Bis[4-(tert-butyl)benzoato-O]hydroxyaluminium

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-05-03 - 2018-06-08

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:

OECD Guideline for the Testing of Chemicals No. 492: Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage, 09 Oct 2017

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM DB-ALM Method Summary No. 164: EpiOcular™ Eye Irritation Test - Summary, 22 July 2015

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: 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, 29 June 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Bis[4-(tert-butyl)benzoato-O]hydroxyaluminium

EC Number:

236-109-7

EC Name:

Bis[4-(tert-butyl)benzoato-O]hydroxyaluminium

Cas Number:

13170-05-3

Molecular formula:

C22H27AlO5

IUPAC Name:

aluminum hydroxide bis(4-tert-butylbenzoate)

Test material form:

solid: particulate/powder

Test animals / tissue source

Species:

human

Strain:

other: not applicable

Remarks:

he EpiOcular tissue construct is a non-keratinized epithelium (0.6 cm²) prepared from normal human keratinocytes (MatTek).

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability : This test uses the three-dimensional RhCE EpiOcular™ (MatTek). It consists of normal, human-derived epidermal keratinocytes and mimics the histological, morphological, biochemical and physiological properties of the human corneal epithelium. The MatTek EpiOcular™ model has been widely used as a research and testing model for many years.
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.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: 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.
The EpiOcular™ tissues were provided as kits (e.g. OCL-200-EIT; MatTek), consisting of the following components relevant for this study:
1x sealed 24-well plate containing 24 inserts with EpiOcular™ tissues on agarose
(Lot No.: 27039 for main experiment, 27044 for viable tissue controls, 27019 for killed tissue controls)
1x bottle EpiOcularTM assay medium (Lot No.: 060418ISA, 043018ISA)
1x bottle Ca2+/Mg2+-free DPBS buffer (Lot No.: 092817MGKA)
Source: MatTek Corporation, Ashland MA, U.S.A.

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 mg (83.3 mg/cm2) of the test item were applied directly atop the EpiOcular™ tissue using an application spoon avoiding compression of the test item. The test item was spread to match size of the tissue.
After washing, it was not possible to completely wash off the test material resulting in little remaining test material on the skin tissue surface.

Duration of treatment / exposure:

6 ± 0.25 h

Duration of post- treatment incubation (in vitro):

18 ± 0.25 h

Number of animals or in vitro replicates:

The test was performed on a total of 2 tissues per dose group.

Details on study design:

- 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 16 - 24 h.
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. While the test item was applied, the tissue inserts were placed on a sterile surface. After dosing, the inserts were placed back into the culture medium. Then the 6-well plate(s) 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. 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 25 ± 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 18 ± 0.25 h at 37 ± 2°C, 5.0% CO2 / 95% air.

- RhCE tissue construct used, including batch number :
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.
The EpiOcular™ tissues were provided as kits (e.g. OCL-200-EIT; MatTek), consisting of the following components relevant for this study:
1x sealed 24-well plate containing 24 inserts with EpiOcular™ tissues on agarose (Lot No.: 27039 for main experiment, 27044 for viable tissue controls, 27019 for killed tissue controls)
1x bottle EpiOcularTM assay medium (Lot No.: 060418ISA, 043018ISA)
1x bottle Ca2+/Mg2+-free DPBS buffer (Lot No.: 092817MGKA

- Doses of test chemical and control substances used :
Approximately 50 mg (83.3 mg/cm2) of the test item were applied directly atop the EpiOcular™ tissue. Controls were set up in parallel to the test item cultures in order to confirm the validity of the test.
Negative Control: Distilled water (Aqua dest.; Sigma, Lot No.: RNBG3520)
Positive Control: Methyl acetate (CAS No. 79-20-9; Merck, Lot No.: S6943111)

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable) :
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 h in a humidified incubator at 37 ± 2 °C, 5.0% CO2 / 95% air. If the mixture turned blue/purple, the test item was presumed to have reduced MTT. The part of absorption due to the non-specific reduction of MTT (NSMTT) was determined by using killed tissues if the mean relative tissue viability of the test item treated tissues (TM) was above the 60% threshold value. For quantitative correction of results, two killed tissues were treated with 50 mg of the test item (KT) and one tissue was treated with 50 µl of the negative control (Aqua dest.; KU), respectively. NSMTT was calculated relative to the negative control of living tissues (NK) according to the following formula:
NSMTT [%] = [(ODKT - ODKU)/ODNK] * 100
If the viability difference of the two identically treated killed test item treated tissues (KT) was > 20% the killed control was considered as non-qualified.
If NSMTT was ≤ 60% relative to the negative control of living tissues it was used for quantitative correction of the results by calculation of the killed control corrected viability (KCCV), which was considered for classification of the test item according to the following formula:
KCCV [%] = viabilityTM – NSMTT
If NSMTT was > 60% relative to the negative control of living tissues the results obtained should be taken with caution as this is the cut-off used to distinguish classified from not classified test items.
If uncorrected ODKT of the tissue extracts fell outside the linear range of the spectrophotometer the test item was considered as incompatible with the test method.
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 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. If 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 test was performed using two additional living tissues treated with 50 mg of the test item (TVT) if the mean relative tissue viability of the test item treated tissues (TM) was above the 60% threshold value. All steps were performed exactly as described in the chapter below, except for the MTT-staining of the test item treated with tissues, which was incubated in medium without MTT. The non-specific colour of additional viable tissues (NSCliving) was then calculated according to the following formula:
NSCliving [%] = [ODTVT/ODNK]*100
If the viability difference of the two identically treated additional viable test item treated tissues (TVT) was > 20% the colour control was considered as non-qualified.
If NSCliving was ≤ 60% relative to the negative control of living tissues the mean relative tissue viability of the test item treated tissues (TM) was corrected to the NSC-corrected mean relative tissue viability (NSCCV), which was considered for classification of the test item according to the following formula:
NSCCV [%] = viabilityTM [%] – NSCliving [%]
If NSCliving was > 60% relative to the negative control of living tissues the results obtained should be taken with caution as this is the cut-off used to distinguish classified from not classified test items.
If uncorrected ODTVT of the tissue extracts fell outside the linear range of the spectrophotometer the test item was considered as incompatible with the test method.
For test items which act as non-specific MTT-reducers and show non-specific colouring of living tissues, a third control for non-specific colour in killed tissues (NSCkilled) was performed to avoid a possible double-correction for colour interference. Therefore, two killed tissues were treated with 50 mg of the test item (TKT). All steps were performed exactly as described in the chapter below, except for the MTT-staining of the test item treated with tissues, which were incubated in medium without MTT. The non-specific colour of additional killed tissues (NSCkilled) was then calculated according to the following formula:
NSCkilled [%] = [ODTKT/ODNK]*100
The true tissue viability was then calculated as the percent tissue viability obtained with living tissues minus NSMTT minus NSCliving plus NSCkilled.

- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable) : 2

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer) :
570 nm using a filter band pass of maximum ± 30 nm in a plate spectrophotometer using isopropanol as a blank.

- Description of the method used to quantify MTT formazan :
After the 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 6-well “extraction plates“, containing 2 mL of isopropanol to extract only the bottom of the tissues. The extraction plates were sealed to inhibit isopropanol evaporation. Extraction was carried out either after storage overnight in the dark at 2 - 8 °C (additional controls) or immediately by shaking on an orbital plate shaker for 2 - 3 h at room temperature (main experiment). At the end of the extraction period the tissues were not pierced to avoid contamination of the extract with remaining test item.
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.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the 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. The test item is considered to be irritant to the eye but it cannot be differentiated between UN GHS “Category 1” or “Category 2”, if the relative tissue viability is less or equal to 60%. The test item is considered to be non-irritant in accordance with UN GHS “No Category” if relative tissue viability is higher than 60%.

- Acceptability 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%.

Results and discussion

In vitro

Results

Irritation parameter:

other: Ocular irritation potential of the test item was predicted from the relative mean tissue viabilities

Run / experiment:

1

Value:

111.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none stated

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
The controls confirmed the validity of the study. The mean absolute OD570 of the two negative control tissues was > 0.8 and < 2.5 (1.764, 1.751). The mean relative tissue viability (% negative control) of the positive control was < 50% (24.9%). The maximum inter tissue difference of replicate tissues of all dose groups was < 20% (7.5%).

Any other information on results incl. tables

Results of the Pre-Experiments

The mixture of 50 mg test item per 1 mL MTT medium showed reduction of MTT as compared to the solvent. The mixture turned blue/purple. Since the mean relative tissue viability of the test item treated tissues (TM) was above the 60% threshold value, killed tissue controls were performed for quantitative correction of results.

NSMTT [%] = [(OD_KT- OD_KU)/OD_NK] * 100 = -0.3%

Difference of NSMTT of the two duplicate tissues must be < 20%, otherwise not accepted.

NSM_TT1 [%] = [mean OD_KT1 - OD_KU)/OD_NK] * 100 = -0.6%

NSM_TT2 [%] = [mean OD_KT2 - OD_KU)/OD_NK] * 100 = -0.1%

NSM_TT1 - NSMTT2 =±0.5%

NSM_TT was ≤ 60% (-0.3%) relative to the negative control of living epidermis and could therefore be used for determination of the killed control corrected viability (KCCV) according to the following formula:

KCCV [%] = viability_TM– NSM_TT = 109.1% - (-0.3%) = 109.4%

 

The mixture of 50 mg test item per 1 mL Aqua dest. showed no colouring as compared to the solvent. The mixture of 50 mg test item per 2 mL isopropanol showed colouring as compared to the solvent. Since the mean relative tissue viability of the test item treated tissues (TM) was above the 60% threshold value, coloured tissue controls were performedfor quantitative correction of results.

NSC_living[%] = [OD_TVT/OD_NK] * 100 = 0.3%

Difference of NSC_livingof the two duplicate tissues must be < 20%, otherwise not accepted.

NSC1 [%] = [OD_TVT1/OD_NK] * 100 = 0.3%

NSC2 [%] = [OD_TVT2/OD_NK] * 100 = 0.3%

NSC1 – NSC2 = ± 0.0%

NSC_livingwas ≤ 60% (0.3%) 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 [%] = viability_TM[%] – NSC_living[%] = 109.4% - 0.3% = 109.1 %

 

Since the test item showed non-specific MTT-reduction and non-specific colouring of living tissues, a third control for non-specific colour in killed tissues (NSC_killed) was performed to avoid a possible double-correction for colour interference.

The non-specificcolour of additional killed tissues (NSC_killed) was calculated according to the following formula:

NSC_killed[%] = [OD_TKT/OD_NK]*100 = 2.8 %

The true tissue viability was then calculated as the percent tissue viability obtained with living tissues minus NSM_TT minus NSC_livingplus NSC_killed.

True Tissue Viability = [%] mean Tissue viability – NSMTT - NSC_living+NSC_killed

        = 109.1% + 2.8% = 111.9%

Results of the Main-Experiment

Result of the Test Item Bis[4-(tert-butyl)benzoato-O]hydroxyaluminium

Name	Negative Control		Positive Control		Test item
Tissue	1	2	1	2	1	2
OD570values	1.890	1.678	0.478	0.450	1.921	1.949
	1.767	1.720	0.503	0.458	1.919	1.889
OD570values (blank-corrected)	1.846	1.634	0.434	0.406	1.877	1.904
	1.723	1.675	0.459	0.414	1.875	1.844
mean of the duplicates	1.784	1.655	0.447	0.410	1.876	1.874
mean OD	1.719*		0.428		1.875
TODTT- NSMTT	-		-		1.881
TODTTNSMTT and NSCliving	-		-		1.877
mean sd OD	0.092		0.026		0.001
tissue viability [%]	103.8	96.2	26.0	23.8	109.1	109.0
relative tissue viability difference [%]^***	7.5		2.1		0.1
mean tissue viability [%]	100.0		24.9**		109.1
mean tissue viability [%] - NSMTT corrected	-		-		109.4
mean tissue viability [%] - NSMTT and NSClivingcorrected	-		-		109.1
True Tissue Viability	-		-		111.9

^{* :} 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%

Result of the NSMTT control

NSMTT	KU		KT		Negative Control
Tissue	1	2	1	2	1	2
absolute OD570 -values	0.076	0.075	0.066	0.074	1.926	1.679
	0.077	0.076	0.066	0.074	1.900	1.678
OD570(Blank Corrected)	0.031	0.030	0.021	0.029	1.881	1.635
	0.033	0.032	0.022	0.030	1.855	1.634
mean OD570 (mean of 2 aliquots)	0.032	0.031	0.022	0.030	1.868	1.634
total mean OD570 (mean of the replicate tissues)	0.032		0.026		1.751
SD OD570(of the replicate tissues)	0.001		0.006		0.165
NSMTT [%]	-0.3				-
Relative Tissue Viability [%]	-				100.0	87.5
Mean Relative Tissue Viability [%]	-				93.7
SD Tissue Viability [%]	-				8.9
CV [% Viabilities]	-				9.4

Result of the NSC_livingcontrol

NSCliving	TVT		Negative Control
Tissue	1	2	1	2
absolute OD570 -values	0.049	0.048	1.926	1.679
	0.048	0.049	1.900	1.678
absolute OD570 - Blank corrected values	0.005	0.004	1.881	1.635
	0.004	0.005	1.855	1.634
mean OD570 (mean of 2 aliquots)	0.004	0.004	1.868	1.634
total mean OD570 (mean of replicate tissues)	0.004		1.751
SD OD570 (of the 2 replicate tissues)	0.000		0.165
NSCliving[%]	0.3		-
Relative Tissue Viability [%]	-		106.7	93.3
Mean Relative Tissue Viability [%]	-		100.0
SD Tissue Viability [%]	-		9.4
CV [% Viabilities]	-		9.4

Result of the NSC_killedcontrol

NSCkilled	TKT		Negative Control
Tissue	1	2	1	2
absolute OD570 -values	0.048	0.049	1.926	1.679
	0.048	0.050	1.900	1.678
absolute OD570 - Blank corrected values	0.048	0.049	1.881	1.635
	0.048	0.050	1.855	1.634
mean OD570 (mean of 3 aliquots)	0.048	0.049	1.868	1.634
total mean OD570 (mean of replicate tissues)	0.049		1.751
SD OD570 (of the 2 replicate tissues)	0.001		0.165
NSCkilled[%]	2.8		-
Relative Tissue Viability [%]	-		106.7	93.3
Mean Relative Tissue Viability [%]	-		100.0
SD Tissue Viability [%]	-		9.4
CV [% Viabilities]	-		9.4

 Test Acceptance Criteria

	Value	Cut off	pass/fail
Mean Absolute OD570 nmNK	1.764 (main experiment), 1.751 (additional controls)	0.8 < NK < 2.5	pass
Mean Relative Viability PC [%]	24.9	< 50%	pass
Max. Difference of % Viability [%]	7.5	< 20%	pass

Historical Data

	Absolute OD570 ± 30 nmNK	Relative Viability PC [%]	Difference of Viability [%]
Mean	1.687	24.6	6.9
SD	0.272	12.3	9.9
n	44	44	178

Historical control data were generated from 2012 – 2018.

Discussion

The potential of the test item to induce eye irritation was analysed by using the three-dimensional human corneal epithelium model EpiOcularä, consisting of normal, human-derived epidermal keratinocytes mimicking characteristics of the corneal epithelium.

In the present study Bis[4-(tert-butyl)benzoato-O]hydroxyaluminium was applied topically to the EpiOcular tissue for 6 h followed by 25 min post-soaking incubation after removal of the test item. After a 18 h post-treatment period cytotoxic effects were determined via MTT reduction assay.

Ocular irritation potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with Aqua dest.

 

The mixture of 50 mg test item per 1 mL MTT medium showed reduction of MTT as compared to the solvent. The mixture turned blue/purple. Since the mean relative tissue viability of the test item treated tissues (TM) was above the 60% threshold value, killed tissue controls were performed for quantitative correction of results.

NSMTT [%] = [(OD_KT- OD_KU)/OD_NK] * 100 = -0.3%

Difference of NSMTT of the two duplicate tissues must be < 20%, otherwise not accepted.

NSMTT1 [%] = [meanODKT1 - ODKU)/ODNK] * 100 = -0.6%

NSMTT2 [%] = [meanODKT2 - ODKU)/ODNK] * 100 = -0.1%

NSMTT1 - NSMTT2 =±0.5%

NSMTT was ≤ 60% (-0.3%) relative to the negative control of living epidermis and could therefore be used for determination of the killed control corrected viability (KCCV) according to the following formula:

KCCV [%] = viability_TM– NSMTT = 109.1% - (-0.3%) = 109.4%

 

The mixture of 50 mg test item per 1 mL Aqua dest. showed no colouring as compared to the solvent. The mixture of 50 mg test item per 2 mL isopropanol showed colouring as compared to the solvent. Since the mean relative tissue viability of the test item treated tissues (TM) was above the 60% threshold value, coloured tissue controls were performed for quantitative correction of results.

NSC_living[%] = [OD_TVT/OD_NK] * 100 = 0.3%

Difference of NSC_livingof the two duplicate tissues must be < 20%, otherwise not accepted.

NSC1 [%] = [OD_TVT1/OD_NK] * 100 = 0.3%

NSC2 [%] = [OD_TVT2/OD_NK] * 100 = 0.3%

NSC1 – NSC2 = ± 0.0%

NSC_livingwas ≤ 60% (0.3%) 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 [%] = viability_TM[%] – NSC_living[%] = 109.4% - 0.3% = 109.1 %

 

Since the test item showed non-specific MTT-reduction and non-specific colouring of living tissues, a third control for non-specific colour in killed tissues (NSC_killed) was performed to avoid a possible double-correction for colour interference.

The non-specific colour of additional killed tissues (NSC_killed) was calculated according to the following formula:

NSC_killed[%] = [OD_TKT/OD_NK]*100 = 2.8 %

The true tissue viability was then calculated as the percent tissue viability obtained with living tissues minus NSMTT minus NSC_livingplus NSC_killed.

True Tissue Viability = [%] mean Tissue viability – NSMTT - NSC_living+NSC_killed

        = 109.1% + 2.8% = 111.9%

 

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was> 60% (111.9%NSMTT-,NSC_living-corrected).

 

The controls confirmed the validity of the study. The mean absolute OD₅₇₀of the two negative control tissues was> 0.8 and < 2.5 (1.764, 1.751). The mean relative tissue viability (% negative control) of the positive control was < 50% (24.9%). The maximum inter tissue difference of replicate tissues of all dose groups was < 20% (7.5%).

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The study was conducted under GLP according to OECD guideline 492 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the irritating potential of the test substance to the eye in vitro.
In the present OECD 492 test, eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The mean relative tissue viability (% negative control) was > 60% (111.9% NSMTT-, NSCliving-corrected) after 6 ± 0.25 hours exposure period and 18 ± 0.25 hours post-treatment period. Since the mean relative tissue viability for Bis[4-(tert-butyl)benzoato-O]hydroxyaluminium was above 60%, the test item is classified as “non-irritant“ in accordance with UN GHS “No Category”.

Executive summary:

In the present study the eye irritating potential of Bis[4-(tert-butyl)benzoato-O]hydroxyaluminium was analysed. Since irritant substances are cytotoxic to the corneal epithelium after a short time exposure the cytotoxic effects of the test item on EpiOcular^TM, a reconstituted three-dimensional human corneal epithelium model, were determined. Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 6 h exposure period and 18 h post-treatment period and compared to those of the concurrent negative controls.

The mixture of 50 mg test item per 1 mL MTT medium showed reduction of MTT as compared to the solvent. The mixture turned blue/purple. Since the mean relative tissue viability of the test item treated tissues (TM) was above the 60% threshold value, killed tissue controls were performed for quantitative correction of results.

NSMTT [%] = [(OD_KT- OD_KU)/OD_NK] * 100 = -0.3%

Difference of NSMTT of the two duplicate tissues must be < 20%, otherwise not accepted.

NSMTT1 [%] = [meanODKT1 - ODKU)/ODNK] * 100 = -0.6%

NSMTT2 [%] = [meanODKT2 - ODKU)/ODNK] * 100 = -0.1%

NSMTT1 - NSMTT2 =±0.5%

NSMTT was ≤ 60% (-0.3%) relative to the negative control of living epidermis and could therefore be used for determination of the killed control corrected viability (KCCV) according to the following formula:

KCCV [%] = viability_TM– NSMTT = 109.1% - (-0.3%) = 109.4%

 

The mixture of 50 mg test item per 1 mL Aqua dest. showed no colouring as compared to the solvent. The mixture of 50 mg test item per 2 mL isopropanol showed colouring as compared to the solvent. Since the mean relative tissue viability of the test item treated tissues (TM) was above the 60% threshold value, coloured tissue controls were performed for quantitative correction of results.

NSC_living[%] = [OD_TVT/OD_NK] * 100 = 0.3%

Difference of NSC_livingof the two duplicate tissues must be < 20%, otherwise not accepted.

NSC1 [%] = [OD_TVT1/OD_NK] * 100 = 0.3%

NSC2 [%] = [OD_TVT2/OD_NK] * 100 = 0.3%

NSC1 – NSC2 = ± 0.0%

NSC_livingwas ≤ 60% (0.3%) 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 [%] = viability_TM[%] – NSC_living[%] = 109.4% - 0.3% = 109.1 %

 

Since the test item showed non-specific MTT-reduction and non-specific colouring of living tissues, a third control for non-specific colour in killed tissues (NSC_killed) was performed to avoid a possible double-correction for colour interference.

The non-specific colour of additional killed tissues (NSC_killed) was calculated according to the following formula:

NSC_killed[%] = [OD_TKT/OD_NK]*100 = 2.8 %

The true tissue viability was then calculated as the percent tissue viability obtained with living tissues minus NSMTT minus NSC_livingplus NSC_killed.

True Tissue Viability = [%] mean Tissue viability – NSMTT - NSC_living+NSC_killed

        = 109.1% + 2.8% = 111.9%

 

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 60% (111.9%,NSMTT-,NSC_living-,NSC_killed- corrected).

In conclusion the test item showed no irritant effects under the given conditions. The test item is classified as “non-irritant“ in accordance with UN GHS "No Category".