Naphthenic acids, zinc salts

Endpoint summary

Description of key information

Skin irritation/skin corrosion

Eurling (2017) is a GLP-compliant study following the OECD Guideline 431. The study is assigned a Klimisch score of 1 (reliable with restrictions). The test item had a mean tissue viability of 60% after a 3-minute application and a mean tissue viability of 83% in the 1-hour application. Therefore, the test item to be considered as non-corrosive to the skin.

Eurling (2018) is a GLP-compliant study following the OECD Guideline 439. The study is assigned a Klimisch score of 1 (reliable with restrictions). The test item had a mean tissue viability of 86%. Therefore, the test item to be considered as non-irritant to the skin.

On the basis of the results of the OECD 431 and OECD 439 studies, naphthenic acids, zinc salts is considered not to meet the criteria for classification under EU CLP for skin irritation or skin corrosion.

Eye irritation/eye damage

Eurling (2017) is a GLP-compliant study following the OECD Guideline 437. The study is assigned a Klimisch score of 1 (reliable with restrictions). The test item had a meanin vitroirritancy score of 10 after 240 minutes of treatment. Since naphthenic acids, zinc salts induced an IVIS > 3 ≤ 55, no prediction on the classification can be made.

Eurling (2018) is a GLP-compliant study following the OECD Guideline 492. The study is assigned a Klimisch score of 1 (reliable with restrictions). The test item had a relative mean tissue viability of 19% so is considered to be potentially irritant or corrosive to the eye and potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1).

On the basis of the results of the OECD 437 and OECD 492 studies, naphthenic acids, zinc salts is considered to meet the criteria for classification under EU CLP as H319: Causes serious eye irritation.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 November 2017 - 01 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test"

Version / remarks:

Official Journal of the European Union No. L142, 31 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Source and lot/batch No.of test material: A036/99
- Expiration date of the lot/batch: 31 August 2018
- Storage condition of test material: Room temperature

Test system:

human skin model

Source species:

other: Reconstructed human epidermis (RHE)

Cell type:

non-transformed keratinocytes

Cell source:

other: Epiderm Skin Model

Source strain:

other: Epiderm Skin Model

Details on animal used as source of test system:

Not applicable

Justification for test system used:

The test is based on the experience that corrosive chemicals show cytotoxic effects following short term exposure to the stratum corneum of the epidermis. The test is designed to predict and classify the skin corrosion potential of a test item by assessment of its effect on a three-dimensional human epidermis model.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model (EPI-200)
- Tissue batch number(s): 27631
- Production date: Not specified
- Shipping date: Not specified
- Delivery date: Not specified.
- Date of initiation of testing: 27/11/17

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37.0 ± 1.0°C (actual range 36.2 - 37.3°C).
- Temperature of post-treatment incubation (if applicable): 37.0 ± 1.0°C (actual range 36.2 - 37.3°C).

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test item.
- Observable damage in the tissue due to washing: None noted
- Modifications to validated SOP: None

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 5 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570nm
- Filter and bandwidth: Not specified
- Linear OD range of spectrophotometer: Not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA: Not specified

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: Not applicable

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: An excess amount of the solid test item applied directly on top of the skin tissue using a nylon mesh disk. The mesh disk matched the size of the tissue completely, resulting in a fully covered tissue with test item.
- Concentration: Test material was applied neat.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration: Not applicable. Milli-Q water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration: 8N KOH

Duration of treatment / exposure:

3 minutes and 1 hour.

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Run 1 / 3 minute application

Value:

60

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Run 2/ 1 hour application

Value:

83

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Because the solutions did not turn blue / purple nor a blue / purple precipitate was observed it was concluded that the test item did not interfere with the MTT endpoint.
Because the mean relative tissue viability for Naphthenic acids, zinc salts was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment Naphthenic acids, zinc salts is considered to be not corrosive.
The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit =<2.8) and the laboratory historical control data range. The mean relative tissue viability following the 1-hour exposure to the positive control was 7.6% (acceptance limit =< 15%). In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was =< 28% (acceptance limit =< 30%), indicating that the test system functioned properly.

Table1          
Mean Absorption in the in vitro Skin Corrosion Test with Naphthenic acids, zinc salts

	3-minute application					1-hour application
	A (OD570)	B (OD570)	Mean (OD570)		SD	A (OD570)	B (OD570)	Mean (OD570)		SD
Negative control	1.885	1.816	1.851	±	0.049	2.092	1.724	1.908	±	0.260
Test item	1.184	1.055	1.119	±	0.091	1.540	1.626	1.583	±	0.061
Positive control	0.422	0.304	0.363	±	0.083	0.162	0.128	0.145	±	0.024

SD = Standard deviation

Duplicate exposures are indicated by A and B.

In this table the values are corrected for background absorption (0.0414). Isopropanol was used to measure the background absorption.

Table2          
Mean Tissue Viability in the in vitro Skin Corrosion Test with Naphthenic acids, zinc salts

	3-minute application viability (percentage of control)	1-hour application viability (percentage of control)
Negative control	100	100
Test item	60	83
Positive control	20	7.6

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion the test item is not corrosive in the in vitro skin corrosion test under experimental conditions.

Executive summary:

Eurling (2017) is a GLP-compliant study following the OECD Guideline 431. The study is assigned a Klimisch score of 1 (reliable with restrictions). The mean tissue viability in the in vitro skin corrosion test had a 100% tissue viability for the negative control after a 3 minute and 60-minute application indicated no potential for skin corrosion which was expected. The positive control had a mean tissue viability of 20% for the 3-minute application and 7.6% mean tissue viability after a 1-hour application, which was a positive indication of skin corrosion. The test item had a mean tissue viability of 60% after a 3-minute application and a mean tissue viability of 83% in the 1-hour application. Therefore, the test item to be considered as non-corrosive to the skin.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 - 29 January 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.46 “In vitro Skin Irritation: Reconstructed Human Epidermis Model Test ".

Version / remarks:

Official Journal of the European Union No. L142; Amended by EC No. 640/2012 OJ No. L193, 20 July 2012.

Deviations:

no

GLP compliance:

yes

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: Room temperature in the dark

Test system:

human skin model

Remarks:

EPISKIN Small Model (EPISKIN-SM)

Source species:

other: adult human-derived epidermal keratinocytes

Cell type:

non-transformed keratinocytes

Cell source:

other: EPISKIN Small Model (EPISKIN-SM)

Source strain:

other: EPISKIN Small Model (EPISKIN-SM)

Details on animal used as source of test system:

EPISKIN Small Model(TM) (EPISKIN-SM (TM)) - This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes are cultured for 13-days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small Model (EPISKIN-SM), 0.38 cm2.
- Tissue batch number(s): 18-EKIN-004
- Production date: Not specified
- Shipping date: Not specified
- Delivery date: Not specified
- Date of initiation of testing: 23 January 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C (actual range 36.5 - 37.3°C)
- Temperature of post-treatment incubation: 37°C (actual range 36.5 - 37.3°C)

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Thoroughly rinsed with phosphate buffered saline (PBS)
- Observable damage in the tissue due to washing: None
- Modifications to validated SOP: None

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm
- Filter and bandwidth: Not specified
- Linear OD range of spectrophotometer: Not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Yes
- Reproducibility: Standard deviation for negative control 0.18 (n=174). Standard deviation for positive control 0.08 (n=173)

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Naphthenic acids, zinc salts was checked for possible direct MTT reduction and color interference in the Skin corrosion test using EpiDerm as a skin model (Test Facility Study No. 20137507). Because no color changes were observed, it was concluded that Naphthenic acids, zinc salts did not interact with the MTT endpoint.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the Relative mean viability of 3 individual tissues after 15 minutes of exposure and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
- The test substance is considered to be non-irritant to skin if the Relative mean viability of 3 individual tissues after 15 minutes of exposure and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 622.4 mg
- Concentration: Applied neat

VEHICLE
- Amount(s) applied (volume or weight with unit): None

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 µL PBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 µL SDS
- Concentration: 5%

Duration of treatment / exposure:

15 ± 0.5 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Value:

86

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Since the mean relative tissue viability for Naphthenic acids, zinc salts was above 50% Naphthenic acids, zinc salts is considered to be non-irritant.
The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 7.5% (acceptability criteria = <50%). The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was 14% (acceptability criteria ≤ 18%), indicating that the test system functioned properly.

Table1          
Mean Absorption in the In Vitro Skin Irritation Test with Naphthenic acids, zinc salts

	A (OD570)	B (OD570)	C (OD570)	Mean (OD570)		SD
Negative control	0.824	0.815	0.867	0.836	±	0.028
Naphthenic acids, zinc salts	0.745	0.816	0.519	0.717	±	0.115
Positive control	0.075	0.063	0.051	0.063	±	0.012

OD = optical density

SD = Standard deviation

Triplicate exposures are indicated by A, B and C.

In this table the values are corrected for background absorption (0.0412). Isopropanol was used to measure the background absorption.

 

Table2          
Mean Tissue Viability in the In Vitro Skin Irritation Test with Naphthenic acids, zinc salts

	Mean tissue viability (percentage of control)	Standard deviation (percentage)
Negative control	100	3.4
Naphthenic acids, zinc salts	86	14
Positive control	7.5	1.4

Interpretation of results:

GHS criteria not met

Conclusions:

Naphthenic acids, zinc salts is non-irritant in the in vitro skin irritation test under the experimental conditions described and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations

Executive summary:

Naphthenic acids, zinc salts, neutral is considered to be non-irritant using the EPISKIN (TM) human epidermis model. The skin irritation potential of Naphthenic acids, zinc salts, neutral was evaluated using EPISKIN (TM) reconstructed human epidermis model after a treatment period of 15 minutes, followed by a post-exposure period of 42 hours, using a colourimetric MTT reduction assay following OECD guideline 439 in an experimental proprietary study (CRL 2018). The quality criteria required for the acceptance of results in the test were satisfied and the study is considered relevant and reliable for use.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 November 2017- 28 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

Adopted October 9 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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: Room temperature in the dark

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Slaughterhouse (Vitelco’s, Hertogenbosch, The Netherlands)
- Number of animals: Not specified.
- Characteristics of donor animals: Young cattle.
- Storage, temperature and transport conditions of ocular tissue: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- Time interval prior to initiating testing: The eyes were excised by a slaughterhouse employee as soon as possible after slaughter. The eyes were checked for unacceptable defects and the isolated corneas were incubated for a minimum of 1 hour.
- Indication of any existing defects or lesions in ocular tissue samples: The eyes were checked for unacceptable defects,such as opacity, scratches, pigmentation and neovascularization, by removing them from the physiological saline and holding them up to the light. Those exhibiting defects were discarded.
- Indication of any antibiotics used: Not reported

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: The test item was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (a filtration paper with an excessive amount of test item on it).
- Concentration: Test material was tested neat.
- Application method: It was not possible to pipet the test item due to physical properties. Since no workable suspension of naphthenic acids, zinc salts in physiological saline could be obtained, the test item was used as delivered by the sponsor and added pure on top of the corneas using a filtration paper with an excessive amount of test item on it. The test item was spread on a filter paper, which was placed upside down on the cornea in order to cover the cornea completely with test item. The cornea is not in contact with the paper. Since controls are liquids, it is not possible to apply these in the same way as the test item.

CONTROLS
- Amount(s) applied: 750 μL was introduced onto the epithelium of the cornea

Duration of treatment / exposure:

Corneas were incubated in a horizontal position for 240 ± 10 minutes

Duration of post- treatment incubation (in vitro):

None

Number of animals or in vitro replicates:

Three test replicates

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
- Preparation: The eyes were checked for defects such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. The isolated corneas were then stored in a petri dish with Earle’s Minimum Essential Medium (cMEM) containing 1% (v/v) L-glutamine and 1% (v/v) Foetal Bovine Serum. The isolated corneas were mounted in a corneal holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM at 32 ± 1°C.

QUALITY CHECK OF THE ISOLATED CORNEAS
- Selection: The corneas were incubated for a minimum of 1 hour at 32 ± 1°C. The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: Physiological saline

SOLVENT CONTROL USED: Not applicable

POSITIVE CONTROL USED: Imidazole

APPLICATION DOSE: 750 μL of positive and negative control solutions were applied to the epithelium of the bovine cornea. The test item was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (a filtration paper with an excessive amount of test item on it).

TREATMENT INCUBATION PERIOD: The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32 ± 1°C. After the incubation the solutions and the test compound were removed

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: The epithelium was washed at least three times with MEM with phenol red (Earle’s Minimum Essential Medium Life Technologies). The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM and the opacity determinations were performed.

POST-EXPOSURE INCUBATION: Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Sigma-Aldrich, Germany) was evaluated. The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 mL of 5 mg Na-fluorescein/mL cMEM solution (Sigma-Aldrich Chemie GmbH, Germany). The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 ± 5 minutes at 32 ± 1°C.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter. The opacity value was measured with the device OP-KIT.
- Corneal permeability: The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader).
- Others: After treatment incubation, the solutions and the test compound were removed and the epithelium was washed then each cornea was inspected visually for dissimilar opacity patterns.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
- Calculations: In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)
- Cut-off values: In vitro score range UN GHS: ≤ 3 - No Category; > 3 - ≤ 55 - No prediction can be made; >55 - Category 1

DECISION CRITERIA: The IVIS cut-off values for identifying the test items as inducing serious eye damage (UN GHS Category 1) and test items not requiring classification for eye irritation or serious eye damage (UN GHS No Category) were used.

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

13

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

2

Value:

11

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

3

Value:

6.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

10

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

cornea opacity score

Run / experiment:

Mean

Value:

2.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: Permeability

Run / experiment:

Mean

Value:

0.509

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 168 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.
Naphthenic acids, zinc salts induced ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 10 after 240 minutes of treatment. In conclusion, since naphthenic acids, zinc salts induced an IVIS > 3 ≤ 55, no prediction on the classification can be made.

A summary of Opacity, Permeability and In Vitro Scores

Treatment	Mean Opacity	Mean Permeability	Mean In vitro Irritation Score^1, 2
Negative control	1.9	0.023	2.2
Positive control	142	1.719	168
Test item	2.6	0.509	10

1 Calculated using the negative control mean opacity and mean permeability values for the positive control and test item.

2 In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

Historical Control Data for the BCOP Studies

	Negative control			Positive control
	Opacity	Permeability	In vitro Irritancy Score	In vitro Irritancy Score
Range	-5.4 – 5.2	-0.010 - 0.205	-5.3 – 5.3	86.5 – 211.4
Mean	0.50	0.02	0.72	135.88
SD	1.83	0.02	1.89	26.52
n	136	136	136	137

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of Nov 2014 to Nov 2017.

 

Interpretation of results:

study cannot be used for classification

Conclusions:

In conclusion, no prediction on the classification of the test item can be made. Naphthenic acids, zinc salts induced ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 10 after 240 minutes of treatment. However, since naphthenic acids, zinc salts induced an IVIS > 3 ≤ 55, no prediction on the classification can be made.

Executive summary:

The study is assigned a reliability score of 1 (reliable without restrictions) as it followed OECD Guideline 437: Bovine Corneal Opacity and Permeability Test Method) and is also compliant with GLP (Eurling 2017). The positive control mean value from the study fulfilled the acceptance criteria for the in vitro irritancy score (168) in accordance to the historical control data range (86.5-211.4). The negative control values for opacity and permeability were less than the upper limits of the laboratory historical range. The test item induced an In vitro irritancy score of more than 3 and less than 55, which concludes no prediction on the classification can be made.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 January 2018 - 16 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

Adopted 28 July 2015

Deviations:

no

GLP compliance:

yes

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: Room temperature in the dark

Species:

human

Strain:

not specified

Details on test animals or tissues and environmental conditions:

- Test system : The EpiOcular tissue construct is a non-keratinized epithelium prepared from normal human keratinocytes. It models the cornea epithelium with progressively stratified, but not cornified cells.
- Source: EpiOcular™ (OCL-200-EIT MatTek Corporation, Lot: 27420. Kit A and B), MatTek Corporation, Ashland MA, U.S.A.
- Test area: Non-keratinized epithelium (0.6 cm2)
- Justification: 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 eye irritation tests is the EpiOcular test, which is recommended in international guidelines and scientific publications (e.g. OECD).

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Due to the physical characteristics of the test item, an abundance was applied directly on top of the skin tissue. No correction was made for the purity/composition of the test item.

Duration of treatment / exposure:

After the exposure period with Naphthenic acids, zinc salts (6 hours ± 15 minutes at 37.0 ± 1.0°C), the tissues were thoroughly rinsed with Ca2+ Mg2+-free D-PBS (brought to room temperature) to remove residual test item.

Duration of post- treatment incubation (in vitro):

After rinsing the cell culture inserts were dried and immediately immersed in 5 mL of Assay Medium (room temperature) for a 25 ± 2 minute (Post-Soak). After the Post-Soak period, cell culture inserts were dried and transferred to 1.0 mL of Assay Medium and incubated for 18 hours ± 15 minutes at 37°C.

Number of animals or in vitro replicates:

The test was performed on a total of 2 tissues per test item together with 2 tissues per negative control and positive control.

Details on study design:

- RhCE tissue construct used, including batch number : EpiOcular™ (OCL-200-EIT MatTek Corporation, Lot: 27420. Kit A and B)
- Doses of test chemical and control substances used : Two tissues were treated with 50 µL Milli-Q water (negative control) and 2 tissues with 50 µL Methyl Acetate (positive control) respectively. An abundance of test item was added into the 6-well plates on top of the tissues.
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: Treatments were exposed for 6 hours ± 15 minutes at 37.0 ± 1.0°C, followed by a 25 ± 2 minute Post-Soak and 18 hours ± 15 minutes post-treatment incubation at 37°C.
- Description of any modifications to the test procedure : None
- Number of tissue replicates used per test chemical and controls (positive control, negative control): 2 tissues

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: The test item did not interact with the MTT endpoint and did not induce colour interference.
- Cell viability measurement method: After incubation, cell culture inserts were dried and transferred to 0.3 mL MTT-medium (1.0 mg/mL) then incubated for 180 ± 10 minutes at 37°C. After incubation with MTT-medium, the tissues dried and transferred to 2 mL isopropanol (so no isopropanol flowed into the insert). Formazan was extracted with 2 mL isopropanol for 2 - 3 hours at room temperature with gentle shaking.
- Description of the method used to quantify MTT formazan: The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

- Description of evaluation: Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Eye hazard potential of the test item was classified according to remaining cell viability following exposure of the test item.
- Classification criteria: 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. The test chemical is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post exposure incubation is less than or equal (≤) to 60%.

- Positive control means and acceptance ranges based on historical data : The positive control had a mean cell viability after 6 hours ± 15 minutes exposure of 24%.
- Negative control means and acceptance ranges based on historical data: The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range.
- Acceptable variability between tissue replicates for positive and negative controls : The difference between the percentage of viability of two tissues treated with either the positive or negative control was less than 6%, indicating that the test system functioned properly.
- Acceptable variability between tissue replicates for the test chemical: The difference between the percentages of viability of two tissues treated with the test item was less than 23%, which is above the acceptability criterion of <20%. However, all individual viabilities are below 60% (7% and 30%), and therefore this deviation does not influence the outcome of the test.

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

19

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

- Conclusion: The relative mean tissue viability obtained after 6 hours ± 15 minutes treatment with Naphthenic acids, zinc salts compared to the negative control tissues was 19%. Since the mean relative tissue viability for Naphthenic acids, zinc salts, neutral was below 60% it is considered to be potentially irritant or corrosive to the eye. The test item is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1).

Table 1. Mean Absorption in the EpiOcular™ Test with Naphthenic acids, zinc salts, neutral

	A (OD570)	B (OD570)	Mean (OD570)		SD
Negative control	1.690	1.687	1.689	±	0.002
Naphthenic acids, zinc salts, neutral	0.124	0.505	0.315	±	0.270
Positive control	0.353	0.451	0.402	±	0.069

OD = optical density

SD = Standard deviation

Duplicate exposures are indicated by A and B.

In this table, the values are corrected for background absorption (0.041). Isopropanol was used to measure the background absorption.

 

Table 2. Mean Tissue Viability in the EpiOcular™ Test with Naphthenic acids, zinc salts, neutral

	Mean tissue viability (percentage of control)	Difference between two tissues (percentage)
Negative control	100	0.2
Naphthenic acids, zinc salts, neutral	18.6	22.6
Positive control	23.8	5.8

 

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Naphthenic acids, zinc salts, neutral gave a relative mean tissue viability of 19% so is considered to be potentially irritant or corrosive to the eye and potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1).

Executive summary:

CRL (2018) is a GLP-compliant Reconstructed Human EpiOcular™ Model study on naphthenic acids, zinc salts, neutral, following OECD guideline 492. The study has been assigned a Klimisch score of 1, reliable with restriction. Naphthenic acids, zinc salts, neutral gave a relative mean tissue viability of 19% so is considered to be potentially irritant or corrosive to the eye and potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Naphthenic acids, zinc salts does not meet the criteria for skin irritation or corrosion classification

Naphthenic acids, zinc salts does not meet the criteria for classification for serious eye damage but meets the criteria for eye irritation category 2 (H319: Causes serious eye irritation) under EU CLP.