Naphthenic acids, copper salts

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-06-27 to 2019-06-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

2017-10-09

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol: EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; for use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model

Version / remarks:

2015-06-29

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2018-04-26

Test material

Test material information

Constituent 1

Reference substance name:

Naphthenic acids, copper salts

EC Number:

215-657-0

EC Name:

Naphthenic acids, copper salts

Cas Number:

1338-02-9

IUPAC Name:

copper(2+) bis(3-(3-ethylcyclopentyl)propanoate)

Test material form:

liquid: viscous

Remarks:

paste

Details on test material:

- State of aggregation: liquid, viscous, green paste

Test animals / tissue source

Details on test animals or tissues and environmental conditions:

JUSTIFICATION OF THE TEST METHODS AND CONSIDERATIONS REGARDING APPLICABILITY:
This in vitro method is recommended to identify chemicals that do not require classification for eye irritation or serious eye damage according to UN GHS (UN GHS “No Category”) without further testing within a tiered testing strategy from those requiring classification and labelling (UN GHS categories 1 and 2). Therefore, it can be used for regulatory purposes as an initial step in the bottom-up approach or as one of the last steps in a top-down approach to test eye irritation/corrosion potential. It is not intended to differentiate between UN GHS “Category 1” (serious eye damage) and UN GHS “Category 2” (eye irritation) which would require additional testing. Ocular irritation potential is predicted by the relative viability of the tissue after a single exposure to the test substance. Relative viability is determined by measuring the MTT dye to formazan conversion by the EpiOcular™ tissue construct after topical exposure to the test substance.

RhCE TISSUE CONSTRUCT USED: EpiOcular™ (Lot No.: 27051; kit OCL-200-EIT; source: MatTek Corporation (82105 Bratislava, Slovakia))
The test was carried out with the EpiOcular™ reconstructed human cornea-line epithelium (RhCE) model (MatTek). The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified, highly differentiated squamous epithelium morphologically similar to that found in a human cornea. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinized surface, showing a cornea-like structure analogous to that found in vivo.
Please also refer to the field "Attached background material " below.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL (83.3 µL/cm2) of the test item were dispensed directly atop the EpiOcular™ tissue. The test item was spread to match size of the tissue.

Duration of treatment / exposure:

30 ± 2 min

Observation period (in vivo):

not applicable

Duration of post- treatment incubation (in vitro):

approx. 120 min

Number of animals or in vitro replicates:

Number of EpiOcular tissues:
Test item: duplicates
Negative control: duplicates
Positive control: duplicates

Details on study design:

PRE-TEST FOR DIRECT MTT-REDUCERS AND COLOURING TEST CHEMICALS
TEST FOR MTT INTERFERENCE
To check the non-specific MTT-reducing capability of the test item 50 µL of the test item were mixed per 1 mL MTT medium and incubated for 3 h in a humidified incubator at 37 ± 2 °C, 5.0% CO2 / 95% air. After incubation verification of the colour by the unaided eye.

TEST FOR COLOUR INTERFERENCE
To check the colouring potential of the test item 50 µL of the test item were mixed per 1 mL Aqua dest. and per 2 mL isopropanol each in a 6-well plate. The water solution was incubated for at least 1 h in a humidified incubator at 37 ± 2 °C, 5.0% CO2 / 95% air. The isopropanol solution was shaken on a plate shaker for 2 to 3 h. After the respective incubation period, 2 x 200 µL aliquots per test solution were transferred into a 96-well plate, using 200 µL Aqua dest. and isopropanol as respective blanks and OD was measured in a range of 570 ± 30 nm without reference wavelength in a plate spectrophotometer. The mixture of 50 μL test item per 2 mL isopropanol showed colouring as compared to the solvent. As ODnet(Isopropanol) was >0.08, coloured tissue controls were performed for quantitative correction of results.
For quantitative correction of results, the colorant control test was performed using two additional living tissues treated with 50 µL of the test item (TVT). All steps were performed exactly as described below in "Details on the test procedure used", except for the MTT-staining of the test item treated with tissues, which was incubated in medium without MTT. The percent non-specific colour of additional viable tissues (NSCliving) was calculated.

DETAILS OF THE TEST PROCEDURE USED
- Upon receipt of the EpiOcular™, the tissues were equilibrated in the 24-well shipment plate to room temperature for about 15 min. Then, the EpiOcular™ tissues were transferred into 6-well plates containing 1 mL pre-warmed assay medium per well and incubated for 1 h in a humidified incubator at 37 ± 2 °C, 5.0% CO2 / 95% air. Then the inserts were transferred into new 6-well plates containing 1 mL fresh assay medium per well and pre-incubated in a humidified incubator at 37 ± 2 °C, 5.0% CO2 / 95% air for 24h.
After the overnight incubation the tissues were pre-treated with 20 µL of DPBS-buffer and incubated for 30 ± 2 min in a humidified incubator at 37 ± 2 °C, 5.0% CO2 / 95% air to mimic the wet conditions of the human eye.
Afterwards, the tissues were treated with each dose group in duplicate, starting with the negative and positive control. Then the 6-well plate(s) were incubated for 30 ± 2 min at 37 ± 2 °C, 5.0% CO2 / 95% air. At the end of the exposure period, the test item and control substances were removed by extensively rinsing 3 times the tissue with DPBS. Excess DPBS was removed by decanting the insert and blotting bottom with blotting paper. After rinsing, the inserts were transferred to and immersed in a prepared 12-well “post-soak plate“, containing 2 mL fresh pre-warmed assay medium per well and incubated for 12 ± 2 min at room temperature. Afterwards, the inserts were removed from the assay medium, the medium was decanted off the tissue and the tissues were blotted on blotting paper. The inserts were transferred to a new 6-well plate (post-treatment plate) containing 1 mL pre-warmed assay medium. The tissues were incubated for 120 ± 15 min at 37 ± 2 °C, 5.0% CO2 / 95% air.
- MTT Assay
After this incubation period excess medium was removed by blotting bottom on absorbent paper before the inserts were transferred in a prepared 24-well “MTT assay plate” containing 0.3 mL pre-warmed MTT medium and further incubated for 3 h ± 15 min at 37 ± 2 °C, 5.0% CO2 / 95% air.
After the 3 h MTT incubation period the inserts were removed, the bottom of the inserts blotted on blotting paper, and then transferred into new 24-well “extraction plates“, containing 2 mL of isopropanol. The extraction plates were sealed to inhibit isopropanol evaporation. Extraction was carried out after storage overnight in the dark at 2 - 8 °C. At the end of the extraction period the tissues were pierced and the liquid within each insert was decanted into the well from which it was taken.
Then the inserts were discarded, and the extracts were mixed three times using a pipette. If any visible cell/tissue fragments were in suspension, extracts were centrifuged to eliminate the fragments and avoid further possible interference with the absorbance readings.
For each tissue 2 x 200 µL aliquots of the extract were transferred into a 96-well plate and OD was measured at 570 nm using a filter band pass of maximum ± 30 nm in a plate spectrophotometer using isopropanol as a blank

TEST ACCEPTANCE CRITERIA
The test meets acceptance criteria:
- mean absolute OD570 nm of the negative control is > 0.8 and < 2.5
- mean relative tissue viability of the positive control is < 50%
- relative tissue viability difference of replicate tissues is < 20%
- OD control values are not be below historically established boundaries/ positive and negative control mean values and acceptance ranges based on historical data.

DEMOSTRATING OF PROFICIENCY IN PERFORMING THE TEST METHOD BEFORE ROUTINE USE BY TESTING OF THE PROFICIENCY CHEMICALS
Prior to routine use of EpiOcularTM EIT for regulatory purposes, the laboratory demonstrated technical proficiency by correctly predicting the eye irritation potential of fifteen proficiency chemicals listed in Table 1 of OECD TG 492. The respective proficiency certificate given by MatTek is attached in the field "Attached background material" below.

DESCRIPTION OF DATA EVALUATION
The mean OD of the two negative control tissues will be calculated after blank correction. This value corresponds to 100 % tissue viability in the current test. For each individual tissue treated with the test item or the positive control, the individual relative tissue viability is calculated according to the following formula:
Relative viability (%) = [mean ODtest item/ positive control / ODmean of negative control] * 100
For the test item and the positive control the mean relative viability ± relative standard deviation of the two individual tissues will be calculated and used for classification according to the following prediction model:
Ocular irritation potential of the test item was predicted from the relative mean tissue viabilities obtained after treatment compared to the negative control tissues concurrently treated with Aqua dest.

PREDICTION MODEL
If the mean percent tissue viability after exposure and post-exposure incubation is less than or equal 60% tissue viability, no prediction can be made. In this case, further testing with other test methods will be required. The test chemical is identified as not requiring classification and labelling according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than 60%. In this case no further testing in other test methods is required

Results and discussion

In vitro

Results

Irritation parameter:

other: % tissue viability (mean)

Value:

103.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

PRE-EXPERIMENTS FOR QUANTITATIVE CORRECTION:

TEST FOR MTT INTERFERENCE
The mixture did not turn blue/purple. So, the additional functional test with freeze-killed tissues and the quantitative corrections were not necessary. Therefore, NSMTT equalled 0%.

TEST FOR COLOUR INTERFERENCE
The mixture of test item/Aqua dest. showed no colouring as compared to the solvent, but the mixture of test item/isopropanol showed colouring as compared to the solvent. Therefore, the absorption of the chemical in isopropanol was measured in the range of 570 ± 30 nm. The test item in isopropanol absorbed light in the relevant range:

ODnet(Aqua dest.) = meanODAqua dest. – mean ODBlank(Aqua dest.) = 0.04355 – 0.0484 = 0.0049
ODnet(Isopropanol) = meanODIsopropanol – mean ODBlank(Isopropnaol) = 1.1128 – 0.04335 = 1.06945

As ODnet(Isopropanol) was >0.08, coloured tissue controls were performed for quantitative correction of results:

NSCliving [%] = [ODTVT/ODNC] * 100 = 0.2%

Difference of NSCliving of the two duplicate tissues must be < 20%, otherwise not accepted.
NSC1 [%] = [ODTVT1 /ODNC] * 100 = 0.2%
NSC2 [%] = [ODTVT2 /ODNC] * 100 = 0.2%
NSC1 – NSC2 = ±0.0%

NSCliving was ≤ 60% (0.2%) relative to the negative control of living epidermis and could therefore be used for determination of the NSC-corrected mean relative tissue viability (NSCCV) according to the following formula:
NSCCV [%] = viabilityTM [%] – NSCliving [%] = 104.1% - 0.2% = 103.9%

ACCEPTANCE OF RESULTS:
- mean absolute OD570 nm of the negative control is > 0.8 and < 2.5 (2.429)
- mean relative tissue viability of the positive control is < 50% (28.8%)
- relative tissue viability difference of replicate tissues is < 20% (3.5 - 11.6%)
- OD control values were not be below historically established boundaries/ positive and negative control mean values and acceptance ranges based on historical data

The acceptance criteria were met. Regarding the reproducibility of the data, the absorbance of the negative controls and positive controls is within the historical range of absorbance.

Please also refer for information on the results to the field "Any other information on results incl. tables" below.

Any other information on results incl. tables

Result of the Test Item Copper naphthenate

Name	Negative Control		Positive Control		Test item
Tissue	1	2	1	2	1	2
Absolute OD570values	2.589	2.306	0.798	0.652	2.529	2.597
	2.546	2.276	0.837	0.639	2.523	2.458
Mean Absolute OD570values	2.429****		0.732		2.527
OD570values (blank-corrected)	2.545	2.262	0.754	0.608	2.485	2.553
	2.502	2.231	0.793	0.595	2.479	2.414
Mean OD570of the duplicates (blank-corrected)	2.523	2.247	0.773	0.602	2.482	2.483
Total Mean OD570of 2 Replicate Tissues (blank corrected)	2.385*		0.687		2.483
SD of Mean OD570of the Replicates (Blank Corrected)	0.196		0.121		0.001
Relative tissue viability [%]	105.8	94.2	32.4	25.2	104.1	104.1
Relative tissue viability difference [%]^***	11.6		7.2		0.1
Meanrelative tissueviability [%]	100.0		28.8**		104.1
mean tissue viability [%] - NSClivingcorrected	-		-		103.9

^*             Corrected mean OD₅₇₀of the negative control corresponds to 100% absolute tissue viability

^**            Mean relative tissue viability of the positive control is < 50%

^***           Relative tissue viability difference of replicate tissues is < 20%

^****         Mean absolute OD570 values of the tissues treated with the negative control is >0.8 and <2.5

 

Result of the NSC(living) control

NSCliving	TVT
Tissue	1	2
absolute OD570 -values	0.048	0.049
	0.048	0.048
absolute OD570 -Blank corrected values	0.004	0.005
	0.004	0.004
mean OD570(mean of 2 aliquots)	0.004	0.004
total mean OD570(mean of replicate tissues)	0.004
SD OD570(of the 2 replicate tissues)	0.000
NSCliving[%]	0.2

Historical Data

	Mean Absolute OD570±30nmNC	Mean Absolute OD570±30nmPC	Mean Relative Viability [%] PC	SD Viability [%] NC, PC, TI
Mean	1.686	0.423	23.4	6.0
SD	0.269	0.205	12.4	5.7
Range of LCL – UCL	1.147 – 2.224	0.012 – 0.833	0.0 – 48.1	0.0 – 17.3
n	25	25	25	114

LCL:      Lower control limit (95%, mean – 2*SD)

UCL:     Upper control limit (95%, mean + 2*SD)

n:           number of control values

Historical control data were generated from 2017 – 2018.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this study, under the reported conditions of the Reconstructed human Cornea-like Epithelium test (RhCE), the test item showed no irritant effects. Thus, in accordance with OECD 492 the test item is identified as not requiring classification and labelling according to UN GHS/ EU CLP (No Category).