 and 772 % in experiment 2) were clearly exceeded.

Key result

Group:

test chemical

Run / experiment:

other: 174.93 µg/mL (1)

Parameter:

RFI CD54>150 [442E]

Value:

348 %

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

Corresponding CV was 90.1 %

Key result

Group:

test chemical

Run / experiment:

other: 174.93 µg/mL (2)

Parameter:

RFI CD54>150 [442E]

Value:

446 %

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

Corresponding CV was 84.1 %

Key result

Group:

test chemical

Run / experiment:

other: both experiments

Parameter:

RFI CD86>200 [442E]

Value:

150 %

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

no dose response observed.

Group:

test chemical

Run / experiment:

other: 209.91 µg/mL (1)

Parameter:

RFI CD54>150 [442E]

Value:

1 476 %

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: Corresponding CV was 52.5 %

Remarks:

cytotoxic effects observed

Group:

test chemical

Run / experiment:

other: 209.91 µg/mL (2)

Parameter:

RFI CD54>150 [442E]

Value:

2 198 %

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: Corresponding CV was 48.0 %

Remarks:

cytotoxic effects observed

Group:

test chemical

Run / experiment:

other: mean of two experiments (dose range finding]

Parameter:

CV75 [442D and 442E]

Value:

174.92 µg/mL

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

Lactic acid

Positive controls validity:

valid

Remarks on result:

other: cytotoxicity was observed

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 section “any other details on results incl. tables”.

Reactivity Check of the Cell Stock

Doubling time of the cells was monitored and found to be 40.07 h which is within the doubling time range specified by the manufacturer (35 - 50 h).

Table 1: Results of the Cell Batch Activation Test

Sample	Concentration [µg/mL]	CD86		CD54		Activated
		Cell Viability [%]	RFI	Cell Viability [%]	RFI	yes/no
DNCB	4 µg/mL	85.1	298	86.3	385	Yes
NiSO4	100 µg/mL	90.5	219	91.2	260	Yes
LA	1000 µg/mL	98.2	68	98.1	65	No

Solvent finding

All test item solutions were freshly prepared immediately prior to use. The test item was soluble in 0.9 % NaCl solution (Braun, Lot No.: 140696, 140698, 152418002) at a concentration of 100 mg/mL.

Dose finding assay

The dose finding assay was performed using stock solutions with a concentration of 100 mg/mL.

Table 2: Results of the Dose Finding Assay

	Experiment 1		Experiment 2
Sample	Concentration applied [µg/ml]	Cell Viability [%]	Concentration applied [µg/ml]	Cell Viability [%]
Medium Control (= solvent control)	0.00	98.20	0.00	97.30
Test item	7.81	98.20	7.81	96.50
	15.63	97.40	15.63	96.60
	31.25	97.20	31.25	96.90
	62.50	97.10	62.50	96.80
	125.00	96.50	125.00	95.60
	250.00	62.00	250.00	33.50
	500.00	0.60	500.00	0.80
	1000.00	11.00	1000.00	7.20
Calculated CV75 [µg/mL]	192.53		157.31
Mean CV75 [µg/mL]	174.92
SD CV75 [µg/mL]	24.90

The mean CV75 was derived from two single runs and was found to be 174.92±24.9µg/mL. Based on the mean CV75, the main experiment was performed covering a concentration range from 209.91 – 58.58µg/mL (20.99 – 5.86 mg/mL stock solution).

Results CD54 and CD86 Expression

For determination of the cell surface markers CD54 and CD86 two independent experiments were performed using separate cultivated cells at passage 18 (first experiment) and passage 19 (second experiment). For each experiment separately weighted samples and preparations were used.

Table 3: CD54 and CD86 Expression Experiment 1

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Flourescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control	-	97.8	97.8	97.7	1817	809	546	1271	263	100	100	333	148
DMSO Control	0.20 %	97.6	97.9	97.8	2041	829	484	1557	345	123	131	422	171
DNCB	4.00	84.7	84.6	83.4	4555	2114	627	3928	1487	252	431	726	337
Test item	209.91	52.3	52.5	53.1	1803	4546	665	1138	3881	90	1476	271	684
	174.93	90.4	90.1	92.3	1856	1462	548	1308	914	103	348	339	267
	145.77	95.0	95.0	94.9	1616	948	523	1093	425	86	162	309	181
	121.48	96.6	96.7	96.8	1534	833	526	1008	307	79	117	292	158
	101.23	96.8	96.1	96.8	1370	763	514	856	249	67	95	267	148
	84.36	97.4	97.3	97.4	1400	777	521	879	256	69	97	269	149
	70.30	97.3	97.2	96.9	1429	820	525	904	295	71	112	272	156
	58.58	97.7	97.2	97.5	1444	795	537	907	258	71	98	269	148

Table 4: CD54 and CD86 Expression Experiment 2

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Flourescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	C86	CD54
Medium Control	-	98.1	98.1	98.4	1117	617	498	619	119	100	100	224	124
DMSO Control	0.20 %	98.1	98.3	98.3	1190	633	483	707	150	114	126	246	131
DNCB	4.0	73.4	72.3	72.9	3097	1770	612	2485	1158	351	772	506	289
Test item	209.91	47.9	48.0	46.2	1183	3316	700	483	2616	78	2198	169	474
	174.93	84.1	84.1	83.9	1140	1064	533	607	531	98	446	214	200
	145.77	94.1	94.3	94.4	1013	689	478	535	211	86	177	212	144
	121.48	95.4	95.4	96.0	875	634	476	399	158	64	133	184	133
	101.23	97.6	97.2	97.2	960	617	491	469	126	76	106	196	126
	84.36	97.4	97.4	97.8	930	620	484	446	135	72	114	192	128
	70.30	97.7	97.9	97.8	932	615	489	443	126	72	106	191	126
	58.58	98.0	98.0	97.9	953	622	487	466	135	75	113	196	128

Table 5: Acceptance criteria

Acceptance criterion	range	Experiment 1			pass/fail	Experiment 2			pass/fail
cell viability solvent controls [%]	>90	97.6 – 97.9			pass		98.1 – 98.4		pass
number of test dosed with viability >50% CD86	≥4	8			pass	7			pass
number of test dosed with viability >50% CD54	≥4	8			pass	7			pass
number of test dosed with viability >50% IgG1	≥4	8			pass	7			pass
RFI of positive control of CD86	≥150	252			pass	351			pass
RFI of positive control of CD54	≥200	431			pass	772			pass
RFI of solvent control of CD86	<150	123			pass	114			pass
RFI of solvent control of CD54	<200	131			pass	126			pass
MFI ratio IgG1/CD86 for medium control [%]	>105	333			pass	224			pass
MFI ratio IgG1/CD86 for solvent control [%]	>105	422			pass	246			pass
MFI ratio IgG1/CD54 for medium control [%]	>105	148			pass	124			pass
MFI ratio IgG1/CD54 for solvent control [%]	>105	171			pass				pass

Table 6: Historical data

Criterion	mean	SD	N
cell viability solvent controls [%]	94.7	3.2	70
number of test doses with viability >50 %	-	-	242
RFI of positive control of CD86	292.8	136.2	11
RFI of positive control of CD54	386.0	75.4	11
RFI of solvent control of CD86	109.3	16.2	10
RFI of solvent control of CD54	121.8	15.2	10
MFI ratio IgG1/CD86 for medium control [%]	224.0	77.9	12
MFI ratio IgG1/CD86 for DMSO control [%]	146.3	12.9	12
MFI ratio IgG1/CD54 for medium control [%]	242.5	89.6	12
MFI ratio IgG1/CD54 for DMSO control [%]	156.4	14.2	12

 

 

Interpretation of results:

study cannot be used for classification

Conclusions:

The test item did upregulate one cell surface marker in at least two independent experiments, which is an indication of skin sensitising properties. The data generated with this method is not sufficient to conclude on the skin sensitisation potential of chemicals and will be considered in the context of integrated approach.

Executive summary:

In an in vitro study according to the OECD Draft Proposal for a new test guideline, "in vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)", the test item was dissolved in 0.9 % NaCl. A CV75 of 174.92 ± 24.9 µg/mL was derived in a dose range finding assay. Based on the CV75, the main experiment was performed covering the following concentration steps: 209.91, 174.93, 145.77, 121.48, 101.23, 84.36, 70.30 and 58.58 µg/mL.

Cells were incubated with the test item for 24 h at 37 °C. After exposure, cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.

The relative fluorescence intensity (RFI) of CD86 and/or CD54 was equal or greater than 150% and 200%, respectively, in at least one concentration with a related cell viability of greater than 50% in both independent runs. In conclusion, the test item activated THP-1 cells under the test conditions of this study. Therefore, the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

This information is used in a read-across approach in the assessment of the target substance. For justification of read-across please refer to the attached read-across report (see IUCLID section 13).

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

For details and justification of read-across please refer to the report attached in section 13 of IUCLID.

Reason / purpose for cross-reference:

read-across source

Positive control results:

The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (2.98 (experiment 1), 5.07 (experiment 2); 4.36 (experiment 3)). The calculated EC 1.5 was between 7 and 30 µM (21.66 µM (experiment 1), 14.38 µM (experiment 2); 15.30 µM (experiment 3)). The positive control result is therefore considered valid.

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

Imax [442D]

Value:

6.79

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: Induction of luciferase was observed only at cytotoxic test item concentrations (viability < 70 %) of 125 µM

Group:

test chemical

Run / experiment:

mean

Parameter:

EC 1.5 [442D]

Value:

70.07 µM

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Group:

test chemical

Run / experiment:

mean

Parameter:

IC30 [442D]

Value:

32.27 µM

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Group:

test chemical

Run / experiment:

mean

Parameter:

IC50 [442D]

Value:

65.12 µM

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

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 Table 7 in section “any other details on results incl. tables”.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item did not induce luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiments at non-cytotoxic concentrations. The data generated with this method is 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:

In an in vitro skin sensitisation assay conducted according to OECD 442D, transgenic keratinocytes constitutively expressing an ARE-reporter gene were incubated with Sophorolipids, fermentation products of glucose and fatty acids (dry matter: 44.1% w/w) dissolved in DMSO.Cells were incubated with the test item for 48 h at 37 °C and later checked for luciferase activity. Sensitisation was scored by measuring maximum luciferase activity induction (Imax), cell viability and EC1.5.

In the first experiment, a max luciferase activity (Imax) induction of 7.96 was determined at a test item concentration of 125.00 μM. The corresponding cell viability was 21.8% indicating severe cytotoxicity. The lowest tested concentration with a significant luciferase induction >1.5 was the identical to that of Imax. The calculated EC1.5 was < 1000 μM (67.67 μM).

In the second experiment, a max luciferase activity (Imax) induction of 4.63 was determined at a test item concentration of 125.00 μM. The corresponding cell viability was 24.0% indicating severe cytotoxicity. The lowest tested concentration with a significant luciferase induction >1.5 was the identical to that of Imax. The calculated EC1.5 was < 1000 μM (73.70 μM).

In the third experiment, a max luciferase activity (Imax) induction of 7.78 was determined at a test item concentration of 125.00 μM. The corresponding cell viability was 18.0%. The lowest tested concentration with a significant luciferase induction >1.5 was the identical to that of Imax. The calculated EC1.5 was < 1000 μM (68.85 μM).

Since the induction of the luciferase was observed only at cytotoxic test item concentrations (viability < 70%) the effect cannot be considered for sensitization evaluation. Additionally, no clear dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.

Under the condition of this study the test item is therefore considered as non sensitiser at non-cytotoxic concentrations.

This information is used in a read-across approach in the assessment of the target substance. For justification of read-across please refer to the attached read-across report (see IUCLID section 13).

Reference 3

Endpoint:

skin sensitisation: in chemico

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

For details and justification of read-across please refer to the report attached in section 13 of IUCLID.

Reason / purpose for cross-reference:

read-across source

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 %.

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

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

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”.

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

 

Interpretation of results:

GHS criteria not met

Conclusions:

The test item showed minimal reactivity towards peptides in a direct peptide reactivity assay. The data generated with this method is 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:

In an in chemico direct peptide reactivity assay (DPRA) for skin sensitisation (OECD TG 442C), 100 mM of the test item (total dry matter: 44.1% w/w) were incubated with the cysteine and lysine peptide solutions at ratios of peptide to test item of 1:10 (cysteine peptide Ac-RFAACAA) and 1:50 (lysine peptide Ac-RFAAKAA). The reaction solutions were incubated in the dark at 25 ± 2.5 °C for 24 ± 2 h before running the HPLC analysis.

After incubation and prior to the HPLC analysis, precipitation was observed for the test item samples, all reference controls, positive control and for the two highest concentrations used for the standard curve of the cysteine run. Slight phase separation was observed for the positive control and the respective co-elution control of the lysine run.

After the HPLC run 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 run. 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%.

In this study test item can be considered as “non-sensitizer”.

This information is used in a read-across approach in the assessment of the target substance. For justification of read-across please refer to the attached read-across report (see IUCLID section 13).

Reference 4

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

The substance described by Ikeda et al. (1986), polyoxypropylene (12) [2'-0-beta-D-glucopyranosyl-beta-D-glucopyranosyl) oxy-] fatty acid ester-], is a glycolipide derivative and an acetylated form of the sophorolipids. The underlying generic structure of the main component of the target substance is identical to the source substance. Therefore, it is expected that these substances have similar physicochemical properties, and thus, there should be no differences in the absorption into the body and in the toxicity profile between source and target substance.

Reason / purpose for cross-reference:

read-across source

Species:

guinea pig

Strain:

Hartley

Reading:

other: open challenge

Hours after challenge:

24

Group:

test chemical

Dose level:

10 and 50%

No. with + reactions:

0

Total no. in group:

15

Remarks on result:

no indication of skin sensitisation

Reading:

other: open challenge

Hours after challenge:

48

Group:

test chemical

Dose level:

10 and 50%

No. with + reactions:

0

Total no. in group:

15

Remarks on result:

no indication of skin sensitisation

Reading:

other: open challenge

Hours after challenge:

72

Group:

test chemical

Dose level:

10 and 50%

No. with + reactions:

0

Total no. in group:

15

Remarks on result:

no indication of skin sensitisation

Reading:

other: occlusive (24 hour) challenge

Hours after challenge:

24

Group:

test chemical

Dose level:

50%

No. with + reactions:

0

Total no. in group:

15

Remarks on result:

no indication of skin sensitisation

Reading:

other: occlusive (24 hour) challenge

Hours after challenge:

48

Group:

test chemical

Dose level:

50%

No. with + reactions:

0

Total no. in group:

15

Remarks on result:

no indication of skin sensitisation

Reading:

other: occlusive (24 hour) challenge

Hours after challenge:

72

Group:

test chemical

Dose level:

50%

No. with + reactions:

0

Total no. in group:

15

Remarks on result:

no indication of skin sensitisation

No sensitising reaction was observed in a GPMT in either the open nor the 24-hour closed application for Sophorolipid (C16-C18).

Interpretation of results:

study cannot be used for classification

Conclusions:

No sensitising reaction was observed in a GPMT in either the open or the 24-hour closed application of the test material for Sophorolipid (C16-C18).

Executive summary:

In a GPMT according to Magnusson and Kligman (1969), Sophorolipid (C16-18) did not show a sensitising reaction in either the open nor the 24-hour closed application.

This information is used in a read-across approach in the assessment of the target substance.

The substance described by Ikeda et al. (1986), polyoxypropylene (12) [2'-0-beta-D-glucopyranosyl-beta-D-glucopyranosyl) oxy-] fatty acid ester-], is a glycolipide derivative and an acetylated form of the sophorolipids. The underlying generic structure of the main component of the target substance is identical to the source substance. Therefore, it is expected that these substances have similar physicochemical properties, and thus, there should be no significant differences in the absorption into the body and in the toxicity profile between source and target substance.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available data, classification for skin sensitization is not warranted according to CLP Regulation 1272/2008 and ECHAs 2o3 guideline for defined approaches for skin sensitization.