Sodium propionate

Endpoint summary

Description of key information

The test item showed minimal reactivity towards peptides (OECD 442C) (reference 7.4.1 -1) and does not activate keratinocytes (OECD 442D) (reference 7.4.1 -2). An in silico assessment of the test item did not indicate a skin sensitisation potential (reference 7.4.1 -3). Furthermore, guinea pig maximisation test showed no skin sensitisation potential (reference 7.4.1 -4). Therefore the test item is considered not to be a skin sensitiser.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

27 April 2017 - 17 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

Skin sensitisation (In chemico test system)

Synthetic peptides used:
- cysteine peptide with an amino acid sequence of Ac-RFAACAA, JPT Peptide Technologies GmbH; > 95%; Lot. No.: 260515HS_DWW1115
- lysine peptide with an amino acid sequence of Ac-RFAAKAA, JPT Peptide Technologies GmbH; > 95%; Lot. No.: 220114HSD_WW0117

Controls used:
- Positive control: Cinnamic aldehyde 100 mM in acetonitrile
- Co-elution control: test item or positive control without cysteine or lysine peptide
- Reference controls: cysteine or lysine peptide in acetonitrile with and without test item

Test substance preparation:
- The test substance was prepared as a 100 mM stock solution in acetonitrile.

Peptide stock solution preparation:
- 19.33 mg cysteine peptide with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (37.535 mL) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM.
- 19.85 mg lysine peptide with an amino acid sequence of Ac-RFAAKAA were pre-weighed in a vial and dissolved in a defined volume of ammonium acetate buffer with pH 10.2 (37.083 mL) to reach a concentration of 0.667 mM.

Experimental procedure:
Samples of the test substance in acetonitrile were incubated with each peptide for 24 ± 2 h at 25 ± 2.5 °C in the dark. After the incubation period the test item was analysed in triplicate for both peptides by HPLC. Reference controls, co-elution controls and positive control were set up in parallel.
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.

Sample preparation:
The test item solutions were incubated with the cysteine and lysine peptide solutions in glass vials using defined ratios of peptide to test item (1:10 cysteine peptide, 1:50 lysine peptide).

HPLC conditions:
Peptide depletion was monitored by HPLC coupled with an UV detector at A = 220 nm using a reversed-phase HPLC column (Zorbax SB-C-18 2.1 mm x 100 mm x 3.5 micron) as preferred column. The entire system was equilibrated at 30 °C with 50% phase A and 50% phase B for at least 2 hours before running the analysis sequence. The HPLC analysis was performed using a flow rate of 0.35 mL/min and a linear gradient from 10% to 25% acetonitrile over 10 minutes, followed by a rapid increase to 90% acetonitrile. The column was re-equilibrated under initial conditions for 7 minutes between injections. Equal volumes of each standard, sample and control were injected.
HPLC analysis for the cysteine and lysine peptide was performed concurrently (if two HPLC systems were available) or on separate days. If analysis was conducted on separate days all test chemical solutions were freshly prepared for both assays on each day.
The analysis was timed to assure that the injection of the first sample started 22 to 26 hours after the test chemical was mixed with the peptide solution. The HPLC run sequence was set up in order to keep the HPLC analysis time less than 30 hours.

Calculation and data evaluation:
The concentration of the cysteine and lysine peptide was determined in each sample form absorbance at A = 220 nm, measuring the area of the appropriated peaks (peak area (PA)) and calculating the concentration of peptide using the linear calibration curves derived from the standard solutions.
PPD = (1- (Peptide Peak Area in the Replicate Injection / Mean Peptide Peak Area in Refrence Control C)) * 100

Acceptance criteria:
The run meets the acceptance criteria if:
- the standard calibration curve has a r2 > 0.99,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 60.8% and 100% for the cysteine peptide and the maximum standard deviation (SD) for the positive control replicates is < 14.9%,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 40.2% and 69.0% for the lysine peptide and the maximum SD for the positive control replicates is < 11.6%,
- the mean peptide concentration of the three reference controls A replicates is 0.50 ± 0.05 mM,
- the coefficient of variation (CV) of peptide peak areas for the six reference control B replicates and three reference control C replicates in acetonitrile is < 15.0%.
The results of the test item meet the acceptance criteria if:
- the maximum standard deviation (SD) for the test chemical replicates is < 14.9% for the cysteine percent depletion (PPD),
- the maximum standard deviation (SD) for the test chemical replicates is < 11.6% for the lysine percent depletion (PPD),
- the mean peptide concentration of the three reference controls C replicates in the appropriate solvent is 0.50 ± 0.05 mM.

Evaluation of results:
Sensitising potential of the test item is predicted from the mean cysteine and lysine PPD value. The test item is considered positive to be a skin sensitiser in accordance with UN GHS "Category 1", if the mean depletion of both peptides exceeds the threshold of the respective prediction model. Negative depletion is considered as "0" when calculating the mean. Sensitizing potential might not be predictable if the test item was incubated using a concentration differently from 100 mM.
By using the prediction model 1 (cysteine 1:10 / lysine 1:50 prediction model) the threshold of 6.38% average peptide depletion was used to support the discrimination between skin sensitisers and non-sensitisers.
By using the prediction model 2 (cysteine 1:10 prediction model) the threshold of 13.89% peptide depletion was used to support the discrimination between skin sensitisers and non-sensitisers.

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 63.25%.

Key result

Run / experiment:

other: cysteine run

Parameter:

other: mean peptide depletion [%]

Value:

3.63

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Run / experiment:

other: lysine run

Parameter:

other: mean peptide depletion [%]

Value:

0.63

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: No precipitation, turbidity or phase separation was observed for the samples of the test item.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

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	4780.4512	0.5340	4444.6675	0.5340
STD2	2343.2332	0.2670	2225.9248	0.2670
STD3	1092.6707	0.1335	1097.2479	0.1335
STD4	339.6955	0.0667	541.0726	0.0667
STD5	194.2570	0.0334	263.3441	0.0334
STD6	115.1963	0.0167	126.4265	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	1441.9841	0.1695	68.28	68.80	0.51	0.74
	1417.9863	0.1668	68.81
	1395.7390	0.1644	69.30
Test Item	4159.9043	0.4679	5.99	3.63	2.04	56.13
	4318.1738	0.4853	2.41
	4314.2832	0.4848	2.50

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	1756.5852	0.2118	57.37	57.71	0.46	0.80
	1749.9872	0.2110	57.53
	1720.8181	0.2075	58.24
Test Item	4113.1968	0.4941	0.75	0.63	0.13	20.02
	4118.4595	0.4947	0.63
	4123.6211	0.4953	0.50

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 ¹

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

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.

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	2.13	Minimal Reactivity	no sensitiser	3.63	Minimal Reactivity	no sensitiser
Positive Control	63.25	High Reactivity	sensitiser	68.80	Moderate Reactivity	sensitiser

Interpretation of results:

GHS criteria not met

Conclusions:

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

Executive summary:

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 the test substance was dissolved in water, based on the results of the pre-experiments.The test item was completely soluble and the resulting solution was used for further testing. Based on a molecular weight of 96.6 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.

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

Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed precipitation and turbidity in these samples was regarded as insignificant.

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

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% (2.13%). Based on the prediction model 1 (Cysteine 1:10/ Lysine 1:50 Prediction Model) 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 63.25%.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

08 May 2017 to 11 September July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155, July 1st, 2015

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

Skin sensitisation (In vitro test system)
Cell line used: KeratinoSensTM (Givaudan, Switzerland)

Technical material and conditions:
- Maintenance Medium: D-MEM (GlutaMAXTM) with 1.0 g/L D-glucose and Na-Pyruvate + 10 % FBS + 1 % Geneticin (final concentration: 500 µg/mL)
- Assay Medium: D-MEM (GlutaMAXTM) with 1.0 g/L D-glucose and Na-Pyruvate + 10 % FBS
- Test Item Exposure Medium: D-MEM with 1.0 g/L D-glucose and Na-Pyruvate + 1 % FBS
- Luciferace reagent: Luciferase Assay Substrate (Promega, Cat. No.: E1501)
- Assay Buffer: Luciferase Assay Buffer (Promega, Cat. No.: E1501)
- Lysisbuffer: Luciferase Cell Culture Lysis (Promega Cat. No.: E1531)
- MTT Solution: MTT stock solution: 5 mg/mL in DPBS
- SDS solution: 10% (w/v) SDS in dist. water
- DPBS solution: DPBS solution (without Ca2+/Mg2+)

Controls used:
- Vehicle control: DMSO: 1% (v/v) in test item exposure medium
- Positive control: Cinnamic aldehyde in DMSO, 4 µM, 8 µM, 16 µM, 32 µM, 64 µM
- Blank control: vehicle control without cells

Test procedure:
Each concentration step of the test item (12 concentrations, 0.98-2000 µM) and the positive control (5 concentrations, 4-64 µM) was assessed in three replicates in every independent run. The negative control was assessed using six replicates per plate in every independent run. The test substance was incubated with a luciferase reporter cell line (LuSens cells) for ca. 48 h at 37 °C and antioxidant response element (ARE) dependent luciferase activity was measured in a plate reader. Cell viability was determined by a MTT assay. The test substance was incubated for 4 h at 37 °C ± 1 °C and 5% CO2. Afterwards the medium was removed and replaced by 10% SDS solution the plate was incubated at 37 °C ± 1 °C and 5% CO2 overnight. The OD was measured at a wavelength of 600 nm.

Data analysis:
For each test item two independent repetitions using separately prepared test item solutions and independently harvested cells are necessary to derive a prediction. Each independent run consists of three replicates for every concentration step of the test item and the positive control. In case of discordant results a third independent run should be performed.

Acceptance criteria:
- The luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations.
- The average induction in the three technical replicates for the positive control at a concentration of 64 µM is between 2 and 8.
- The EC1.5 value of the positive control is within two standard deviations of the historical mean.
- The average coefficient of variation (CV; consisting of 6 wells) of the luminescence reading for the negative (solvent) control DMSO is <20% in each repetition.

Evaluation of results:
The test item is considered positive if the following conditions were met in at least two independently prepared test repetitions:
- Imax is >1.5 fold increased and statistically significant (p <0.05) compared to the negative control
- cell viability is >70% at the lowest concentration with an induction of luciferase activity >1.5
- EC1.5 value is <1000 µM
- an apparent overall dose-response for luciferase induction
If in a given repetition, all of the three first conditions are met but a clear dose-response for the luciferase induction cannot be observed, the result of that repetition is considered as inconclusive and further testing may be required. In addition, a negative result obtained with concentrations <1000 µM is considered as inconclusive.

Positive control results:

The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (5.70 (experiment 1) and 6.31 (experiment 2)). Therefore, the positive control fullfilled the validity criteria of the test.

Key result

Run / experiment:

other: 1

Parameter:

other: Imax

Value:

1.43

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Run / experiment:

other: 1

Parameter:

other: EC1.5 [µM]

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

not determinable

Key result

Run / experiment:

other: 2

Parameter:

other: Imax

Value:

0.96

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Run / experiment:

other: 2

Parameter:

other: EC1.5 [µM]

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

not determinable

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for solvent control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Table1: Results of the Cytotoxicity Measurement

	Concentration [µM]	Cell Viability [%]
		Experiment 1	Experiment 2	Mean	SD
Solvent Control	-	100.0	100.0	100.0	0.0
Positive Control	4.00	102.4	94.3	98.3	5.7
	8.00	105.5	102.2	103.8	2.3
	16.00	112.2	102.4	107.3	6.9
	32.00	106.2	105.8	106.0	0.3
	64.00	121.1	106.7	113.9	10.2
Test Item	0.98	94.9	101.3	98.1	4.6
	1.95	106.5	101.6	104.0	3.4
	3.91	101.9	104.7	103.3	2.0
	7.81	111.5	97.0	104.2	10.3
	15.63	107.6	96.9	102.3	7.6
	31.25	112.4	103.8	108.1	6.1
	62.50	100.4	109.8	105.1	6.6
	125.00	106.8	108.1	107.5	0.9
	250.00	112.4	105.4	108.9	4.9
	500.00	102.0	102.9	102.5	0.6
	1000.00	119.9	107.9	113.9	8.4
	2000.00	116.0	116.2	116.1	0.1

Table 2: Induction of Luciferase Activity Experiment 1

Experiment 1	Concentration [µM]	Fold Induction					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.17	1.33	1.21	1.24	0.08
	8.00	1.35	1.34	1.08	1.25	0.15
	16.00	1.46	1.55	1.27	1.43	0.14
	32.00	2.11	1.98	1.64	1.91	0.24	*
	64.00	6.96	5.89	4.26	5.70	1.36	*
Test Item	0.98	1.11	0.99	1.01	1.03	0.07
	1.95	1.55	1.43	1.32	1.43	0.11
	3.91	0.98	1.04	0.91	0.98	0.06
	7.81	1.03	1.09	0.94	1.02	0.08
	15.63	1.08	1.00	0.91	1.00	0.08
	31.25	1.07	1.09	0.88	1.01	0.12
	62.50	1.31	0.96	0.88	1.05	0.23
	125.00	1.02	1.08	0.92	1.01	0.08
	250.00	0.88	1.00	0.79	0.89	0.10
	500.00	0.75	1.01	0.70	0.82	0.16
	1000.00	0.77	0.88	0.62	0.76	0.13
	2000.00	0.82	0.90	0.66	0.79	0.12

* = significant induction according to Student’s t-test, p<0.05

Table 3: Induction of Luciferase Activity Experiment 2

Experiment 2	Concentration [µM]	Fold Induction					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.19	1.24	1.06	1.16	0.09
	8.00	1.16	1.12	1.32	1.20	0.11
	16.00	1.37	1.55	1.59	1.50	0.12	*
	32.00	2.02	2.27	2.16	2.15	0.13	*
	64.00	6.51	5.74	6.68	6.31	0.50	*
Test Item	0.98	0.93	0.80	1.11	0.95	0.16
	1.95	0.85	0.82	0.96	0.87	0.07
	3.91	0.95	1.05	0.88	0.96	0.09
	7.81	0.91	0.83	0.85	0.86	0.04
	15.63	0.68	0.72	0.71	0.70	0.02
	31.25	0.90	0.93	0.89	0.91	0.02
	62.50	1.06	0.93	0.76	0.92	0.15
	125.00	0.93	1.00	0.96	0.96	0.03
	250.00	0.76	0.82	0.79	0.79	0.03
	500.00	0.73	0.79	0.78	0.77	0.03
	1000.00	0.76	0.68	0.59	0.68	0.08
	2000.00	1.02	0.93	0.89	0.95	0.06

* = significant induction according to Student’s t-test, p<0.05

Table 4: Induction of Luciferase Activity – Overall Induction

	Concentration [µM]	Fold Induction				Significance
		Experiment 1	Experiment 2	Mean	SD
Solvent Control	-	1.00	1.00	1.00	0.00
Positive Control	4.00	1.24	1.16	1.20	0.05
	8.00	1.25	1.20	1.23	0.04
	16.00	1.43	1.50	1.47	0.05
	32.00	1.91	2.15	2.03	0.17	*
	64.00	5.70	6.31	6.01	0.43	*
Test Item	0.98	1.03	0.95	0.99	0.06
	1.95	1.43	0.87	1.15	0.39
	3.91	0.98	0.96	0.97	0.01
	7.81	1.02	0.86	0.94	0.11
	15.63	1.00	0.70	0.85	0.21
	31.25	1.01	0.91	0.96	0.07
	62.50	1.05	0.92	0.98	0.09
	125.00	1.01	0.96	0.98	0.03
	250.00	0.89	0.79	0.84	0.07
	500.00	0.82	0.77	0.79	0.04
	1000.00	0.76	0.68	0.72	0.06
	2000.00	0.79	0.95	0.87	0.11

* = significant induction according to Student’s t-test, p<0.05

 

Table 5: Additional Parameters

Parameter	Experiment 1	Experiment 2	Mean	SD
EC1.5[µM]	n.a.	n.a.	-	-
Imax	1.43	0.96	1.20	0.33
IC30[µM]	n.a.	n.a.	-	-
IC50[µM]	n.a.	n.a.	-	-

n.a. = not applicable

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered as non sensitiser.
The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Executive summary:

In the present study the test item was dissolved in distilled water. Based on a molecular weight of 96.06 g/mol a stock solution of 200 mM was prepared. Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium. The following concentration range was tested in the assay: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM. Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.

In the first experiment, no significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC_1.5 value could be calculated. In the second experiment, no significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC_1.5 value could be calculated. No 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. The controls confirmed the validity of the study.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

Please refer to the QMRF and QPRF files provided under the section attached justification.

Qualifier:

no guideline available

Principles of method if other than guideline:

Estimates the skin sensitising properties of chemicals using structural alert relationships.

GLP compliance:

no

Specific details on test material used for the study:

[Na+].CCC(=O)[O-]

Key result

Parameter:

other: alerts

Value:

0

Remarks on result:

no indication of skin sensitisation

Remarks:

QSAR predicted value. The substance is within the applicability domain of the model.

Interpretation of results:

other: Derek result: no alerts matched.

Conclusions:

Using Derek Nexus v5.0, no skin sensitising properties of the test item were estimated. The substance is within the applicability domain of the model. Thus the estimation can be regarded as accurate.

Executive summary:

The skin sensitising properties were estimated using Derek Nexus v5.0. No skin sensitising properties were estimated based on the described QSAR method (Derek, 2017).

The adequacy of a prediction depends on the following conditions:

a) the (Q)SAR model is scientifically valid: the scientific validity is established according to the OECD principles for (Q)SAR validation;

b) the (Q)SAR model is applicable to thequery chemical: a (Q)SAR is applicable if the query chemical falls within the defined applicability domain of the model;

c) the (Q)SAR result is reliable: a valid (Q)SAR that is applied to a chemical falling within its applicability domain provides a reliable result;

d) the (Q)SAR model is relevant for theregulatory purpose.

For assessment and justification of these 4 requirements the QMRF and QPRF files were developed and attached to this study record.

 

Description of the prediction Model

The prediction model was descripted using the harmonised template for summarising and reporting key information on (Q)SAR models. For more details please refer to the attached QSAR Model Reporting Format (QMRF) file. 

 

Assessment of estimation domain

The assessment of the estimation domain was documented in the QSAR Prediction Reporting Format file (QPRF). Please refer to the attached document for the details of the prediction and the assessment of the estimation domain.

Reference 4

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

other: summary

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Remarks:

primary source unclear (stated: REACH), limited documentation

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

GLP compliance:

not specified

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The endpoint was addresed by the current data requirements stated in Annex VII, Section 8.3.1 of REACH (in vitro/in chemico battery). According to this, the LLNA is not the standard method anymore.In addition, QSAR calculation was performed. Data on a GPMT are public available and are therefore additonally provided in the dossier.

Species:

guinea pig

Details on test animals and environmental conditions:

TEST ANIMALS
- Microbiological status of animals: albino specific pathogen-free guinea pigs

Route:

intradermal and epicutaneous

Vehicle:

other: intradermal: water, epicutaneous: vaseline

Concentration / amount:

intradermal: 0.1 mL of the chemical at 2 % concentration in distilled water
epicutaneous: 20 % of the chemical in vaseline

Day(s)/duration:

epicutaneaous application: one week after intradermal application, exposure duration 48 hours

Adequacy of induction:

not specified

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

not specified

Concentration / amount:

20 % of the chemical

Day(s)/duration:

24 hours

Adequacy of challenge:

not specified

Key result

Reading:

other: not specified

Group:

test chemical

Remarks on result:

no indication of skin sensitisation

Interpretation of results:

GHS criteria not met

Conclusions:

In a guinea pig maximisation test according to OECD TG 406 the test chemical did not show a skin sensitisation potential.

Executive summary:

The skin sensitisation potential of the test chemical was investigated in a guinea pig maximisation test according to OECD TG 406. Sensitisation was induced by intradermal injections (two 0.1 mL of Freund's complete adjuvant, two of 0.1 mL of the chemical at 2 % concentration in distilled water, and two of a 1:1 mixture of the chemical and adjuvant) on the shoulder of albino specific pathogen-free guinea pigs. One week after the injection, 20 % of the chemical in vaseline was applied occlusively for 48 hours to the same area. Two weeks later the animals were challenged by an occlusive dermal application of 20 % of the chemical for 24 hours. The test chemical did not cause positive responses at challenge.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

For the evaluation of the skin sensitisation potential of the test substance a Weight of Evidence approach was used. The protein reactivity of the test item was assessed in a Direct Peptide Reactivity Assay (DPRA). A Keratinocyte Activation Assay (LuSens) was performed to evaluate the keratinocyte activation potential. Furthermore, an in silico assessment using Derek Nexus v5.0 was carried out and a guinea pig maximisation test was present.

 

DPRA (reference 7.4.1 -1)

An in chemico direct peptide reactivity assay (DPRA) according to OECD 442C was performed (reference 7.4.1 -1). In the study the test substance was dissolved in water, based on the results of the pre-experiments.The test item was completely soluble and the resulting solution was used for further testing. Based on a molecular weight of 96.6 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. After the 24 ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation.For the 100 mM solution of the test item no turbidity or precipitation was observed when diluted with the cysteine or lysine peptide solution. Precipitation was observed for the samples of the positive control. Samples were not centrifuged prior to the HPLC analysis. Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed precipitation and turbidity in these samples was regarded as insignificant. No co-elution of test item with the peptide peaks was observed. Sensitising 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. 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% (2.13%). Based on the prediction model 1 (Cysteine 1:10/ Lysine 1:50 Prediction Model) 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 63.25%.

LuSens (reference 7.4.1-2)

The keratinocyte activating potential of test substance was evaluated in a LuSens Assay according to OECD 442D (reference 7.4.1 -2). The test item was dissolved in distilled water. Based on a molecular weight of 96.06 g/mol a stock solution of 200 mM was prepared. Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium. The following concentration range was tested in the assay: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM. Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.

In the first experiment, no significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated. In the second experiment, no significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated. No 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. The controls confirmed the validity of the study. 

In silico assessment(reference 7.4.1 -3)

For in silico assessment, Derek Nexus v5.0 was used (reference 7.4.1 -3). No skin sensitising properties of the test item were estimated. The substance is within the applicability domain of the model. Thus the estimation can be regarded as accurate.

GPMT (reference 7.4.1-4)

The skin sensitisation potential of the test chemical was investigated in a guinea pig maximisation test according to OECD TG 406 (reference 7.4.1 -4). Sensitisation was induced by intradermal injections (two 0.1 mL of Freund's complete adjuvant, two of 0.1 mL of the chemical at 2 % concentration in distilled water, and two of a 1:1 mixture of the chemical and adjuvant) on the shoulder of albino specific pathogen-free guinea pigs. One week after the injection, 20 % of the chemical in vaseline was applied occlusively for 48 hours to the same area. Two weeks later the animals were challenged by an occlusive dermal application of 20 % of the chemical for 24 hours. The test chemical did not cause positive responses at challenge.

Conclusion

The test item showed minimal reactivity towards the peptides (DPRA, OECD 442C) (reference 7.4.1 -1) and does not activate keratinocytes (LuSens, OECD 442D) (reference 7.4.1 -2). In addition, an in silico assessment (reference 7.4.1 -3) and guinea pig maximisation test (reference 7.4.1 -4) also showed no indication of skin sensitization. Therefore, the test item is considered not to have a skin sensitising potential (UN GHS: no category).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. Based on this data, the substance is not considered to be classified for skin sensitisation under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EC) No 2017/776.