Potassium hydrogen sulphite

Endpoint summary

Description of key information

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

eye irritation/corrosion: no prediction can be made (OECD TG 437; GLP)

eye irritation/ corrosion: no prediction can be made (OECD TG 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

Study period:

2004-04-13 to 2004-04-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

2002-04-24

Deviations:

yes

Remarks:

, occlusive dressing (not considered to influence the results of the study)

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS - albino rabbits
- Source: Allevamento "Conelli" - Arona (NO)
- Weight at study initiation: 2820 - 2950 g
- Housing: individual housing in cages (NORYL; polyphenylenoxid; 48.2 cm x 63 cm x 37 cm)
- Diet: standard pellets (Harlan Italy)
- Water (ad libitum): cleaned water
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

occlusive

Preparation of test site:

shaved

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 mL

Duration of treatment / exposure:

4 hours

Observation period:

60 minutes as well as 24, 48, and 72 hours after application

Number of animals:

3 male rabbits

Details on study design:

TEST SITE
- Area of exposure: 24 hours before the test item application the backs and the hips of the rabbits were shaved (area: 240 cm²). The test item was applied onto a gauze patch (2.5 cm x 2.5 cm), which was placed onto the shaved skin and secured with a plaster.

REMOVAL OF TEST SUBSTANCE
- Washing: after exposure the bandage and plaster were removed. The test substance was removed from the skin with physiological solution.

SCORING SYSTEM: according to the Draize scale

OBSERVATIONS:
- clinical signs were observed daily

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritant / corrosive response data:

None of the rabbits showed any signs of skin irritation or oedema.

Interpretation of results:

GHS criteria not met

Conclusions:

Potassium bisulfite solution is not irritating to the skin of the rabbits.
According to the Regulation (EC) No 1272/2008 and subsequent regulations, the test item should not be classified as irritating to the skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2021-04-26 to 2021-04-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

2020-06-26

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2019-10-23

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: AB Schlachthof GmbH & Co. KG, 63739 Aschaffenburg, Germany
- Characteristics of donor animals: 14 – 24 month old donor cattle
- Storage, temperature and transport conditions of ocular tissue: Freshly isolated bovine eyes of donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were stored in HBSS containing 1% (v/v) penicillin/streptomycin (100 units/mL penicillin and 100 µg/mL streptomycin) in the cooled slaughter-house and during transportation on the same morning to the laboratory using a Styrofoam box.
- Time interval prior to initiating testing: The corneae were isolated on the same day after delivery of the eyes.

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): 0.75 mL of the undiluted test item

Duration of treatment / exposure:

10 minutes ± 30 seconds

Observation period (in vivo):

not applicable

Duration of post- treatment incubation (in vitro):

2 hours

Number of animals or in vitro replicates:

Number of bovine corneae per dose (test item, negative and positive control): triplicate

Details on study design:

INCUBATION MEDIUM
The incubation medium consists of MEM, supplemented with 1.1 g sodium bicarbonate, 5 mL L-glutamine, 5 mL penicillin/streptomycin per 500 mL medium (final concentration of 100 units penicillin per mL medium, and 100 µg streptomycin per mL medium). Immediately before starting the test, MEM was supplemented with 1% fetal calf serum (FCS). MEM containing all supplements is called cMEM.
The OECD guideline 437 recommends the use of EMEM which is in composition and osmolarity equivalent to the MEM, thus MEM can be used without restriction.

PREPARATION OF CORNEAS
- each isolated cornea was mounted according to the description given in OEDC guideline 437, i.e. in a specially designed cornea holder that consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. Both compartments of the holder were filled with incubation medium cMEM (MEM, supplemented with sodium bicarbonate, L-glutamine, penicillin/streptomycin and 1 % fetal calf serum).
- for equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath.

QUALITY CHECK OF THE ISOLATED CORNEAS
- all eyes were carefully examined macroscopically for defects before removing the cornea. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.
- at the end of the equilibration period of the corneae in the holder, the basal opacity was determined (t0).
- only cornea with a value of the basal opacity < 7 were used

APPLICATION DOSE AND EXPOSURE TIME
- the anterior compartment received the test item, the negative control and the positive control at a volume of 0.75 mL each on the surface of the corneae.
- corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath for ten minutes ± 30 seconds.
- after exposure of the test item or control items to the corneae, they were rinsed off from the application sides with EMEM containing phenol red at least three times or more if phenol red is still discoloured (yellow or purple), or the test item is still visible.
- then corneas were finally rinsed with cMEM without phenol red.
- fresh cMEM was added into the anterior compartment. Then the corneae were incubated at 32 ± 1 °C for further 2 hours (post-incubation) in a vertical position, followed by a second opacity reading (t130).
- permeability of the corneae was determined.

METHODS FOR MEASURED ENDPOINTS
OPACITY MEASUREMENT
- Corneal opacity: the opacitometer (OP_KiT opacitometer (Electro Design, 63-Riom France)) was calibrated and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
Evaluation of opacity:
- the change of opacity value of each treated cornea or of the positive and negative control corneae is calculated by subtracting the initial basal opacity from the post treatment opacity reading (t130 – t0), for each individual cornea.
- the average change in opacity of the negative control corneae is calculated and this value is subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.

PERMEABILITY DETERMINATION
Following the opacity readings, the permeability endpoint was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the incubation medium was removed from both chambers. The posterior chamber was filled with fresh cMEM first. Then the anterior compartment was filled with 1 mL of a 0.4% (w/v) sodium fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 ± 5 minutes in a water-bath at 32 ± 1 °C. Complete medium from the posterior compartment was removed, well mixed and transferred into a 96 well plate and the optical density at 490 nm (OD490) was measured with a microplate reader (Versamax® Molecular Devices). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).
Evaluation permeability:
- the corrected OD490 value of each cornea treated with positive control or test item is calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The following formula is used to determine the IVIS of the negative control:
IVIS = opacity value + (15 x OD490 value)
The following formula is used to determine the IVIS of the positive control and the test item:
IVIS = (opacity value – corrected opacity value mean negative control) + (15 x (corrected OD490 value)
The mean IVIS value of each treated group is calculated from the respective individual IVIS values.
Depending on the score obtained, the test item is classified into the following category according to OECD guideline 437.

DECISION CRITERIA:
The test will be acceptable if:
- the positive control gives an IVIS that falls within two standard deviations of the current historical mean (updated every three months), and if
- the negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.

Irritation parameter:

in vitro irritation score

Value:

17.03

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: Optical evaluation of the test item treated corneas with the unaided eye revealed visible damage in turbidity form.

CONTROLS:
For the negative controls (saline and deionised water), neither an increase of opacity nor permeability of the corneas could be observed (mean IVIS = 1.35 and 0.82) corresponding to no classification (UN GHS No category).
The positive controls (2-Ethoxyethanol and Dimethylformamide) were tested undiluted and showed clear opacity and distinctive permeability of the corneas (mean IVIS = 99.15 and 103.66) corresponding to a classification as serious eye damaging (UN GHS Category 1).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
opacity and permeability of the negative control are less than the respective established upper limits for background opacity and permeability.
- Acceptance criteria met for positive control:
the IVIS of the positive control falls within two standard deviations of the current historical mean.

Please also refer to the field "Overall remarks, attachments" below.

Interpretation of results:

study cannot be used for classification

Conclusions:

In conclusion, according to the current valid OECD 437 study and under the experimental conditions reported, a prediction of the damage hazard to eyes cannot be made (GHS) for potassium hydrgensulfite.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2022-08-16 to 2022-08-19

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 2019-10-23

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:

Amount / concentration applied:

TEST MATERIAL
On the day of the experiments right before application, the test item was dissolved in physiological saline (0.9% (w/v) 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 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. 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 obtained with the positive control (0.01% SLS) should be within two standard deviations of the historical mean.
- 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:

112.55

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:

5.66

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 (0.474 - 0.832).
- Viability of the solvent control relative to the medium control was higher than 80% (86.04 - 93.40).
- The cell viability obtained with the positive control (36.38) was within two standard deviations of the historical mean (20.23% - 47.71%).
- 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 (1.8 and 7.0).
The acceptance criteria were met. Please also refer to the field "Overall remarks, attachments" below.

Interpretation of results:

study cannot be used for classification

Conclusions:

In conclusion, in the described STE study under the experimental conditions reported, no prediction of the eye irritation potential of potassium hydrogensulfite can be made.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Read-across concept for sulfites, hydrogensulfites, metabisulfites, dithionites and thiosulfates:

A comprehensive read-across concept has been developed for sulfites, hydrogensulfites and metabisulfites, based on the pH-dependant equilibrium in aqueous solutions which is summarised in the following equations:^[1],[2]

           SO₂+ H₂O <->`H₂SO₃´         H₂SO₃<->H⁺+ HSO₃^-<->2H⁺+SO₃^2-    2HSO₃^-<->H₂O +S₂O₅^2-

Since the nature of the cation (i.e., sodium, potassium, ammonium…) is not assumed to contribute substantially to differences in toxicity and solubility (all compounds are very soluble in water), only the chemical and biological properties of the anion are considered as relevant determinants. Based on the described equilibrium correlations, unrestricted read-across between the groups of sulfites, hydrogensulfites and metabisulfites is considered justified.

 

Additionally, it is known that sodium dithionite disproportionates in water to form sodium hydrogen sulfite and sodium thiosulfate (equation II)^2,[1], so that this substance can also be considered to be covered by the read-across concept described above. Since it can easily be anticipated that the substance is not stable enough under physiological conditions to fulfil the requirements of study guidelines, instead the products of decomposition have to be considered:

 

       2 S₂O₄^2-+ H₂O→2HSO₃^-+ S₂O₃^2-

 

Not fully covered by this read-across concept is the substance class of thiosulfates: although the thiosulfates are also well known to disproportionate in aqueous solution to form polythionic acids and SO₂(HSO₃^-), this requires somewhat different, more acidic conditions. Therefore, read-across to sulfites is primarily restricted to appropriate physiological conditions, i.e. oral administration where the gastric passage with the strongly acidic conditions in the stomach will facilitate the chemical disproportionation described above:

 

       HS₂O₃^-+ H₂S₂O₃→HS₃O₃- + SO₂+ H₂O

 

[1]Hollemann Wiberg, Lehrbuch der Anorganischen Chemie, 101.Auflage

[2]Handbook of Chemistry and Physics, Ed. Lide, DR, 88^thedition, CRC Press

Skin irritation:

One in vivo study on dermal irritation of potassium hydrogensulfite was identified and used as key study. The study was conducted according to OECD guideline 404. No erythema or oedema were seen in any of the rabbits at 24, 48, 72 hours after the beginning of the study.

Based on the outcome of these studies potassium hydrogensulfite is considered as not irritating to the skin.

Several further studies performed with sodium sulfite, ammonium hydrogensulfite, sodium hydrogensulfite, sodium metabisulfite and potassium metabisulfite equivalent or similar to OECD 404, used for read-across, are available which describe the test item as not irritating to the skin.

Based on the outcome of the key study and the read-across studies, it can be concluded that potassium hydrogensulfite has no skin irritating properties.

 

Eye irritation:

No conclusive information can be obtained from substance specific in-vitro studies according to OECD 491 an OECD 437.

An in-vivo study based on read-across to sodium sulfite equivalent or similar to OECD guideline 405 is available.

In a conservative approach and based on the assumption that potassium hydrogensulfite acts as a weak acid in aqueous solution, potassium hydrogen sulfite is considered as irritating to the eye.

Justification for classification or non-classification

Skin irritation:

(for additional information, see `discussion´)

References Consonni (2004) is considered as key study for skin irritation and will be used for classification.

According to the EC Regulation No. 1272/2008 and subsequent regulations, the test item is not classified as skin irritant.

Based on the outcome of the read-across studies, it can be concluded, that potassium hydrogen sulfite is not irritating to the skin and has not to be classified according to the criteria specified by Directive 67/548/EEC and subsequent regulations and according to the EC Regulation No. 1272/2008 and subsequent regulations.

Eye irritation:

No conclusive information can be obtained from in-vitro studies according to OECD 491 an OECD 437. Nevertheless, according to the EC Regulation No. 1272/2008 and subsequent regulations, the test item is considered as eye irritant due to its weak acidic properties.

Respiratory irritation:

The classification as respiratory irritant is covered under the endpoint specific target organ toxicity- single exposure and repeated exposure. Please refer to the endpoint summaries on acute toxicity (endpoint 7.2) and repeated dose toxicity (endpoint 7.5) for further information.