Reaction mass of butanoic acid, 2-ethyl-2,3,3-trimethyl-, ethyl ester and butanoic acid, 2,3-dimethyl-2-(1-methylethyl)-, ethyl ester and pentanoic acid, 2,2,4,4-tetramethyl-, ethyl ester and ethyl 2,2,3,3-tetramethylpentanoate and ethyl 2,2,3,4-tetramethylpentanoate

Endpoint summary

Description of key information

In an in vitro skin sensitization test strategy, the test substance was not peptide reactive and did not activate keratinocytes in a Direct Peptide Reaction Assay (OECD guideline 442C, DPRA) and the ARE-Nrf2 luciferase test method (OECD guideline 442D) and was therefore predicted not to be a skin sensitizer.

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

18 June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

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

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

TEST SYSTEM
Cysteine- (C-) containing peptide: Ac-RFAACAA-COOH (MW=751.9 g/mol)
Lysine- (K-) containing peptide: Ac-RFAAKAA-COOH (MW=776.2 g/mol)

MATERIAL AND TECHNICAL EQUIPMENT
- See table 2

CONTROLS
- Negative control (=vehicle control): Acetonitrile
- Positive control: Ethylene glycol dimethacrylate prepared as a 50 mM solution in acetonitrile
- Co-elution control: Sample prepared of the respective peptide buffer and the test substance but without peptide

TEST SUBSTANCE PREPARATION:
The test substance was prepared as a 100 mM preparation in acetonitrile. After stirring the test substance was soluble in the vehicle.

EXPERIMENTAL PROCEDURE
- Replicates: 3
- Solubility: A suitable non-reactive, water-miscible solvent which dissolves the test substance completely should be used. The preferred solvent was acetonitrile. When not soluble in acetonitrile solutions in water, isopropanol, acetone, propanol, methanol or mixtures of these solvents were tried.

- Preparation of peptide stock solutions: Concentration of 0.667 mM were prepared in pH 7.5 phosphate buffer (C-containing peptide) or pH 10.2 ammonium acetate buffer (K-containing peptide). The peptide stock solution was used for preparing the calibration samples and the test substance and control samples.

- Preparation of calibration samples: 6 calibration samples were prepared from the peptide stock solutions in 20% acetonitrile in the respective buffer (= dilution buffer) using serial dilution: 0.534 mM, 0.267 mM, 0.134 mM, 0.067 mM, 0.033 mM and 0.017 mM peptide + dilution buffer (0 mM peptide).

- Preparation of the test-substance samples: The samples were prepared in triplicates for each peptide according to the pipetting scheme given in tabe 1. The samples were prepared in suitable tubes, capped tightly and incubated at 25°C ± 2.5°C in the dark for 24 ± 2 hours. Visual inspection for solubility was performed directly after sample preparation and prior to HPLC analysis. Unsolved samples were centrifuged and/or filtrated prior to injection into the HPLC in order to remove any unsolved particles. The HLPC analysis of the batch of samples started about 24 hours after sample preparation and the analysis time itself did not exceed 30 hours.

- Preparation of the vehicle controls: As described above but with the vehicle (acetonitrile) instead of the test substance:
Set A: Analyzed together with the calibration samples without incubation, serves as a performance control.
Set B (x2): Incubated with the samples. Sets B were placed at the very start and ending of the sample list and serves as stability control of the peptide over the analysis time.
Set C: Analyzed with the samples and serves for calculation of the peptide depletion of any chemical formulated in the vehicle.

- Preparation of the co-elution control: As described above but without the peptides. Instead the respective peptide buffer was used. The samples were analyzed together with the calibration samples. Samples which were visually turbid or display precipitates were centrifuged and/or filtrated prior to injection into the HPLC in order to remove any unsolved particles.

- Measurement of peptide concentrations: The analyses of the samples were performed via HPLC under the conditions shown in table 2.

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

- Calculation of the peptide concentrations: For each peptide a calibration curve is generated from the measured peak areas of the calibration samples of known peptide concentration. The peptide concentration of the samples is calculated with the respective calibration curve using linear regression (b = axis intercept; m = slope).
Peptide conc. [mM] = (peak area at 220 nm [mAU x s] – b) / m

- Calculation of the peptide depletion:
Peptide depletion of a sample = (1 – (peptide conc. sample [mM] / mean peptide conc. NC [mM])) x 100 [%]

The mean peptide depletion for each of the two peptides is calculated as the mean value of the three samples conducted for each peptide and test substance (C-containing and Kcontaining peptide depletion; example calculation for C-containing peptide):
C-cont. peptide depletion of a test substance [%] = mean [C-cont. peptide depletion of samples 1 – 3] [%]

When a negative value for C- or K-containing peptide depletion is obtained the value is considered zero for calculation of the mean peptide depletion. The mean peptide depletion of a test substance is calculated as the mean value of C-containing peptide depletion and Kcontaining peptide depletion:
Mean peptide depletion [%] = (C-cont. peptide depletion [%] + K-cont. peptide depletion [%]) / 2

For evaluation criteria see table 3+4

Key result

Run / experiment:

other: mean of three runs

Parameter:

other: mean of both cysteine and lysine peptide depletion [% of negative control]

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

Negative depletions were considered to be “zero” for calculation of the mean peptide depletion, which was thus calculated to be 0.00%.

Key result

Run / experiment:

other: mean of three runs

Parameter:

other: mean lysine peptide depletion [% of negative control]

Value:

-1.62

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: mean of three runs

Parameter:

other: mean cysteine peptide depletion [% of negative control]

Value:

-1.64

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Acceptance criteria met for variability between replicate measurements: Yes.

The samples of the test substance with the peptides were solutions at the time of preparation. Visual observation after the 24-hour incubation time did not reveal precipitates in any samples of the test substance with the peptides.
No co-elution of test substance and peptides was present.

Table 1: DPRA. Peak area, peptide concentration and peptide depletion of NC, PC and the test substance for cysteine-peptide.

Reaction with cysteine-peptide	peak area [mAU*min] at 220 nm			peptide concentration [mM]
	sample 1	sample 2	sample 3	sample 1	sample 2	sample 3	mean	SD
NC: ACN	7.09793	7.39951	7.26572	0.492	0.513	0.504	0.503	0.011
Test item	7.42260	7.36746	7.32974	0.515	0.511	0.508	0.511	0.003
PC: EGDMA in ACN	3.38995	3.25257	3.06537	0.234	0.225	0.212	0.223	0.011

 

 

Reaction with cysteine-peptide	peptide depletion [%]
	sample 1	sample 2	sample 3	mean	SD
NC: ACN	2.16	-2.01	-0.16	0.00	2.09
Test item	-2.33	-1.56	-1.04	-1.64	0.65
PC: EGDMA in ACN	53.44	55.34	57.93	55.57	2.25

 

Table 2: DPRA. Area ratio 220 nm/258 nm of NC, PC and the test substance for cysteine-peptide.

 

Reaction with cysteine-peptide	peak area [mAU*min] at 258 nm			area ratio 220 nm/258 nm
	sample 1	sample 2	sample 3	sample 1	sample 2	sample 3
NC: ACN	0.23974	0.25042	0.24790	29.6	29.5	29.3
Test item	0.24790	0.24957	0.24644	29.9	29.5	29.7
PC: EGDMA in ACN	0.11544	0.11499	0.10889	29.4	28.3	28.2

 

The mean area ratio 220 nm/ 258 nm of the 9 vehicle control samples of sets B and C was calculated to be 29.6. Hence, the area ratio 220 nm/ 258 nm of the test-substance samples correspond to 99.7% to 101.2% of the mean of the vehicle controls, demonstrating the absence of interference.

 

Table 3: Peak area, peptide concentration and peptide depletion of NC, PC and the test substance for lysine-peptide.

Reaction with cysteine-peptide	peak area [mAU*min] at 220 nm			peptide concentration [mM]
	sample 1	sample 2	sample 3	sample 1	sample 2	sample 3	mean	SD
NC: ACN	7.48937	7.55902	7.56081	0.526	0.531	0.531	0.529	0.003
Test item	7.68490	7.63913	7.64901	0.540	0.536	0.537	0.538	0.002
PC: EGDMA in ACN	6.76893	6.62330	6.55198	0.475	0.464	0.459	0.466	0.008

 

 

Reaction with cysteine-peptide	peptide depletion [%]
	sample 1	sample 2	sample 3	mean	SD
NC: ACN	0.63	-0.30	-0.33	0.00	0.54
Test item	-1.99	-1.37	-1.51	-1.62	0.32
PC: EGDMA in ACN	10.26	12.21	13.17	11.88	1.48

 

 

Table 4: DPRA. Area ratio 220 nm/258 nm of NC, PC and the test substance for lysine-peptide.

 

Reaction with lysine-peptide	peak area [mAU*min] at 258 nm			area ratio 220 nm/258 nm
	sample 1	sample 2	sample 3	sample 1	sample 2	sample 3
NC: ACN	0.25407	0.25196	0.25018	29.5	30.0	30.2
Test item	0.25675	0.25898	0.26086	29.9	29.5	29.3
PC: EGDMA in ACN	0.21729	0.21318	0.21157	31.2	31.1	31.0

 

Table 5: DPRA. Mean peptide depletions of Cysteine, Lysine and both peptides.

	Cysteine-Peptide		Lysine-Peptide		mean of both depletions [%]
	mean depletion [%]	SD [%]	mean depletion [%]	SD [%]
Test item	-1.64	0.65	-1.62	0.32	0.00
PC: EGDMA in ACN	55.57	2.25	11.88	1.48	33.73

Interpretation of results:

GHS criteria not met

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU Method B.60 In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

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

Type of study:

activation of keratinocytes

Details on the study design:

CONCENTRATION SELECTION
In order to determine the concentrations suitable for the main experiment a pre-test (experimental conduct in accordance with GLP but without a GLP status) was performed. Cells
were exposed to 9 concentrations of the test-substance preparation and cytotoxicity was determined by MTT assay. No decrease in cell viability below 75% was observed.

PREPARATION OF TEST ITEMS
- Test item concentrations
Pre-experiment (cytotoxicity assay): 9 concentrations of the test substance between 8.1 μM and 2069 μM in DMSO
Main experiments: 577, 693, 831, 998, 1197, 1437, 1724, 2069 µM in DMSO

PREPARATION OF POSITIVE/NEGATIVE CONTROLS
- Positive control: Ethylene glycol dimethacrylate (EGDMA), 90.8 μM (= 18 μg/mL) in 1% DMSO in culture medium 3
- Negative Control: DL-Lactic acid, 5000 μM in 1% DMSO in culture medium 3
- Vehicle control: 1% (v/v) DMSO in culture medium 3
- Blank control: Culture medium 3 without cells
- Basal control: Culture medium 3 with cells

EXPERIMENTAL PROCEDURE
- Cell culture medium used:
Culture medium 1: D-MEM + 10 % FBS + 1 % Penicillin / Streptomycin, Puromycin dihydrochloride 25 μL (Sigma)
Culture medium 2: D-MEM + 10 % FBS
Culture medium 3: D-MEM + 1 % FBS

- Replicates: 2 experiments with three replicates of each test substance concentration
- Incubation: Cells were grown for 24h ± 1h in cell culture medium 2 at 37°C ± 1 °C and 5% CO2. Thereafter, the medium was discarded and replaced by 150 µL culture medium 3. 50 µl of the corresponding test item concentration was then added per well and incubated for 48h ± 1h.

- Measurement of luciferase activity: Cells were washed twice with 300 µL PBS. Subsequently, 200 µL of One-Glo-preparation (= 100 μL One-Glo- Mix and 100 μL PBS (without Ca2+/Mg2+)) was added into each well and the plate was incubated at room temperature in the absence of light on a shaker for 10 min. After the incubation the luminescence was measured in the luminometer.

- Cell viability: 200 μL of a 0.5 mg/mL thiazolyl blue tetrazolium bromide (MTT) solution (prepared 1:10 from a 5 mg/mL (MTT) stock solution in PBS (without Ca2+/Mg2+) and culture medium 3) was added to each well. The plate was incubated for 2h at 37°C ± 1°C and 5% CO2. Afterwards the medium was removed and replaced by 100 µL lysis solution (99.6 mL DMSO; 10 g sodium dodecyl sulfate, SDS; and 0.4 mL glacial acetic acid). Absorbance was measured at 570 nm with reference wavelength 690 nm using a spectral-photometer.

Key result

Run / experiment:

other: Run 1

Parameter:

other: Relative luciferase fold induction

Value:

0.95

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: Run 2

Parameter:

other: Relative luciferase fold induction

Value:

1.05

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Acceptance criteria met for variability between replicate measurements: Yes.

Table 1: LuSens. Results of preliminary cytotoxicity assessment. The final test substance concentrations were calculated considering a purity/contents of 96.65%.

Concentration	Concentration	mean OD570-690	mean rel.
(final test substance)	(test substance)	of 3 replicates	viability
[µM]	[µM]		[%]
VC	VC	0.430	100
8	8	0.372	87
16	16	0.389	91
31	32	0.381	89
63	65	0.365	85
125	129	0.355	83
250	259	0.356	83
500	517	0.359	83
1000	1035	0.364	85
2000	2069	0.362	84

 

Table 2: LuSens. Mean values and standard deviations of luciferase induction and rel. viability as well as p-values of t-test (experiment 1). Concentrations with fold inductions above 1.50 with rel. viability ≥70% and with statistical significance are indicated in bold and in grey when < 70% viability.

Concentration			1^st experiment
(test substance)	fold induction		rel. viability [%]		t-test
[µM]	mean	SD	mean	SD	p-value	markers
577	1.02	0.13	90	6	0.404	n.s.
693	0.95	0.21	91	3	0.376	n.s.
831	1.04	0.20	93	6	0.369	n.s.
998	1.03	0.15	90	2	0.369	n.s.
1197	1.07	0.10	90	2	0.182	n.s.
1437	1.02	0.15	93	6	0.404	n.s.
1724	1.07	0.11	91	4	0.187	n.s.
2069	1.08	0.17	91	1	0.249	n.s.
VC	1.00	0.08	100	8	-	-
EGDMA 90.8 µM	4.20	0.48	78	3	0.000	**
LA 5000 µM	0.87	0.09	86	4	0.014	*

 

Table 3: LuSens. Mean values and standard deviations of luciferase induction and rel. viability as well as p-values of t-test (experiment 2). Concentrations with fold inductions above 1.50 with rel. viability ≥70% and with statistical significance are indicated in bold and in grey when < 70% viability.

Concentration			2^nd experiment
(test substance)	fold induction		rel. viability [%]		t-test
[µM]	mean	SD	mean	SD	p-value	markers
577	1.16	0.17	95	8	0.124	n.s.
693	1.28	0.29	89	6	0.121	n.s.
831	1.05	0.11	105	6	0.248	n.s.
998	1.24	0.13	100	8	0.039	*
1197	1.09	0.20	95	5	0.253	n.s.
1437	1.20	0.11	106	8	0.034	*
1724	1.06	0.15	96	4	0.294	n.s.
2069	1.06	0.14	96	6	0.264	n.s.
VC	1.00	0.09	100	8	-	-
EGDMA 90.8 µM	4.64	0.54	92	4	0.000	**
LA 5000 µM	1.13	0.12	116	8	0.029	*

 

Interpretation of results:

GHS criteria not met

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In the chosen key study for skin sensitization according to OECD 422C/D and GLP (BASF SE 2020; 67V0493/19V067), Neova was tested in a combination of three in vitro methods, addressing key events of the adverse outcome pathway (AOP) for skin sensitization (OECD, 2012) as defined by the OECD. This in vitro Skin Sensitization Turnkey Testing Strategy addresses protein reactivity (DPRA), the activation of keratinocytes (LuSens), and the activation of dendritic cells (h-CLAT). However, in the current case for Neova the results derived with DPRA and LuSens were sufficient for a final assessment. Therefore, further testing was not considered necessary.

In the DPRA, the first key events of the adverse outcome pathway (AOP) for skin sensitization was addressed and the reactivity of Neova towards synthetic cysteine (C)- or lysine (K)-containing peptides was evaluated. For this purpose Neova was incubated with synthetic peptides for approx. 24 hours at approx. 25°C and the remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and UV-detection at 220 nm. Three samples were incubated with each peptide in ratios of 1:10 (for C-containing peptide) or 1:50 (for K-containing peptide). Additionally, triplicates of the concurrent vehicle control (= VC) were incubated with the peptides. Neova was dissolved in acetonitrile at a concentration of 100 mM and visual observation after the 24-hour incubation time did not reveal precipitates with the peptides. No co-elution of Neova and peptides was present. The mean C-peptide depletion, caused by Neova was determined to be -1.64%. The mean K-peptide depletion, caused by Neova was determined to be -1.62%. Negative depletions were considered to be “zero” for calculation of the mean peptide depletion, which was thus calculated to be 0.00%. Based on the observed results, it was concluded that Neova shows minimal or no chemical reactivity in the DPRA under the test conditions chosen.

In the ARE-Nrf2 luciferase test method (LuSens), the keratinocyte activating potential of Neova was evaluated. For this purpose, Neova was incubated with a luciferase reporter cell line (LuSens cells) for ca. 48 hours at 37°C and antioxidant response element (ARE) dependent luciferase activity was measured in a luminometer. In order to determine the concentrations suitable for the main experiment a pre-test (non-GLP) was performed. In the main test luciferase activity was measured after 48-hour exposure and a MTT assay was performed in parallel to assess cytotoxicity. A total of 2 valid experiments were performed. The concentrations tested ranged from 577 to 2069 µM Neova. Neova was soluble in the vehicle DMSO at the concentrations used and no precipitates were noticed in any concentration after 48 hours. The mean fold induction ranged between 0.95 - 1.08 in the 1^stexperiment and 1.05 – 1.28 in the 2^ndexperiment compared to the vehicle control in all concentrations tested. Thus, the calculation of an EC1.50 (the concentration resulting in a 1.50-fold luciferase induction) was not applicable. The relative viability was found to be above 70% in all concentrations tested. In summary, after 48 hours of exposure to Neova, luciferase activity in LuSens cells was not induced in at least two consecutive concentrations with statistical significance affording at least 70% viability in at least two independent experiments. From this, it is concluded, that Neova does not have a keratinocyte activating potential.

Based on these results, Neova is not peptide reactive and does not activate keratinocytes. When applying the Adverse Outcome Pathway-based "2 out of 3" integrated testing strategy approach to skin hazard identification, Neova is predicted not to be a skin sensitizer.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

No data available

Justification for classification or non-classification

The present data on dermal sensitization do not fulfill the criteria laid down in regulation (EU) 1272/2008, and therefore, a non-classification is warranted.