Benzenesulfonic acid, 4-dodecyl-, cerium(4+) salt, basic

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-06-27 to 2018-06-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

29-06-2015

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2018-04-26

Test material

Test material information

Constituent 1

Reference substance name:

Benzenesulfonic acid, 4-dodecyl-, cerium(4+) salt, basic

EC Number:

282-500-0

EC Name:

Benzenesulfonic acid, 4-dodecyl-, cerium(4+) salt, basic

Cas Number:

84238-45-9

IUPAC Name:

Benzenesulfonic acid, 4-dodecyl-, cerium(4+) salt, basic

Test material form:

solid: particulate/powder

Details on test material:

- State of aggregation: brown powder

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, in a sealed container

Test animals / tissue source

Details on test animals or tissues and environmental conditions:

JUSTIFICATION OF THE TEST METHODS AND CONSIDERATIONS REGARDING APPLICABILITY:
The EpiOcular™ Eye Irritation Test (EIT) was validated by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) and Cosmetics Europe from 2008 to 2013. From this validation study and its independent peer review it was concluded that the EpiOcular™ EIT is able to correctly identify chemicals (both substances and mixtures) not requiring classification and labelling for eye irritation or serious eye damage according to UN GHS (i.e. “No Category”), and the test method was recommended as scientifically valid for this purpose. 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.

RhCE TISSUE CONSTRUCT USED: EpiOcular™ (Lot No.: 27051; source: MatTek (Bratislava, Slovakia))
The test was carried out with the EpiOcular™ reconstructed human cornea-line epithelium (RhCE) model. 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): approx. 50 mg (83.3 mg/cm²) of the test item

Duration of treatment / exposure:

6 ± 0.25 hours

Observation period (in vivo):

not applicable

Duration of post- treatment incubation (in vitro):

18 ± 0.25 hours

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 mg of the test item were mixed per 1 mL MTT medium and incubated for 3 hours 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 mg 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 hour 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 hours. After the 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. Since one of the two ODnet was > 0.08, or if the intrinsic colour of the test item is blue, black or dark-purple, the test item was checked for its tissue-colouring potential. For quantitative correction of results, the colorant control test was performed using two additional living tissues treated with 50 mg 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.

EXPERIMENT - DETAILS ON 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 minutes.
- then, the EpiOcular™ tissues were transferred into 6-well plates containing 1 mL pre-warmed assay medium per well and incubated for 1 hour in a humidified incubator at 37 ± 2 °C, 5.0% CO2 / 95% air.

- next, 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 23.5 hours.
- after the overnight incubation the tissues were pre-treated with 20 µL of DPBS-buffer and incubated for 30 ± 2 minutes in a humidified incubator at 37 ± 2 °C, 5.0% CO2 / 95% air.

- afterwards, the tissues were treated with each dose group in duplicate, starting with the negative and positive control follwoed by the test item.
- after dosing, the inserts were placed back into the culture medium and were incubated for 6 ± 0.25 h 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 the tissue with DPBS.
- complete test material could not be washed off resulting in little remaining test material on the skin tissue surface. As this little residues were only at the edge of the tissues, the impact on the results are presumably negligible.
- after rinsing the inserts were transferred to and immersed in a prepared 12-well plate, containing fresh pre-warmed assay medium per well and incubated for 25 ± 2 minutes at room temperature.
- then, the inserts were removed from the assay medium, the medium was decanted off the tissue and the tissues were blotted.
- the inserts were transferred to a new 6-well plate containing pre-warmed assay medium and the tissues were incubated for 18 ± 0.25 hours at 37 ± 2°C, 5.0% CO2 / 95% air.

MTT ASSAY
- after the incubation period excess medium was removed and the inserts were transferred in a prepared 24-well plate containing 0.3 mL pre-warmed MTT medium and further incubated for 3 hours ± 15 minutes at 37 ± 2 °C, 5.0% CO2 / 95% air.
- following the MTT incubation period, the inserts were removed, the bottom of the inserts blotted and transferred into new 6-well plates, containing 2 mL of isopropanol to extract only the bottom of the tissues.
- 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 not pierced to avoid contamination of the extract with remaining test item.
- 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 optical density (OD) was measured at 570 nm using a filter band pass of maximum ± 30 nm in a plate spectrophotometer using isopropanol as a blank.

DATA ANALYSIS
- mean optical density (OD) of the two negative control tissues will be calculated after blank correction (corresponds to 100 % tissue viability).
- 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 a prediction model.

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.
- if the mean percent tissue viability after exposure and post-exposure incubation is ≤ 60% tissue viability, no prediction can be made.
- if the mean percent tissue viability after exposure and post-exposure incubation is > 60%, the test chemical is identified as not requiring classification and labelling according to UN GHS (No Category).

TEST ACCEPTANCE CRITERIA:
The test meets acceptance criteria if:
- 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 should not be below historically established boundaries/ positive and negative control mean values and acceptance ranges based on historical data.

Results and discussion

In vitro

Results

Irritation parameter:

other: % tissue viability (mean)

Value:

1.3

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

PRE-EXPERIMENT
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.

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.) = 0.0509 – 0.0429 = 0.008
ODnet(Isopropanol) = meanODIsopropanol – mean ODBlank(Isopropnaol) = 1.4277 – 0.4405 = 1.38365
As ODnet(Isopropanol) was >0.08, coloured tissue controls were performed for quantitative correction of results.

The non-specific colour of additional viable tissues (NSCliving) was calculated according to the following formula:
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 [%] = 1.5% - 0.2% = 1.3%

EXPERIMENT
After treatment with the test item, the mean relative tissue viability compared to the relative mean tissue viability of the negative control was reduced to ≤ 60% (1.3%, NSCliving-corrected) after 6 hours treatment and 18 hours post-incubation. Since the mean percent tissue viability after exposure and post-exposure incubation is less than 60% tissue viability, no prediction of the ocular irritation potential of the test item can be made.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: mean absolute OD570 of the two negative control tissues was > 0.8 and < 2.5 (1.566)
- Acceptance criteria met for positive control: mean relative tissue viability (% negative control) of the positive control was < 50% (23.8%).
- The maximum inter-tissue difference of replicate tissues of all dose groups was < 20% (0.1 - 11.6%).
- The absorbance values were not below historically established boundaries.

Any other information on results incl. tables

Table 1: Result of the test item benzenesulfonic acid, 4-dodecyl-, cerium(4+) salt, basic

Name	Negative Control		Positive Control			Test item
Tissue	1	2	1		2	1	2
Absolute OD570values	1.665	1.474	0.423		0.393	0.068	0.067
	1.645	1.481	0.421		0.392	0.068	0.065
Mean Absolute OD570values	1.566****		0.407			0.067
OD570values (blank-corrected)	1.620	1.429	0.378		0.348	0.023	0.022
	1.600	1.437	0.377		0.347	0.023	0.021
Mean OD570of the duplicates (blank-corrected)	1.610	1.433	0.377		0.347	0.023	0.021
Total Mean OD570of 2 Replicate Tissues (blank corrected)	1.521*		0.362			0.022
SD of Mean OD570of the Replicates (Blank Corrected)	0.125		0.021			0.001
Relative tissue viability [%]	105.8	94.2	24.8	22.8		1.5	1.4
Relative tissue viability difference [%]^***	11.6		2.0			0.1
Mean of relative tissue viability [%]	100.0		23.8**			1.5
Mean Relative Tissue Viability [%] - NSClivingCorrected	-		-			1.3

^*            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 OD₅₇₀of the negative control tissues is > 0.8 and < 2.5.

Table 2: Result of the NSCliving control

NSCliving	TVT
Tissue	1	2
absolute OD570 -values	0.048	0.048
	0.048	0.048
absolute OD570 - Blank corrected values	0.003	0.003
	0.003	0.003
mean OD570 (mean of 2 aliquots)	0.003	0.003
total mean OD570 (mean of replicate tissues)	0.003
SD OD570 (of the 2 replicate tissues)	0.000
NSCliving[%]	0.2
Relative Tissue Viability [%]	-
Mean Relative Tissue Viability [%]	-
SD Tissue Viability [%]	-
CV [% Viabilities]	-

Table 3: Historical data (from 2017 - 2018)

	Mean Absolute OD570±30nmNC	MeanAbsoluteOD570±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

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

According to the reconstructed human cornea-like epithelium (RhCE) test method, since the mean percent tissue viability after exposure and post-exposure incubation is less than 60% tissue viability (1.3 %, NSCliving-corrected), no prediction of the ocular irritation potential of the benzenesulfonic acid, 4-dodecyl-, cerium(4+) salt, basic can be made.