Potassium sulphite

Endpoint summary

Description of key information

skin irritation/ corrosion: not irritating, No Category (OECD 404; GLP)

eye irritation/ corrosion: not irritating, No Category (OECD 491; GLP)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Minor deviations with no effect on the results: - Purity and stability were not stated. - According to guideline, the observations should be made at 30-60 minutes and at 24, 48, and 72 hours after patch removal and not 24 hours, 48 hours and 72 days after start of exposure. - A modified Draize scoring system was used in this study. - According to the guideline the test substance should be moistened with water. In this study the test patch was moistend with water for application instead of the test substance. - The water supply was not unrestricted. - in the study report it was stated that observations were made at 30-60 minutes after patch removal. In the result table data were listed for an observation at 4-hours.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

1981-05-12

Deviations:

yes

Remarks:

, see "rationale for reliability"

GLP compliance:

not specified

Specific details on test material used for the study:

not specified

Species:

rabbit

Strain:

Vienna White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Savo; Med. Versuchstierzuchten GmbH; 7964 Kisslegg/Allgäu FRG
- Weight at study initiation: mean weight 2.56 kg (Individual weights of the three test animals:2.78 kg, 2.22 kg, and 2.68 kg)
- Housing: The animals were individually housed. The animals were housed in fully air-conditioned rooms. Cage made of stainless steel with wire mesh walk floors. Floor area: 40 cm X 51 cm. No bedding in the cages.Sawdust in the waste trays.
- Diet: Kliba 341, 4 MM; Firma Klingentalmuehle AG, CH- 4303 Kaiseraugst, Switzerland (about 130 g per animal per day)
- Water: About 250 ml tap water per animal per day
- Acclimation period: At least 8 days before the beginning of the study; same housing conditions as during the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24 °C
- Relative humidity: 30 - 70 %
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

water

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): A dose of 0.5 g of the unchanged test substance.

Duration of treatment / exposure:

4 hours

Observation period:

72 hours; Readings of skin irritation at 30 - 60 minutes after removal of the test patches and 24 h, 48 h and 72 h after the beginning of application.

Number of animals:

3 male rabbits

Details on study design:

TEST SITE
The fur of the rabbits was clipped at least 15 hours before the beginning of the study.The application area was 2.5 cm X 2.5 cm on the upper third of the back or flanks. The test patch (2.5 cm X 2.5 cm) was covered with a dose of 0.5 g of the unchanged test substance and has been moistened with aqua dest.. Test patches were secured in position with a porous dressing (four layers of absorbant gauze and porous bandage). The untreated skin sites of the same animals was used as negative control.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The test substance was removed with lutrol and lutrol/water (1:1).
- Time after start of exposure: At the end of the exposure period

SCORING SYSTEM: Modified Draize scoring system
Evaluation of erythema (R) and oedema (ED):
0 = none
1 = very slight
2 = well-defined
3 = moderate to severe
4 = severe to very severe

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: The readings were made 24 h, 48 h and 72 h after the beginning of application

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: The readings were made 24 h, 48 h and 72 h after the beginning of application

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: The readings were made 24 h, 48 h and 72 h after the beginning of application

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: The readings were made 24 h, 48 h and 72 h after the beginning of application

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: The readings were made 24 h, 48 h and 72 h after the beginning of application

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritant / corrosive response data:

Very slight erythema was observed in two animals at the 4-hour reading, which had disappeared at the 24 hour reading.

Interpretation of results:

GHS criteria not met

Conclusions:

The substance is not irritating to the skin based on an in vivo study (OECD 404).
According to the EC Regulation No. 1272/2008 and subsequent regulations, the test item is not classified as skin irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-07-29 to 2021-12-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

signed 2022-11-01

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.

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

other: Medium Control: complete medium (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)

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). The test item was prepared freshly prior to each repetition.

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 is day 0 and the day of treatment are included in the calculation of the days for the cell cultivation: e.g. seeding on Friday of 6000 cells/well and treatment on Tuesday (four days) or seeding on Friday of 3000 cells/well and treatment on Wednesday (five days). The plates will be incubated at 37 ± 1.5 °C and 5.0 ± 0.5% carbon dioxide atmosphere. Cells should have reached a confluence of more than 80% at the time of testing.
NUMBER OF REPETITIONS AND REPLICATES
The test item was tested in six independent repetitions with different cell cultures and/or on different days. All dose groups were tested in triplicates in each repetition. The first three independent repetitions were not valid and had to be repeated. Only the three valid repetitions were reported.

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. In addition, in one dose group empty wells without any cells were treated with the test item and treated exactly the same as the other groups.

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) correspond to Table 2 of the OECD TG 491.

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 comparable run acceptance criteria are attached in the field "Overall remarks, attachments" below.

TEST ACCEPTANCE CRITERIA
According to OECD TG 491:
- Optical density of the medium control 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.

Irritation parameter:

other: Mean Cell Viability [%]

Run / experiment:

Test Item 0.05%

Value:

91.76

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:

78.1

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 (0.381 - 0.759).
- Viability of the solvent control relative to the medium control was higher than 80% (93.4 - 98.5).
- Standard deviation of the final cell viability derived from three independent repetitions was less than 15% for both 5% and 0.05% test item concentrations (0.5 and 6.8).
The above acceptance criteria were met.
- The cell viability of the positive control (16.49%) was not within the upper and lower acceptance boundaries established by the method developer (21.1% and 62.3%) in compliance with the OECD TG 491. Regarding this acceptance criteria the positive control was too low. From a scientific point of view this does not affect the validity of the study, especially since a negative result was obtained for the test item. A repetition should provide higher OD values for the positive control that would finally lead to an increase of the OD level of the test item treated group, too. Higher OD values for the test item still would result in “No category”. Therefore, it can be assumed that a too low positive control stands for a sensitive cell passage and therefore the result for the test item is convincing and reliable.
Please also refer to the field "Overall remarks, attachments" below.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, in the described STE study under the experimental conditions reported, the test item potassium sulfite 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

The substance was not observed to be a skin irritant in a reliable in vivo skin irritation study according to OECD 404.

Eye irritation:

The substance was not observed to be an eye irritant in a reliable in vitro eye irritation study according to OECD 491.

Justification for classification or non-classification

Skin irritation:

The substance does not possess a skin irritation potential based on an in vivo OECD 404 test and does not require classification as skin irritant according to Regulation (EC) No 1272/2008 and its subsequent adaptations.

 

Eye irritation:

The substance does not possess an eye irritation potential based on an in vitro OECD 491 test and does not require classification as eye irritant according to Regulation (EC) No 1272/2008 and its subsequent adaptations.