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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2021-05-10 to 2022-11-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2023
Report date:
2023

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 491 (Short Time Exposure In Vitro 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:
2020-06-26
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
GLP certificate signed 2022-11-01

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium hydrogensulfite
EC Number:
231-548-0
EC Name:
Sodium hydrogensulfite
Cas Number:
7631-90-5
Molecular formula:
NaHSO3
IUPAC Name:
sodium hydrogensulfite
Test material form:
other: aqueous solution

Test animals / tissue source

Details on test animals or tissues and environmental conditions:
CELL CULTURE
- Cell Line SIRC: The rabbit corneal cell line SIRC (Statens Seruminstitut Rabbit Cornea) was used for performing the STE test method. SIRCs are growing as confluent monolayers.
- Large stocks of the SIRC cell line (supplied by ATCC) were stored in liquid nitrogen in the cell bank of ICCR-Roßdorf GmbH allowing the repeated use of the same cell culture batch in experiments. Therefore, the parameters of the experiments remain similar, because of the reproducible characteristics of the cells.
- Thawed stock cultures were propagated at 37 ± 1.5 °C in plastic flasks containing a culture medium comprising Eagle’s minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine and 100 units/mL penicillin and 100 µg/mL streptomycin.
- The cells were sub-cultured twice weekly. The cell cultures were incubated at 37 ± 1.5 °C and 5.0 ± 0.5% carbon dioxide atmosphere. Cells were propagated 2 to 3 passages in a culture flask before being employed for testing and did not exceed 25 passages from thawing.

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
yes, concurrent positive control
other:
Amount / concentration applied:
TEST MATERIAL
On the day of the experiments right before application, the test item was dissolved in physiological saline (0.9% NaCl in deionised water) to reach a final concentration of 5% (w/w). Next, this solution was diluted by serial 10 fold dilution with the respective solvent to reach final concentrations of 0.5% (w/w) and 0.05% (w/w).

VEHICLE
physiological saline (0.9% NaCl in deionised water)
Duration of treatment / exposure:
5 minutes at room temperature
Observation period (in vivo):
not applicable
Duration of post- treatment incubation (in vitro):
not applicable
Number of animals or in vitro replicates:
triplicate
Details on study design:
CELL LINE
Please refer to the field „Details on test animals or tissues and environmental conditions“.

SEEDING OF THE CULTURES
Exponentially growing stock cultures more than 50% confluent were rinsed with PBS and treated with Trypsin at 37 ± 1.5 °C for 5 minutes. Then the enzymatic digestion was stopped by adding complete medium and a single cell suspension was prepared.
Individual wells of a 96-well tissue-culture microtiter plate were inoculated with 0.2 mL complete medium containing approximately 3 x 10E4 cells/mL (6000 cells per well) in case that the cells were seeded four days prior to the treatment and 1.5 x 10E4 cells/mL (3000 cells per well) in case that the cells were seeded 5 days prior to the treatment. The seeding day was day 0: e.g. seeding on Friday of 6000 cells/well and treatment on Tuesday (four days ≙ 96 h) or seeding on Friday of 3000 cells/well and treatment on Wednesday (five days ≙ 120 h). The plates were incubated at 37 ± 1.5 °C and 5.0 ± 0.5% carbon dioxide atmosphere. The cells reached a confluence of more than 80% at the time of testing.

NUMBER OF REPETITIONS AND REPLICATES
The test item was tested in three independent repetitions with different cell cultures and/or on different days. All dose groups were tested in triplicates in each repetition.

TREATMENT
For the treatment the complete medium was removed, and the cells were re-fed with 200 µL treatment solution containing medium, solvent and positive control as well as the two different concentrations of the test item (5% and 0.05%) and the complete medium blank, respectively.

CELL VIABILITY MEASUREMENT
After exposure, cells were washed twice with 0.2 mL of calcium- and magnesium-free PBS and 0.2 mL MTT solution (0.5 mg MTT/mL of MEM) was added. After a two-hour ± 15 minutes reaction time at 37 ± 1.5 °C and 5.0 ± 0.5% carbon dioxide atmosphere the MTT solution was decanted. MTT formazan was extracted by adding 0.2 mL of 0.04 N hydrochloric acid – isopropanol for at least 60 minutes (not longer than 120 minutes) in the dark at room temperature, and the absorbance of MTT formazan solution was measured with a microplate reader (Versamax® Molecular Devices) at 570 nm (without a reference). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

DECISION CRITERIA
The cell viability cut-off values for identifying test items inducing serious eye damage (UN GHS category 1) and test items not requiring classification for eye irritation or serious eye damage (UN GHS no category) corresponds to Table 2 of the OECD TG 491.

TEST ACCEPTANCE CRITERIA
According to OECD 491:
- Optical density of the medium control (exposed to culture medium) should be 0.3 or higher after subtraction of blank optical density.
- Viability of the solvent control should be 80% or higher relative to the medium control.
- The cell viability of the positive control should be within the upper and lower acceptance boundaries established by the method developer (21.1% and 62.3%) in compliance with the OECD TG 491.
- Standard deviation of the final cell viability derived from three independent repetitions should be less than 15% for both 5% and 0.05% concentrations of the test chemical.

DEMOSTRATING OF PROFICIENCY IN PERFORMING THE TEST METHOD BEFORE ROUTINE USE BY TESTING OF THE PROFICIENCY CHEMICALS
Prior to routine use for regulatory purposes, the laboratory demonstrated technical proficiency by correctly predicting the eye irritation potential of the proficiency substances listed in Table 1 of OECD TG 491. The respective proficiency data are attached in the field "Overall remarks, attachments" below.

REFERENCE TO HISTORICAL POSITIVE CONTROL MEAN AND STANDARD DEVIATION (SD)
Historical data are available to derive compareable run acceptance criteria are attached in the field "Overall remarks, attachments" below.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
other: Mean Cell Viability [%]
Run / experiment:
Test Item 0.05%
Value:
101.88
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: Medium control: valid
Irritation parameter:
other: Mean Cell Viability [%]
Run / experiment:
Test Item 5%
Value:
86.18
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: Medium control: valid
Other effects / acceptance of results:
DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use for regulatory purposes, the laboratory demonstrated technical proficiency by correctly predicting the eye irritation potential of the chemicals listed in Table 1 of OECD TG 491. The respective proficiency data are attached in the field "Overall remarks, attachments" below.

TEST ACCEPTANCE CRITERIA
- Optical density of the medium control after subtraction of blank optical density was higher than 0.3 (0.924 - 1.098).
- Viability of the solvent control relative to the medium control was higher than 80% (100.94 - 107.45).
- The cell viability of the positive control (51.99%) was within the upper and lower acceptance boundaries established by the method developer (21.1% and 62.3%) in compliance with the OECD TG 491.
- Standard deviation of the final cell viability derived from three independent repetitions was less than 15% for both 5% and 0.05% test item concentrationsl (3.52 and 2.35).
The acceptance criteria were met. Please also refer to the field "Overall remarks, attachments" below.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, in the described STE study under the experimental conditions reported, the test item sodium hydrogensulfite is not eye irritating and does not require classification and labelling for eye irritation or serious eye damage according to UN GHS and EU CLP.