Naphthenic acids, zinc salts

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6 April 2018 - 4 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of study:

other: KeratinoSens Assay

Justification for non-LLNA method:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need for in vivo testing. One of the validated in vitro skin sensitization tests is the KeratinoSensTM assay, which is recommended in international guidelines (e.g. OECD 442D).

Test material

Test material information

1

Reference substance name:

Naphthenic acids, zinc salts

EC Number:

234-409-2

EC Name:

Naphthenic acids, zinc salts

Cas Number:

12001-85-3

Molecular formula:

C20H34O4Zn

IUPAC Name:

Naphthenic acids, zinc salts

Test material form:

solid: bulk

Details on test material:

- Description: Zinc salts of naphthenic acids, consisting of an acidic (naphthenic) fraction (70-95%) composed mainly of C8-C20 and 0-3 rings and a non-acidic (petroleum) fraction (5-30%), composed mainly of C12-C22, <10% aromatics, with a boiling range of approximately 160-350°C.
- Starting materials: Naphthenic acids (EC 215-662-8; CAS 1338-24-5); Zinc oxide (EC 215-222-5; CAS 1314-13-2, 7440-66-6)
- Manufacturing method: Zinc oxide and the naphthenic acids, with or without water, are reacted together in a solvent or vehicle, such as base oil to form zinc naphthenate. Sufficient amount of zinc oxide is added to the naphthenic acids to completely neutralise all the naphthenic acids, without significant excess of either starting material. The mixture is heated to complete the reaction then cooled. The amount of zinc oxide is calculated depending on the Total Acid Number of the naphthenic acid starting material. This calculation ensures that no significant excess unreacted zinc oxide remains. The zinc naphthenate produced is therefore considered to be the neutral form.
- Sample preparation: Sample used for hazard testing was prepared as a neat substance without base oil.

Specific details on test material used for the study:

- Source and lot/batch No. of test material: A036/99 (Supplier batch number: SCC-1709-0300)
- Expiration date of the lot/batch: 31 August 2018
- Storage condition of test material: At room temperature protected from light

In vitro test system

Details on the study design:

- Test system: A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSens™ cell line). The KeratinoSens™ cell line was generated by and obtained from Givaudan (Duebendorf, Switserland). Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock. Cells from this original stock can be propagated up to a maximum passage number from the frozen stock (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.

- Cell sub-culture: Cells were subcultured upon reaching 80-90% confluency. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock, and were not cultured for more than 25 passages from the frozen stock (P+25).
- Cell culture, basic medium: Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.
- Cell culture, maintenance medium: Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum and geneticin (500 µg/mL).
- Cell culture, exposure medium: Dulbecco’s minimal (DMEM glutamax) supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.

- Dose formulation: In the main experiments, the test item was suspended in dimethyl sulfoxide (DMSO) at 40 mg/mL. From this stock, 11 spike solutions in DMSO were prepared (2-fold dilution series). The stock and spike solution were diluted 25-fold with exposure medium. These solutions were diluted 4-fold in the assay, resulting in final test concentrations 400, 200, 100, 50, 25, 13, 6.3, 3.1, 1.6, 0.78, 0.39 and 0.20 µg/mL (final concentration DMSO of 1%). All concentrations of the test item were tested in triplicate. Test item concentrations were used within 3 hours after preparation.
- Solution appearance: All formulations formed a clear solution, except at 40 mg/mL where it formed a suspension. No droplet formation was observed in the plates. No precipitation was observed at the start and end of the incubation period in the 96-well plates.

- Environmental conditions: All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 70-100%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.7 – 36.3°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature and humidity occurred due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

- Plating of cells: For testing, cells were 80-90% confluent. One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight in the incubator. The passage number used was P+9 in experiment 1 and P+12 in experiment 2.

- Treatment of cells: The medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control items were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were covered with foil and then incubated for about 48 hours at 37±1.0°C in the presence of 5% CO2. Initially, experiment 2 did not pass all the acceptability criteria and therefore this part of the study was repeated. In total, 2 valid experiments were performed.

- Luciferase Activity measurement: The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 µL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 3 minutes at room temperature. Plates with the cell lysates were placed in the TECAN Infinite® M200 Pro Plate Reader to assess the quantity of luciferase (integration time two seconds).

- Cytotoxicity assessment: Medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and cells were incubated for 3 - 4 hours at 37°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.

- Vehicle control: The vehicle control was 1% DMSO in exposure medium. Eighteen wells were tested per plate.
- Blank control: On each plate three blank wells were tested (no cells and no treatment).
- Positive control: Ethylene dimethacrylate glycol

- Parameters: The following parameters are calculated in the KeratinoSensTM test method:
• The maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the tested chemical and positive control
• The EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) was obtained
• The IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability

Results and discussion

Positive control results:

- Experiment 1: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.73 and the EC1.5 75 µM.
- Experiment 2: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.49 and the EC1.5 54 µM.

In vitro / in chemico

Results

Results 1

Run / experiment:

other: 1

Parameter:

other: EC1.5 (µg/mL)

Value:

31.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Run / experiment:

other: 2

Parameter:

other: EC1.5 (µg/mL)

Value:

21.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Run / experiment:

other: 1

Parameter:

other: Imax

Value:

2.15

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Run / experiment:

other: 2

Parameter:

other: Imax

Value:

5.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 5

Run / experiment:

other: 1

Parameter:

other: IC30 (µg/mL)

Value:

124

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 6

Run / experiment:

other: 2

Parameter:

other: IC30 (µg/mL)

Value:

74

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 7

Run / experiment:

other: 1

Parameter:

other: IC50 (µg/mL)

Value:

146

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 8

Run / experiment:

other: 2

Parameter:

other: IC50 (µg/mL)

Value:

82

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Experiment 1
• No precipitation was observed at the start and end of the incubation period in the 96-well plates.
• The test substance showed toxicity. The calculated IC30 was 124 µg/mL and the calculated IC50 was 146 µg/mL.
• A dose related luminescence activity induction was observed after treatment with the test substance. The Imax was 2.15 and the EC1.5 was 32 µg/mL.

Experiment 2
• No precipitation was observed at the start and end of the incubation period in the 96-well plates.
• The test substance showed toxicity. The calculated IC30 was 74 µg/mL and the calculated IC50 was 82 µg/mL.
• A dose related luminescence activity induction was observed after treatment with the test substance. The Imax was 5.10 and the EC1.5 was 22 µg/mL.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration (from 7.8 to 250 µM). The EC1.5 of the positive control was between 5 and 125 µM (75 µM and 54 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.73-fold and 2.49-fold in experiment 1 and 2, respectively).
- Acceptance criteria met for variability between replicate measurements: The average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (7.1% and 11% in experiment 1 and 2, respectively).

Any other information on results incl. tables

Table 1. Overview Luminescence Induction and Cell Viability of the test substance in Experiment 1 and 2

Concentration (µg/mL)	0.20	0.39	0.78	1.6	3.1	6.3	13	25	50	100	200	400
Exp 1 luminescence	1.09	1.10	1.12	1.03	1.09	1.07	1.08	1.38	1.85^***	2.15	0.00	0.00
Exp 1 viability (%)	109.3	105.8	89.3	86.1	78.8	73.7	78.1	92.8	117.1	92.3	-0.2	-0.2
Exp 2 luminescence	1.05	1.06	0.98	0.93	1.01	1.02	1.04	1.66^***	5.10^**	0.00	0.00	0.00
Exp 2 viability (%)	103.8	98.0	103.5	103.3	104.4	101.5	110.7	137.5	133.9	1.0	3.1	-0.9

^**p<0.01 Student’s t test

^***p<0.001 Student’s t test

 

Table 2. Overview Luminescence Induction and Cell Viability Positive Control EDMG in Experiment 1 and 2

Concentration (µM)	7.8	16	31	63	125	250
Exp 1 luminescence	1.05	1.05	1.27	1.41	1.88^***	2.73^***
Exp 1 viability (%)	100.8	104.6	103.9	109.9	110.9	106.2
Exp 2 luminescence	1.05	1.09	1.24	1.61^***	1.80^***	2.49^***
Exp 2 viability (%)	98.9	101.2	104.8	110.8	102.6	121.5

^***p<0.001 Student’s t test

 

Table 3. Overview EC_1.5, I_max, IC₃₀ and IC₅₀ Values

	EC1.5(µg/mL)	Imax	IC30(µg/mL)	IC50(µg/mL)
Test item Experiment 1	31.5	2.15	124	146
Test item Experiment 2	21.9	5.10	74	82
	EC1.5(µM)	Imax	IC30(µM)	IC50(µM)
Pos Control Experiment 1	74.9	2.73	NA	NA
Pos Control Experiment 2	53.5	2.49	NA	NA

NA = Not applicable

 

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The test item showed toxicity (IC30 values of 124 µg/mL and 74 µg/mL and IC50 values of 146 µg/mL and 82 µg/mL in experiment 1 and 2, respectively). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 values of 32 µg/mL and 22 µg/mL in experiment 1 and 2, respectively) was measured in both experiments. The maximum luciferase activity induction (Imax) was 2.15-fold and 5.10-fold in experiment 1 and 2 respectively. The test item is classified as positive in the KeratinoSensTM assay since positive results (>1.5-fold induction) were observed at test concentrations < 200 µg/mL with a cell viability of >70% compared to the vehicle control. No adverse interference on the study results of the droplets observed in the dose formulation is expected since the test item is classified as positive, and if interference would be present, it would only lead to an under prediction.

Executive summary:

The skin sensitisation of the test item was assessed in a KerationSens Assay with procedure based on the OECD TG 442D guideline, in two independent experiments in triplicate. The test item was suspended in the vehicle solvent, dimethyl sulfoxide (DMSO), prior to dilution with test media and the experiment was performed at final test concentrations 400, 200, 100, 50, 25, 13, 6.3, 3.1, 1.6, 0.78, 0.39 and 0.20 µg/mL (final concentration DMSO of 1%). All formulations formed a clear solution, except at 40 mg/mL were it formed a suspension. No droplet formation was observed in the plates. No precipitation was observed at the start and end of the incubation period in the 96-well plates. Alongside the test item, a solvent (negative control) of DMSO and positive control of Ethylene dimethacrylate glycol (EDMG) were tested. For the positive control, a 2-fold dilution series ranging from 0.78 to 25 mM was prepared in DMSO and diluted, so that the final concentration of the positive control ranges from 7.8 to 250 µM (final concentration DMSO of 1%).

 

The test substance showed toxicity (IC₃₀ values of 124 µg/mL and 74 µg/mL and IC₅₀ values of 146 µg/mL and 82 µg/mL in experiment 1 and 2, respectively). A biologically relevant, dose-related induction of the luciferase activity (EC_1.5 values of 32 µg/mL and 22 µg/mL in experiment 1 and 2, respectively) was measured in both experiments. The maximum luciferase activity induction (I_max) was 2.15-fold and 5.10-fold in experiment 1 and 2 respectively. The test item is classified as positive in the KeratinoSens assay since positive results (>1.5-fold induction) were observed at test concentrations < 200 µg/mL with a cell viability of >70% compared to the vehicle control.