Ammonium hydrogensulphite

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 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:

Ammonium bisulfite solution 70% 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

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% 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 experiments/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 TG 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 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.

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 chemicals 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.

Irritation parameter:

other: Mean Cell Viability [%]

Run / experiment:

Test Item 0.05%

Value:

115.56

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:

63.18

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 proficiency 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 (exposed to culture medium) should be 0.3 or higher after subtraction of blank optical density (0.474 – 0.832).
- Viability of the solvent control should be 80% or higher relative to the medium control (86.04 – 93.40).
- The cell viability of the positive control (36.38%) is within two standard deviations of the historical mean (20.01% - 44.31%).
- 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 (9.4 and 10.1).
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 ammonium hydrogensulfite can be made.

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 TG 404.

Eye irritation:

A reliable in vitro test according to OECD TG 491 provided the result "No prediction can be made".

Justification for classification or non-classification

Skin irritation:

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

Eye irritation:

A reliable in vitro test according to OECD TG 491 provided the result "No prediction can be made".