Naphthenic acids, copper salts

Endpoint summary

Description of key information

Skin corrosion/irritation: not irritating (OECD 404; GLP)

Eye irritation: not irritating (OECD 492; GLP)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-08-27 to 2018-09-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

2015-07-28

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2018-04-26

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: -15°C to 40°C

Species:

rabbit

Strain:

New Zealand White

Remarks:

Crl:KBL (NZW)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, 97633 Sulzfeld, Germany
- Age at study initiation:
animal no. 1 approx 19-20 weeks
animal no. 2 approx. 20-21 weeks
- Weight at study initiation: 3.4 – 3.8 kg
- Housing: individually housed in ABS-plastic or Noryl rabbit cages, floor 4200 cm2
- Diet (ad libitum): autoclaved hay and Altromin 2123 maintenance diet for rabbits, rich in crude fibre
- Water (ad libitum): tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 18 ± 3 °C
- Relative humidity: 55 ± 10 %
- Air changes: at least 10 x / hour
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

water

Remarks:

A small amount of aqua ad injectionem was used for moistening in order to ensure contact between test material and skin

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 g of the test item
In order to ensure good skin contact, it was moistened with aqua ad injectionem.

Duration of treatment / exposure:

initial animal: 3 minutes, 1 hour and 4 hours
confirmatory animal: 4 hours

Observation period:

72 hours

Number of animals:

2 male rabbits

Details on study design:

TEST SITE
- Area of exposure/Type of wrap if used: approx. 24 hours before the test, the fur was removed from the dorsal area of the trunk by using an electric clipper. The test item was applied to a small area (approx. 6 cm²) of skin on one side of the dorsal area and covered with a gauze patch, which was held in place with a non-irritating tape. The untreated other side served as control. The test item was applied to the patch first, moistened with the smallest amount of aqua ad injectionem and then applied to the skin. The patch was fixed with a semi-occlusive dressing. The limits of the application site were marked with an ink marker.

INITIAL AND CONFIRMATORY TESTING
As the test item is expected to produce severe irritancy/corrosion, a single animal test was employed. Up to three test patches were applied sequentially to the animal. The first patch was removed after three minutes. No serious skin reaction was observed, so a second patch was applied at a different site and removed after one hour. As no signs of skin irritation were noted at this stage, the test item was thereafter held in contact with the skin throughout a 4-hour period.

The results of the initial test indicated that the test item is not corrosive to the skin using the procedure described. In order to confirm the reversible irritant response, one additional animal was treated in the same manner. According to OECD 404, section 17, treatment of a third animal can be omitted when animal no. 1 and 2 exhibit the same response. Furthermore, according to the classification directives, the results of animal no. 1 and 2 were sufficient for classification of the test item. Moreover, as the test item showed no signs of corrosion in two animals and full reversibility of the effects, it is considered that adding a third animal would not change the outcome of the study.

REMOVAL OF TEST SUBSTANCE
As the test item showed a water-repellent effect, an appropriate solvent, cottonseed oil (Sigma, lot no. MKCC5905, expiry date: 21/10/2018), was used for rinsing the application site after patch removal. The solvent was chosen in order not to alter the existing response or the integrity of the epidermis.

OBSERVATION TIME POINTS
- initial animal: immediatley and 1 hour, 24, 48,72 and 96 hours after patch removal
- confirmatory animal: 1 hour, 24, 48 and 72 hours after patch removal

SCORING SYSTEM
according to the Draize scale

FURTHER OBSERVATIONS
- body weights: prior to the administration and at the end of the observation period
- local effects such as hyperplasia, scaling, discolouration, fissures and scabs
- systemic effects

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

0.67

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritant / corrosive response data:

INITIAL ANIMAL:
After patch removal during sequential application (3 min, 1 hour) no signs of irritation were noted in animal no. 1. Erythema grade 1 was first observed immediately, 1 hour and 24 hours after 4-hour exposure. The effect was reversible within 48 hours after application of the test material.

CONFIRMATION ANIMAL:
Animal no. 2 showed erythema grade 1 at 1 hour, 24 and 48 hours after patch removal. The effect disappeared within 72 hours after application.

The single dermal application of 0.5 g Copper naphthenate to two rabbits showed irritant (very slight erythema, grade 0.67 and 0.33) but not corrosive effects. In both animals these findings were reversible within the observation time period of 48 hours (animal no. 1) and 72 hours (animal no. 2).

Other effects:

- Neither mortalities nor local or systemic effects were observed.
- There were no significant body weight changes during the observation period
- The body weight development was within the expected range.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is not irritating to the skin.
According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance does not require classification as skin irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

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

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

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

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.

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

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.

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.

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).

Endpoint conclusion

Endpoint conclusion:

no study available

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin corrosion/irritation:

The substance is either corrosive or irritating to the skin (OECD 439; GLP). Since the RhE test methods covered by OECD TG 439 cannot resolve between UN GHS Categories 1 or 2, a follow-up study was conducted according to OECD 435 (GLP). However, the OECD 435 test method was not applicable, since the receptor fluid was not activated by the test substance. Thus, an in vivo skin irritation study (OECD 404; GLP) was conducted. In this study, the substance displayed no irritating properties to the skin.

Eye irritation:

The OECD 437 (GLP) test method was was not applicable. Thus, follow-up test (OECD 492; GLP) was conducted.

The substance was observed to be not irritating to the eyes in an in vitro eye irritation study according to OECD 492.

Justification for classification or non-classification

Skin irritation:

Naphthenic acids, copper salts does not possess a skin irritating potential based on an in vivo OECD 404 test and does not require classification according to Regulation (EC) No 1272/2008 and its subsequent adaptations.

Eye irritation:

Naphthenic acids, copper salts does not possess a serious eye damaging or irritating potential based on an in vitro OECD 492 test and does not require classification according to Regulation (EC) No 1272/2008 and its subsequent adaptations.