Sulphophthalic acid derivatives reaction products of fatty alcohol C12-C14 and sodium hydroxide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August from 16 to November 09, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

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:

28 July 2015

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Dilasoft TF

IUPAC Name:

Dilasoft TF

Test material form:

liquid

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

- Model: EpiOcular™, three-dimensional human cornea model; it is composed of stratified human keratinocytes in a three-dimensional structure, consisting of at least three viable layers of cells.
- Supplier: MatTek In Vitro Life Science Laboratories Mlynské Nivy 73, 821 05 Bratislava, Slovakia.
- Lot No.: 27000.
- Justification for Selection of the Test System: the EpiOcular™ Eye Irritation Test (EIT) was validated by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 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, and the test method was recommended as scientifically valid for that purpose.
- Demonstration of Proficiency: prior to routine use of the method testing laboratory demonstrated the technical proficiency in a separate study using the fifteen Proficiency Chemicals according to OECD Test Guideline No. 492.
- Quality Control: the EpiOcular™ (kits are manufactured according to defined quality assurance procedures. All biological components tissue and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking an MTT cell viability test and a cytotoxicity test with Triton X-100 (100 μl of 0.3 % (v/v) Triton X-100).

TISSUES PREPARATION FOR TREATMENT
- Pre-treatment: after the test kit arrival, the tissues were equilibrated to room temperature for about 15 minutes.
- Plates: the appropriate number of an assay plate wells (6-well plates) was filled with the pre-warmed (to 37 ± 1 °C) medium (1 ml per well).
- Pre-incubation: at 37 ± 1 °C in an incubator with 5 ± 1 % CO2, 90 ± 10 % humidified atmosphere for one hour in the Assay Medium.
- Incubation: after one hour, the Assay Medium was replaced by 1 ml of fresh Assay Medium at 37 °C and the EpiOcular™ tissues was incubated at standard culture conditions overnight (16 - 24 hours).

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

yes, concurrent positive control

Amount / concentration applied:

Approximately 50 µl test item was applied (each test item treated insert) topically onto the EpiOcular™ tissues.
The test item was applied in its original form, no formulation was required.

Duration of treatment / exposure:

plates with the treated tissue were incubated for the exposure time of 30 minutes (± 2 min) at standard culture conditions (37 ± 1 °C in an incubator with 5 ± 1 % CO2, 90 ± 10 % humidified atmosphere).

Number of animals or in vitro replicates:

Two replicates were used for the test item and control(s) respectively.

Details on study design:

PROCEDURES
- Pre-treatment: the tissues were pre-wetted with approximately 20 μl of Ca++Mg++Free-DPBS.
- Incubation: the tissues were incubated at standard culture conditions for 30 ± 2 minutes.
- Rinsing: with Ca++Mg++ Free-DPBS.To assure throughput, the tissues were rinsed two at a time by holding replicate inserts together by their collars using forceps. The process was performed three times in the first beaker; then the tissue was rinsed in the second and third beakers of DPBS three times each. Any remaining liquid was decanted onto the absorbent material.
- Post-Soak and Post-incubation: after rinsing, the tissues were transferred to and immersed in 5 ml of previously warmed Assay Medium (room temperature) for a 12 ± 2 minute immersion incubation at room temperature. At the end of the Post-Soak immersion, the medium was decanted off the tissue, and the insert was blotted on absorbent material, and afterwards transferred to the appropriate well of the pre-labeled 6-well plate containing 1 ml of warm (37 °C) Assay Medium. The tissues were incubated for 120 minutes ±
15 minutes at standard culture conditions.
- MTT Test After Post-incubation: after the post-incubation the EpiOcular™ units were transferred into the MTT filled 24-well plate (300 µl of 1 mg/ml MTT per well) and then incubated for 3 hours (± 10 min).
- Formazan Extraction: each insert was removed from the 24-well plate after 3 hours ± 10 minutes, the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labeled 24-well plate containing 2 ml of isopropanol so that it was flowing into the insert on the tissue surface. The plates were stored overnight at 2-8 °C in the dark. To extract the MTT, the plates were placed on an orbital plate shaker and shaken (150 rpm) for approximately 3 hours at room temperature. At the end of the extraction period, the tissue was pierced and the liquid within each insert was decanted into the well from which it was taken.

CONTROLS
- Negative control: a volume of 50 µl sterile deionized water was applied on the tissues surface by using a suitable pipette.
- Positve control: a volume of 50 µl methyl acetate was applied on the tissues surface by using a suitable pipette.

MTT SOLUTION
- Preparation: after filtration (using a sterile filter) MTT stock solution was diluted with pre-warmed “assay medium” to a final concentration of 1 mg/ml.
- Storage: stored less than 2 hour before use at 2-8 °C, protected from light.

EFFECT MEASURED
Following the formazan extraction, 200 µl sample(s) from each tube (2×200 µl) was placed into the wells of a 96-well plate and read Absorbance / Optical Density of the samples in a 96-well plate spectrophotometer at the wavelength of 570 nm using isopropanol solutions as the blank (8×200 µl).
The cytotoxicity of the test item (and thus the ocular irritation potential) is evaluated by the relative viability of the treated tissues in comparison to the negative control-treated tissues. Viability is determined by the NAD(P)H-dependent microsomal enzyme reduction of MTT (and to a lesser extent, by the succinate dehydrogenase reduction of MTT) in control and test item treated cultures (Berridge, et al., 1996). Data are presented in the form of relative survival (relative MTT conversion).

CHECK OF INTERFERENCE WITH TISSUE VIABILITY
- Solution: approximately 50 µl test item was added to 1 ml MTT 1 mg/ml solution in a tube.
- Incubation: in the dark at 37 ± 1 °C, 5 ± 1 % CO2, 90 ± 10 % humidified atmosphere. The mixture was incubated for approximately three hours.
- Measurement: any colour change of the mixture was observed.

CHECK OF COLOURING POTENTIAL OF TEST ITEM
- Solutions: approximately 50 µl test item was added to 1 ml of water in a tube; approximately 50 µl test item was added to 2 ml isopropanol, the same amount as used for MTT extraction, and was incubated in tubes.
- Incubation: in the dark at 37 ± 1 °C, 5 ± 1 % CO2, 90 ± 10 % humidified atmosphere for one hour; mixture with isopropanol was placed on an orbital plate shaker and shaken (150 rpm) for approximately 2 hours at room temperature and then colour checked.

ACCEPTANCE CRITERIA
- The mean OD value of the two negative control tissues should be between 0.8 and 2.5.
- The acceptable percentage viability for positive control (mean of two tissues) is: 30 minute exposure: below 50 % of control viability (liquids); 6 hours exposure: below 50 % of control viability (solids)
- The difference of viability between the two relating tissues of a single chemical is < 20 % in the same run (for positive and negative control tissues and tissues of single chemicals). This applies also to the killed controls (single chemicals and negative killed control) and the colorant controls which are calculated as percent values related to the viability of the relating negative control.

INTERPRETATION OF RESULTS
The irritancy potential of test items is predicted by the mean tissue viability of tissues exposed to the test item. The test item is identified as 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 % of the negative control. However, this test method (OECD 492) cannot resolve between UN GHS Categories 1 and 2, further testing with other test methods will be required to decide on its final classification.
Depending on the regulatory framework in member countries, the test item 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 with the help of other test methods is required.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Remarks:

percentage of Cell Viability

Run / experiment:

mean of duplicate

Value:

< 60

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The test item showed significantly reduced cell viability in comparison to the negative control (mean viability: 44 %). All obtained test item viability results were below 60 % when compared to the viability values obtained from the negative control.

Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

CHECK OF INTERFERENCE WITH TISSUE VIABILITY and COLOURING POTENTIAL OF TEST ITEM
- Possible direct MTT reduction with test item: no colour change was observed after three hours of incubation during the check for possible direct MTT reduction by the test item (see section 5.5.1). The test item did not interact with MTT, therefore additional controls and data corrections were not necessary. A false estimation of viability can be precluded.
- Colouring potential of test item: the test item showed no ability to become coloured in contact with water and isopropanol. Additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

VALIDITY OF THE TEST
- The mean OD value of the two negative control tissues was: 1.951
- The positive control result showed 7 % viability at 30 minutes exposure.
- The difference of viability between the two tissue replicates: 1 % to 12 %

All validity criteria were within acceptable limits and therefore the study can be considered as valid.

Any other information on results incl. tables

OD values and viability percentages of the test item and controls

Test substance	Optical Density (OD)		Viability (%)	Δ%
Negative Control, terile deionized water	1	1.835	94	12
	2	2.066	106
	mean	1.951	100	-
Test item	1	0.847	43	1
	2	0.871	45
	mean	0.859	44	-
Positive Control, methyl acetate	1	0.048	2	9
	2	0.222	11
	mean	0.135	7	-

Δ %: the difference of viability between the two relating tissues

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

Irritant and/or able to cause serious eye damage.

Executive summary:

The purpose of the study was to determine the acute ocular irritation potential of the test item on three-dimensional RhCE tissue in the EpiOcular™ model in vitro, according to the OECD guideline 492.

At the end of the Post-Soak immersion test item treated tissues were incubated for 120 minutes ± 15 minutes at standard culture conditions (Post-treatment Incubation). Fresh Assay Medium was used during the Post-Soak and Post-incubation. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 ± 1 °C in an incubator with 5 ± 1 % CO₂ protected from light, 90 ± 10 % humidified atmosphere. The precipitated formazan was then extracted using isopropanol and quantified spectrophotometrically. Sterile deionized water and methyl acetate treated tissues were used as negative and positive controls, respectively. The Disks of EpiOcular™ (two units / control) were treated with positive and negative control and incubated for 30 minutes ± 2 minutes.

The test item showed significantly reduced cell viability in comparison to the negative control (mean viability: 44 %). All obtained test item viability results were below 60 % when compared to the viability values obtained from the negative control.

Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

Conclusion

The results obtained from this in vitro eye irritation test, using the EpiOcular™ model, with the test item indicated that the test item is irritant and/or able to cause serious eye damage. However, this test method cannot resolve between UN GHS Categories 1 and 2.