Zinc bis(hydroxymethanesulphinate)

Endpoint summary

Description of key information

Effects on skin and eye irritation were tested according to standard OECD in vitro assays.

The OECD 439 in vitro skin irritation assay concluded that the test substances has to be considered as non-irritant to skin.

Both the OECD 438 and 492 in vitro eye irritation assays did identify effects, but were not conclusive in the final classification. Based on the weight of evidence, a classification as eye irritant cat. 2 was assigned to zinc formaldehyde sulfoxylate.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6-14 June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch no: 515

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Reconstructed Human epidermis (SkinEthic RHE® model, Episkin SA, RHE/S/17)
- Tissue batch number(s): 18-RHE-064
- Production date: not reported
- Shipping date: not reported
- Delivery date: 12 June 2018
- Date of initiation of testing: 12 June 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: not reported
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 42 minutes after the test item application, the human epidermis were washed with 25 x 1 mL of DPBS (Dutscher - Batch No. 9120318).
- Observable damage in the tissue due to washing: none
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: ELx800 absorbance microplate reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
There is no direct interaction between the test item and MTT and there is no need to add non- specific coloration controls to the study.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test item is considered as non-irritant to skin in accordance with UN GHS No Category: if the mean percent viability after 42 minutes exposure and 42 hours of post-treatment incubation is > 50%.
- The test item is identified as requiring classification and labelling according to UN GHS (Category 2): if the mean percent tissue viability after 42 minutes exposure and 42 hours of post-treatment incubation is ≤ 50% and the result of a skin corrosion test is “non corrosive”.
- The test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1): if the mean percent tissue viability after 42 minutes exposure and 42 hours of post-treatment incubation is ≤ 50% and in absence of information on a skin corrosion test.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

16 mg/0.5 cm2

Duration of treatment / exposure:

42 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

81.3

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

85.5

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

87

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Mean tissue viability test item: 84.6 %
Mean tissue viability negative control: 100%
Mean tissue viability positive control (5% sodium dodecyl sulfate): 1.3%

Interpretation of results:

GHS criteria not met

Conclusions:

In accordance with the Regulation EC No. 1272/2008, the test item ZINC FORMALDEHYDE SULFOXYLATE has to be considered as Non-irritant to skin. It corresponds to UN GHS No Category.

Executive summary:

The aim was to evaluate the possible irritating effects of the test item ZINC FORMALDEHYDE SULFOXYLATE after topical application on in vitro human reconstructed epidermis (SkinEthic RHE model) in accordance with OECD 439 and Test Method B.46 of Council regulation No 761/2009).

The test item was applied as supplied, at the dose of 16 mg to 3 living Reconstructed Human epidermis during 42 minutes. The application was followed by a rinse with 25 mL of DPBS and a 42 hours post-incubation period at 37°C, 5% CO2. Cell viability was then measuered by enzymatc conversion of the vital dye MTT into a blue formazan salt that was quantitatively meausured after extraction from tissues.

The mean percent viability of the treated tissues was 84.6%, versus 1.3% in the positive control (5% SDS).

In accordance with the Regulation EC No. 1272/2008, the test item ZINC FORMALDEHYDE SULFOXYLATE has to be considered as Non-irritant to skin. It corresponds to UN GHS No Category.

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:

key study

Study period:

9 July 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch no 515

Species:

chicken

Details on test animals or tissues and environmental conditions:

Eyes collected from chickens obtained from a slaughterhouse (Etablissement Brun, 33820 Etauliers, France) where they were killed for human consumption have been used for this assay. Heads have been removed immediately after sedation of the chickens by electric shock, and incision of the neck for bleeding. The intact heads were transported from the slaughterhouse at ambient temperature in plastic boxes humidified with towels moistened with physiological saline. The eyes were enucleated at Phycher afterwards.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

30 mg per eye

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

240 minutes

Number of animals or in vitro replicates:

3 eyes for the test item, 1 eye for negative control, 3 eyes for positive controls

Details on study design:

Damages by the test item were assessed by determination of corneal swelling, opacity, and fluorescein retention at 30, 75, 120 180 and 240 minutes post-dose.

Irritation parameter:

cornea opacity score

Run / experiment:

Mean

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class I

Irritation parameter:

fluorescein retention score

Run / experiment:

Mean

Value:

1.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class III

Irritation parameter:

percent corneal swelling

Run / experiment:

Mean

Value:

3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class I

Other effects / acceptance of results:

The combination the three endpoints for the test item was 1 x III, 2 x I.

The combination of the three endpoints for the positive control, Sodium hydroxide, was 3 x IV. Therefore, the positive control is classified as "Corrosive/Severe Irritant", as expected.

The combination of the three endpoints for the negative control, physiological saline, was 3 x I. Therefore, the negative control is classified as "No Category", as expected.

Interpretation of results:

other: no prediction can be made

Conclusions:

In accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions lead to the category "no prediction can be made", as defined by the OECD Guideline No 438. Therefore the test item is not predicted as causing serious eye damage (Category 1) or as not classified for eye irritation/serieous eye damage (No category) with the Isolated Chicken eye test method.

Executive summary:

The aim of the study was to evaluate the possible ocular corrosive or severe irritating effects of the test item after administration on enucleated chicken eyes.

The test item ZINC FORMALDEHYDE SULFOXYLATE was applied, after being reduced in fine powder, at the dose of 30 mg, to 3 enucleated chicken eyes, during 10 seconds. Then the eyes were rinsed twice with 10 mL of physiological saline. Three eyes were treated in the same manner with a positive control and one eye with a negative control. Damages by the test item were assessed by determination of corneal swelling, opacity, and fluorescein retention at 30, 75, 120 180 and 240 minutes post-dose. The test experimental protocol was established in accordance with OECD 438 and test method B.48.

The ocular reactions observed in eyes treated with the test item were:

- maximal mean score of corneal opacity: 0.0, corresponding to ICE Class I;

- mean score of fluorescein retention: 1.7, corresponding to ICE Class III;

- maximal mean corneal swelling: 3%, corresponding to ICE Class I.

The combination the three endpoints for the test item was 1 x III, 2 x I.

The combination of the three endpoints for the positive control, Sodium hydroxide, was 3 x IV. Therefore, the positive control is classified as "Corrosive/Severe Irritant", as expected.

The combination of the three endpoints for the negative control, physiological saline, was 3 x I. Therefore, the negative control is classified as "No Category", as expected.

In accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions lead to the category "no prediction can be made", as defined by the OECD Guideline No 438. Therefore the test item is not predicted as causing serious eye damage (Category 1) or as not classified for eye irritation/serieous eye damage (No category) with the Isolated Chicken Eye test method.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 September 2018 - 11 October 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch no 515

Species:

human

Details on test animals or tissues and environmental conditions:

EpiOcular OCL-212-ver2.0, supplied by MatTEK Corporation, Reconstructed human Cornea-like Epithelia
Lot number: 27073
Keratinocyte strain: 4F1188

Vehicle:

unchanged (no vehicle)

Remarks:

The test item was used after being reduced in fine powder in the study.

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 mg per DBPS pre-treated RhCE or killed RhCE (0.60 cm2)

Duration of treatment / exposure:

6 hours at 37°C, 5% CO2, 95% humidity

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

2 DPBS pre-treated RhCE and 2 killed RhCE

Details on study design:

The test item was identified as a direct MTT reducer and two killed control tissue models were added to the study which underwent the entire testing procedure to generate a non- specific MTT reduction control.

After the overnight incubation, the tissues were pre-wetted with 20 μL of Ca2+Mg2+Free-DPBS (Dutscher - Batch No. 4080718). The tissues were incubated at standard culture conditions for 30 minutes.
The test item was applied, after being reduced in fine powder, during 6 hours at standard culture conditions, at the dose of 50 mg to the entire surface of 2 living RhCE tissue replicates and 2 killed RhCE (EpiOcularTM tissue model) (NSC control)
In the same experimental conditions, a positive control (Methyl acetate - MatTek Corporation, batch No. 022118ISA), and a negative control (distilled water - VWR - Batch No.1007127) were carried out. The controls were applied, as supplied, at the dose of 50 μL, to the surface of 2 RhCE tissue replicates during 6 hours at standard culture conditions.
After the treatment, the test item and control substances were carefully washed from the RhCE tissues by extensive rinsing with Ca2+Mg2+Free-DPBS (Dutscher - Batch No. 4080718). The rinsed tissues were checked for any coloration and noted to be of comparable colour with the negative control treated tissues (whitish).
This rinsing step was followed by a 25-minute post-exposure immersion period at room temperature in 5 mL of fresh medium to remove any test item absorbed into the tissue. The RhCE constructs were then incubated for an 18 hours post-exposure incubation at standard culture conditions in 1 mL of fresh medium at 37°C, 5% CO2.

Following the exposure to the test item, viability measurements are performed immediately after the post-exposure incubation period of the rinsed tissues in fresh medium. This period allows both for recovery from weak cytotoxic effects and for appearance of clear cytotoxic effects.
The RhCE tissue viability was measured by enzymatic conversion of the vital dye MTT [3-(4,5- Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1] by the viable cells of the tissue into a blue MTT formazan salt that is quantitatively measured after extraction from tissues.
The RhCE constructs were placed in 300 μL of a MTT solution at 1.0 mg/mL for 3 hours at standard culture conditions.
The precipitated blue formazan product was then extracted from the tissues by placing each insert in 2 mL of isopropanol during 2 hours at room temperature in the dark.
The concentration of formazan was measured by determining the OD at 570 nm, just after dilution of the extractions in isopropanol (1:2).
The OD at 570 nm was measured in triplicate samples of formazan extracts. The measured OD are proportional to the number of living cells.
The measurement of OD was performed using the ELx800 absorbance microplate reader (controlled every year and calibrated if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.

Irritation parameter:

other: % viability

Run / experiment:

second run

Value:

2.67

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

mean tissue viability of postitive control: 42.03%

Interpretation of results:

other: potentially requiring classification and labelling according to UN GHS Category 2 or Category 1

Conclusions:

In accordance with the Regulation EC No. 1272/2008, the test item ZINC FORMALDEHYDE SULFOXYLATE has to be identified as potentially requiring classification and labelling according to UN GHS Category 2 or Category 1.

Executive summary:

The aim of the study was to evaluate the eye hazard potential of the test item ZINC FORMALDEHYDE SULFOXYLATE after topical administration on in vitro reconstructed human cornea-like epithelium tissues (EpiOcular tissue model).

The test item was applied, after being reduced in fine powder, during 6 hours at 37°C, 5% CO2, 95% humidity (standard culture conditions), at the dose of 50 mg to 2 DPBS pre-treated RhCE (EpiOcular tissue model) and 2 killed RhCE (EpiOcular tissue model). The exposure perioed was followed by extensive rinsing with DPBS at room temperature, a 25 minutes post-exposure immersion period at room temperature and 18 hours post-exposure incubation at standard culture conditions. The tissue viability was measured by performing an MTT assay. The experimental protocol was established in accordance with OECD Test Guideline No 492 adopted 09 October 2017.

As the acceptability criteria defined in OECD 492 were not met for the negative control during the 1st run, it was not possible to conclude. A second test was therefore performed under the same experimental conditions to conclude.

Fot the second run, the mean corrected percent tissue viability of the RhCE replicates treated with the test item was 2.67%, versus 42.03% in the positive control (Methyl acetate).

In accordance with the Regulation EC No. 1272/2008, the test item ZINC FORMALDEHYDE SULFOXYLATE has to be identified as potentially requiring classification and labelling according to UN GHS Category 2 or Category 1.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin irritation/corrosion:

The substance is not classified a skin irritant because in the selected test it does not meet the classification criteria of the CLP regulation n. 1272/2008.

Eye irritation:

The substance is classified a eye irritant (cat. 2) based on 2 in vitro tests. The isolated chicken eye (ICE, OECD 438) test results in the category "no prediction can be made", as defined by the OECD Guideline No 438 and the test item is not predicted as causing serious eye damage (Category 1) or as not classified for eye irritation/serieous eye damage (No category). The reconstructed human cornea-like epithelium (RhCE, OECD 492) test method identified the substance as potentially requiring classification and labelling according to UN GHS Category 2 or Category 1. Combining the results of both non-conclusive tests (clear identification of effects but not predicted as eye damage), the substance is classified as eye irritant cat. 2.