Sodium methyl sulphate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Oct 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

other: part of a testing Strategy

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Nov 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

other: part of a testing strategy

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:

26 July 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No. of test material: KIRSCHAZ2-00182
- Content: 99.1 g/100 g
- pH-value: Ca. 6 (undiluted test substance, moistened with de-ionized water; determined in the lab prior to start of the GLP study)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The solid test substance was ground with pestle and mortar before application. The test-substance preparation was produced on a weight per volume (w/v) basis shortly before application by stirring with a magnetic stirrer. After stirring with a magnetic stirrer the test substance was soluble in the vehicle.
- Form of application: 20% (w/v) solution in de-ionized water

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: freshly slaughtered cattle; supplier: Schlachthof Mannheim, Schlachthofstr. 21, 68165 Mannheim, Germany
- Characteristics of donor animals: age of the animals: minimum 12 months, maximum 60 months
- indication of any existing defects or lesions in ocular tissue samples: Corneas free of defects (opacity, scratches, pigmentation etc.)

Vehicle:

water

Remarks:

de-ionized

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 20% (w/v) solution in de-ionized water

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 µL of de-ionized water

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 μL of 20% (w/v) solution of imidazole in de-ionized water

Duration of treatment / exposure:

4 hours

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS: Corneas free of defects (opacity, scratches, pigmentation etc.) were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in cornea holders that consists of anterior and posterior chambers. Both chambers were filled to excess with pre-warmed Eagle’s MEM (without phenol red) and then equilibrated in a vertical position at about 32°C for at least 1 hour. After the equilibration period, the medium in both chambers was replaced by fresh pre-warmed medium and initial corneal opacity readings were taken for each cornea with an opacitometer.

QUALITY CHECK OF THE ISOLATED CORNEAS: Any corneas that showed macroscopic tissue damage or an opacity value < 550 opacity units were discarded. The remaining corneas were then distributed into negative control, positive control and treatment groups.(According to OECD TG 437, corneas that have an opacity value >7 or equivalent for the opacitometer and cornea holders used after an initial one-hour equilibration period are to be discarded. In the test facility´s opacitometer in combination with the used cornea holder set 2013-22 and 2013-24, the maximal initial opacity of >7 arises from I= 550 lux with Io= 641 lux).

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: Yes

POSITIVE CONTROL USED: Yes

APPLICATION DOSE AND EXPOSURE TIME: Application of 750 µL of the 20% (w/v) test-substance preparation, 4 hours exposure time

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least 3times.

- POST-EXPOSURE INCUBATION: no

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Yes.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader: Yes, OD490, SunriseTM Absorbance Reader.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: amendment to TG (refinement of decision criteria).
IVIS: < 1.5, Prediction: No classification for eye irritation.
IVIS: 1.5 – 4.5, Prediction: Borderline.
IVIS: > 4.5, < 45, Prediction: No prediction can be made for eye irritation, further testing with another suitable method is required.
IVIS: 45 - 65, Prediction: Borderline.
IVIS: > 65, Prediction: Ocular corrosive or severe irritant.
The “borderline“ evaluation (IVIS 3.0 ± 1.5 and 55.0 ± 10.0) was statistically determined by using historic test facility data and takes the test facility specific variance of the test method into account. This evaluation is an amendment to the evaluation provided in OECD Guideline 437

Irritation parameter:

in vitro irritation score

Run / experiment:

Run 1, mean of three single corneas

Value:

0.1

Vehicle controls validity:

other: vehicle control = negative control

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: No.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Proficiency given

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Table 1: Results of the BCOP Test, in vitro irritancy score (IVIS) of the test substance, the NC and the PC.

Test substance identification	Cornea- No.	Opacity per cornea	Permeability per cornea	per cornea	IVIS per group
					mean	SD
	7	0.0	0.000	0.0
16/0352-1	8	0.0	0.010	0.1	0.1	0.1
	9	0.0	0.000	0.0
	1	16.5	0.000	16.5
NC	2	7.5	0.000	7.5	12.4	4.6
	3	13.2	0.000	13.2
	4	81.0	2.097	112.4
PC	5	86.0	1.868	114.1	110.6	4.6
	6	71.7	2.247	105.4

Executive summary:

The objective was to assess the eye irritating potential of the test substance. Using the currently available methods a single in vitro assay is not sufficient to cover the full range of eye irritating potential. Therefore, two in vitro assays were part of this in vitro eye irritation test strategy: The Bovine Corneal Opacity and Permeability Test (BCOP Test) and EpiOcular™ Eye Irritation Test.

BCOP:

The potential of the test substance to cause ocular irritation or serious damage to the eyes was assessed by a single topical application of 750 μL of a 20% test-substance preparation in de-ionized water to the epithelial surface of isolated bovine corneas.

Three corneas were treated with the test-substance preparation for an exposure period of 4 hours. In addition to the test substance a negative control (NC; de-ionized water) and a positive control (PC; 20% imidazole in de-ionized water) were applied to three corneas each. Corneal opacity was measured quantitatively as the amount of light transmitted through the cornea. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements were used to calculate an In Vitro Irritancy Score (IVIS) of the test substance.

The BCOP identified the test substance as not corrosive or severe irritant based on a mean IVIS of 0.1.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Sodium methyl sulphate

EC Number:

208-142-7

EC Name:

Sodium methyl sulphate

Cas Number:

512-42-5

Molecular formula:

CH4O4S.Na

IUPAC Name:

sodium methyl sulphate

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No. of test material: KIRSCHAZ2-00182
- Content: 99.1 g/100 g
- pH-value: Ca. 6 (undiluted test substance, moistened with de-ionized water; determined in the lab prior to start of the GLP study)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The solid test substance was ground with pestle and mortar before application. The test substance is applied undiluted, therefore no preparation of the test substance in a vehicle was performed.

Test animals / tissue source

Species:

human

Strain:

other: normal human derived epidermal keratinocytes

Details on test animals or tissues and environmental conditions:

Description of the cell system used: Reconstructed human cornea-like epithelium
- Model used: EpiOcular™ model (OCL-200)
- Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
- Tissue batch number: 23740
- Certificate of authenticity: provided by the supplier
- Verification of tissue functionality and quality (performed by the supplier):
Tissue viability: acceptance criteria met
Barrier function: acceptance criteria met
Sterility: no contamination

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): ca. 50 μL bulk volume (about 26 mg) of the undiluted test substance

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 50 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 50 µL, undiluted

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

2

Details on study design:

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: The tissues were immersed and swiveled three times in each of three beakers filled with sterile PBS.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
A direct interference was not observed.

NUMBER OF REPLICATE TISSUES PER TEST CHEMICAL AND CONTROLS: 2

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg / mL MTT diluent
- Incubation time: 3 hours
- Spectrophotometer: SunriseTM Absorbance Reader
- Wavelength: 570 nm, without reference filter

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to the eye if the mean relative tissue viability with a test material is less than 55%.
- The test substance is considered to be non- irritant to the eye if the mean relative tissue viability with a test material is greater than 65%.
- In case of borderline results such as non-concordant replicate measurements and/or mean percent tissue viability of 55 - 65%, a second test should be considered as well as a third one in case of discordant results between the first two tests.
- Justification for the selection of the cut-off point(s) if different than recommended in TG 492: 4The „borderline“-evaluation (60 ± 5%) was determined statistically using historic data of the test facility and hence considers the variance of the test method. This evaluation is an amendment to the evaluation provided in OECD Guideline 492.

ACCEPTANCE CRITERIA
- Negative control: Tissue viability is acceptable if the mean OD570 of the negative control (NC) is ≥ 0.8. The mean OD570 of the NC should not exceed 2.5.
- Positive control: Methyl acetate used as positive control (PC) usually leads to a tissue viability of approx. 25%. A viability of < 50% is acceptable.
- Variability: Two tissues were treated under the same conditions. A variability between the tissues is considered to be acceptable if the difference of the viability is ≤ 20%.

HISTORICAL DATA (see table 1)

Results and discussion

In vitro

Results

Irritation parameter:

other: mean viability [%]

Run / experiment:

Run 1, mean of 2 replicate tissues

Value:

2.4

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 value of the negative control was slightly outside the range of historical control data. Due to the unambiguous result of the study this deviation was not considered to have any influence on the validity of the data.
- Acceptance criteria met for positive control: yes

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

Any other information on results incl. tables

Table 2: Results of the EpiOcular Test, individual and mean OD₅₇₀ values, individual and mean viability values and inter-tissue variability

Testsubstance identification		tissue 1	tissue 2	mean	Inter-tissue variability [%]
NC	mean OD570	2.429	2.158	2.293
	viability [% of NC]	105.9	94.1	100.0	11.8
16/0352-1	mean OD570	0.048	0.063	0.055
	viability [% of NC]	2.1	2.7	2.4	0.7
PC	mean OD570	0.375	0.473	0.424
	viability [% of NC]	16.3	20.6	18.5	4.3

NC: negative control

PC: positive control

Applicant's summary and conclusion

Executive summary:

The objective was to assess the eye irritating potential of the test substance. Using the currently available methods a single in vitro assay is not sufficient to cover the full range of eye irritating potential. Therefore, two in vitro assays were part of this in vitro eye irritation test strategy: The Bovine Corneal Opacity and Permeability Test (BCOP Test) and EpiOcular™ Eye Irritation Test.

EpiOcular™

The potential of the test substance to cause ocular irritation was assessed by a single topical application of ca. 50 μL bulk volume (about 26 mg) of the undiluted test substance to a reconstructed three-dimensional human cornea model (EpiOcular™).

Two EpiOcular™ tissues were incubated with the test substance for 6 hours followed by an 18-hours post-incubation period.

Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test substance treated epidermal tissues was compared to that of negative control tissues. The ratio of the values indicates the relative tissue viability.

The following results were obtained in the EpiOcular™ eye irritation assay:

The test substance was not able to reduce MTT directly. The mean viability of the test substance treated tissues was 2.4%. Based on these results the test substance was identified as irritant.