Sodium permanganate

Endpoint summary

Description of key information

Skin Irritation

Under the conditions of the study the test material was classified as corrosive, Category 1A.

 

Eye Irritation

In accordance with Column 2 of REACH Annex VII, information requirement section 8.2, this study does not need to be conducted if the substance is classified for skin corrosion leading to classification as serious eye damage (Category 1).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 March 2021 to 12 Match 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Method B.40bis of Commission Regulation (EC) No 440/2008, of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006, 18 December 2006, of the European Parliament and of the Council on REACH

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

18 June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE)
EpiDerm™ Reconstructed Human Epidermis Model Kit was used.
Supplier: MatTek In Vitro Life Sciences Laboratories
Date received: 09 March 2021
EpiDermTM Tissues (0.63 cm^2) lot number: 34132
Assay Medium lot number: 030421LHB

PRE-TEST PROCEDURES
ASSESSMENT OF DIRECT TEST ITEM REDUCTION OF MTT

MTT DYE METABOLISM, CELL VIABILITY ASSAY
The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue/purple formazan salt by mitochondrial succinate dehydrogenase in viable cells. One limitation of the assay is possible interference of the test item with MTT. A test material may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test material is only a problem if at the time of the MTT test (after rinsing) there is still a sufficient amount of the test material present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified

TEST FOR DIRECT MTT REDUCTION
As specified, a test material may interfere with the MTT endpoint, if it was able to directly reduce MTT and at the same time was present on or in the tissues when the MTT viability test was performed. To identify this possible interference, the test material was checked for the ability to directly reduce MTT according to the procedure below:
50 µL of the test material was added to 1 mL of a freshly prepared 1.0 mg/mL MTT solution. The solution was incubated in the dark at 37 °C, 5 % CO2 in air for 60 minutes. Untreated MTT solution was tested concurrently to act as a control.
If the MTT solution containing the test material turned blue/purple relative to the control, the test material was presumed to have reduced the MTT.
An assessment of the test material’s capability to directly reduce MTT was inconclusive due to the intrinsic colour of the test material and therefore, an additional procedure using freeze killed tissues was performed. There was a possibility that if the test material could not be totally rinsed off the tissues, any residual test material present on or in the tissue may directly reduce MTT and could have given rise to a false negative result. Therefore, the determination of skin corrosion potential was performed in parallel on viable and freeze killed tissues.
This step was a functional check which employs freeze killed tissues that possess no metabolic activity but may absorb and bind the test material in the same way as viable tissues.
Freeze killed tissues were prepared prior to the study by placing untreated EPIDERM™ tissues in an empty 12 well plate and storing in a freezer (35 to 10 °C) for a minimum of 24 hours. Before use each tissue was thawed by placing in 0.9 mL of assay medium for approximately 1 hour in an incubator at 37 °C, 5 % CO2.
In addition to the normal test procedure, the test material was applied to two freeze killed tissues per exposure period. In addition, two freeze killed tissues per exposure period remained untreated. The untreated freeze killed control normally show a small amount of MTT reduction due to residual reducing enzymes within the killed tissues.

ASSESSMENT OF COLOR INTERFERENCE WITH THE MTT ENDPOINT
The test material was checked for possible colour interference before the study was started. A test material may interfere with the endpoint if it is coloured or if it becomes coloured when in wet or aqueous conditions. 50 µL of test material was added to 300 µL of sterile water. The solution was incubated in the dark at 37 °C, 5 % CO2 in air for 60 minutes. A visual assessment of the colour was then made.

DOUBLE CORRECTION CHECK
A third set of controls was used to prevent a double correction from a coloured test material that also reduces MTT using killed tissues. Intrinsically coloured test materials may bind to both living and killed tissues and therefore the non‑viable freeze‑killed tissues may not only correct for potential direct MTT reduction by the test material, but also for colour interference arising from the binding of the test material to the killed tissues. This could lead to a double correction for colour interference since the viable colour interference tissues already correct for colour interference arising from the binding of the test material to living tissues. Two freeze‑killed tissues were dosed with the test material and two freeze‑killed tissues remained untreated to act as the negative control for the 3 minute exposure, with the same number being used for the 60 minute exposure group. These tissues were incubated with assay medium instead of MTT post‑exposure.

MAIN TEST
PRE-INCUBATION
The assay medium was brought to room temperature before use. 0.9 mL of this assay medium was pipetted into the appropriate wells of two pre-labelled 6-well plates for both the 3‑Minute and 60‑Minute exposure periods. EpiDerm™ tissues were transferred into the 6‑well plates containing the assay medium. The 6‑well plates containing the EpiDerm™ samples were pre-incubated (37 °C, 5 % CO2) for approximately 1 hour before dosing.

APPLICATION OF TEST ITEM AND RINSING
Before pre-incubation was complete, a 24‑well plate was prepared for use as a “holding plate” for both the 3‑Minute and 60‑Minute exposure periods. This plate was used to maintain the viability of the tissue inserts between rinsing following chemical exposure and MTT-loading. Another 24‑well plate was prepared for the MTT-loading. 300 µL of either pre‑warmed assay medium (holding plate) or MTT medium (MTT-loading plate) was dispensed into each well. The two plates were placed into the incubator until required. After pre‑incubation of the EpiDerm™ tissues, the medium was aspirated and replaced with 0.9 mL of fresh assay medium. The 6-well plate for the 3‑Minute exposure period was returned to the incubator, while the other was being dosed for the 60‑Minute exposure. For the 60‑Minute exposure period, 50 µL of sterile distilled water (negative control) was added to the first two tissues. The tissues were dosed at regular intervals to allow for the time taken to rinse each tissue following exposure and to ensure that each tissue gets an equal exposure time. 50 µL of the test item and 50 µL of 8.0 N Potassium Hydroxide (positive control) were also applied to the corresponding tissues in turn. The plate was returned to the incubator (37 °C, 5 % CO2) for the 60‑Minute exposure period. When dosing for the 60‑Minute exposure period was complete, the same procedure was repeated for the 3‑Minute exposure period. Because the exposure time was so short, the tissues were dosed at regular intervals to ensure that each tissue received an equal exposure time and to allow for the time taken to rinse each tissue following exposure. Rinsing was achieved by filling and emptying each tissue under a constant soft stream of Dulbecco’s Phosphate Buffered Saline (DPBS) (without Ca++ Mg++) for approximately 40 seconds, to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper. Each tissue was placed into the prepared holding plate until all tissues were rinsed. They were then blotted and transferred to the 24‑well plate prepared for MTT-loading. The plate was incubated (37 °C, 5 % CO2) for 3 hours. Once the 60‑Minute exposure period was complete, the same rinsing and MTT-loading procedure was repeated. After the 3‑Hour MTT incubation was complete, the tissue inserts were blotted and transferred to 24‑well plates for formazan (reduced MTT) extraction. The formazan was extracted from the top and bottom of the tissue by completely immersing the tissue insert in 2 mL of isopropanol. The plate was covered with plate sealer, to prevent isopropanol evaporation, and stood overnight at room temperature, to allow extraction to proceed.

ABSORBANCE/OPTICAL DENSITY MEASUREMENTS.
After extraction, each tissue was pierced with a pipette fitted with a 1 000 µL tip and the extraction solution was forced vigorously up and down to form a homogenous solution. 3 x 200 µL aliquots of the extract were transferred to the appropriate wells of a pre‑labelled 96‑well plate. 200 µL of isopropanol alone was added to the three wells designated as blanks. Absorbency at 570 nm (OD570) of each well was measured using the Labtech LT‑4500 microplate reader and LT-com analysis software.

DATA EVALUATION
QUANTITATIVE MTT ASSESSMENT (PERCENTAGE TISSUE VIABILITY)
The corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after the 3 and 60‑Minute exposure periods, compared to the mean of the negative control tissues (n=2) treated with sterile distilled water. The relative mean viabilities were calculated as follows:

Relative mean viability (%) = mean OD570 of test item / mean OD570 of negative control) * 100

Classification of corrosivity potential was based on relative viabilities for both exposure times, according to EU CLP Regulation (EC) No 1272/2008 UN GHS:

STEP 1
< 50 % after 3 min exposure = Corrosive
≥ 50 % after 3 min exposure AND < 15 % after 60 min exposure = Corrosive
≥ 50 % after 3 min exposure AND ≥ 15 % after 60 min exposure = Non-corrosive

STEP 2 for test items identified as corrosive in step 1
< 25 % after 3 min exposure = H314 Sub-category 1A
≥ 25 % after 3 min exposure = H314 Combination of sub-categories 1B/1C

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

50 µL of sterile distilled water (negative control)
50 µL of the test item
50 µL of 8.0 N Potassium Hydroxide (positive control)

Duration of treatment / exposure:

3 Minute and 60 Minute exposure periods

Duration of post-treatment incubation (if applicable):

The assay medium was brought to room temperature before use. 0.9 mL of this assay medium was pipetted into the appropriate wells of two pre-labeled 6-well plates for both the 3 Minute and 60 Minute exposure periods. EpiDerm™ tissues were transferred into the 6 well plates containing the assay medium. The 6 well plates containing the EpiDerm™ samples were pre-incubated (37 °C, 5 % CO2) for approximately 1 hour before dosing.

Number of replicates:

2 per sample

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes

Value:

17.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes

Value:

0.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

Acceptance Criteria:
The mean OD570 for the negative control treated tissues was 1.779 for the 3‑Minute exposure period and 1.666 for the 60‑Minute exposure period.  The negative control acceptance criteria were therefore satisfied.
The relative mean tissue viability for the positive control treated tissues was 3.1 % relative to the negative control following the 60‑Minute exposure period.  The positive control acceptance criterion was therefore satisfied.
In the range 20 to 100 % viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30 %.  The acceptance criterion was therefore satisfied.

Direct MTT Reduction

An assessment found the test item was able to directly reduce MTT.  Therefore, an additional procedure using non-viable, freeze‑killed, tissues was performed.  However, the results obtained showed that no interference due to direct reduction of MTT occurred.  It was therefore considered unnecessary to use the results of the freeze‑killed tissues for quantitative correction of results or for reporting purposes.

 

Assessment of Colour Interference with the MTT endpoint

The solution containing the test material was a purple colour, therefore additional colour correction tissues were incorporated into the testing procedure.  However, the results obtained showed that no colour interference occurred.  It was therefore considered unnecessary to use the results of the colour correction tissues for quantitative correction of results or for reporting purposes.

 

Double Correction Check

The results of the third set of control tissues were not used to adjust the calculated correction factors derived from the colour interference and MTT direct reduction controls due to no interference occurring, therefore the correction was unnecessary.

 

Test Material, Positive Control Item and Negative Control Item

Mean OD570 values and viabilities for the negative control, positive control are reported.

The relative mean viabilities for each treatment group are:

Exposure Period	Percentage Viability
	Negative Control	Positive Control	Test Item
3 minute	100*	2.9	17.2
60 minute	100*	3.1	0.4

*The mean viability of the negative control tissues is set at 100 %.

 

Mean OD₅₇₀ Values and Viabilities for the Negative Control Item, Positive Control Item and Test Material

Tissue	Exposure Period	Mean OD570 of individual tissues	Mean OD570 of duplicate tissues	Standard Deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative Control	3 Minutes	1.808	1.779	0.041	2.3	100*
		1.750
	60 Minutes	1.669	1.666	0.005	0.3
		1.662
Positive Control	3 Minutes	0.051	0.052	0.001	na	2.9
		0.052
	60 Minutes	0.053	0.051	0.003	na	3.1
		0.049
Test Item	3 Minutes	0.237	0.306	0.098	31.9 (na)	17.2
		0.375
	60 Minutes	0.007	0.006	0.001	23.6 (na)	0.4
		0.005

OD = Optical density.

* = The mean percentage viability of the negative control tissue is set at 100 %.

na = Not applicable.

Interpretation of results:

other: Classified as corrosive Category 1A according to EU criteria.

Conclusions:

Under the conditions of the study the test material was classified as corrosive, Category 1A.

Executive summary:

A study was conducted to assess the skin irritation potential of the test material according to OECD Test Guideline 431 and EU Method B.40bis and in compliance with GLP.

The purpose of this test was to evaluate the corrosivity potential of the test material using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes.

Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue.  Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test material treated tissues relative to the corresponding negative control.  Viable cells are able to reduce MTT to formazan whereas non-viable cells cannot.  The results are used to make a prediction of the corrosivity potential of the test material (increased cytotoxicity is indicative of corrosion potential).

Duplicate tissues were treated with the negative control, positive control and test material for exposure periods of 3 and 60 minutes.  The test material was found to have the potential to cause colour interference and therefore additional tissues were incorporated for colour correction purposes.  The test material was found to directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes.  A third set of controls was included, comprising freeze‑killed tissues, in order to prevent a double correction from a coloured test material that also reduces MTT.  At the end of the exposure period the control items and test material were rinsed from the tissues before each tissue was taken for MTT‑loading.  After MTT-loading the tissues were placed into 2 mL of isopropanol for formazan extraction.

At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 mL samples were transferred to the appropriate wells of a pre-labelled 96‑well plate.  The optical density (OD) was measured at 570 nm (OD570).

Data are presented in the form of percentage viability (MTT reduction in the test material treated tissues relative to negative control tissues).

The relative mean viabilities for each treatment group were as follows:

3-minute exposure: The test material percentage viability was 17.2.  The negative and positive controls were 100 and 2.9 % respectively.

60-minute exposure: The test material percentage viability was 0.4.  The negative and positive controls were 100 and 3.1 % respectively.

The criteria required for acceptance of results in the test were satisfied.

Under the conditions of the study the test material was classified as corrosive, Category 1A.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because the substance is classified as skin corrosion, leading to classification as serious eye damage (Category 1)

Justification for type of information:

In accordance with Column 2 of REACH Annex VII, information requirement section 8.2, this study does not need to be conducted if the substance is classified for skin corrosion leading to classification as serious eye damage (Category 1).

Reference 2

Endpoint:

eye irritation: in vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because the substance is classified as skin corrosion, leading to classification as serious eye damage (Category 1)

Justification for type of information:

In accordance with Column 2 of REACH Annex VII, information requirement section 8.2, this study does not need to be conducted if the substance is classified for skin corrosion leading to classification as serious eye damage (Category 1).

Endpoint conclusion

Endpoint conclusion:

no study available

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Irritation

A study was conducted to assess the skin irritation potential of the test material according to OECD Test Guideline 431 and EU Method B.40bis and in compliance with GLP.

The purpose of this test was to evaluate the corrosivity potential of the test material using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes.

Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue.  Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test material treated tissues relative to the corresponding negative control.  Viable cells are able to reduce MTT to formazan whereas non-viable cells cannot.  The results are used to make a prediction of the corrosivity potential of the test material (increased cytotoxicity is indicative of corrosion potential).

Duplicate tissues were treated with the negative control, positive control and test material for exposure periods of 3 and 60 minutes.  The test material was found to have the potential to cause colour interference and therefore additional tissues were incorporated for colour correction purposes.  The test material was found to directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes.  A third set of controls was included, comprising freeze‑killed tissues, in order to prevent a double correction from a coloured test material that also reduces MTT.  At the end of the exposure period the control items and test material were rinsed from the tissues before each tissue was taken for MTT‑loading.  After MTT-loading the tissues were placed into 2 mL of isopropanol for formazan extraction.

At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 mL samples were transferred to the appropriate wells of a pre-labelled 96‑well plate.  The optical density (OD) was measured at 570 nm (OD570).

Data are presented in the form of percentage viability (MTT reduction in the test material treated tissues relative to negative control tissues).

The relative mean viabilities for each treatment group were as follows:

3-minute exposure: The test material percentage viability was 17.2.  The negative and positive controls were 100 and 2.9 % respectively.

60-minute exposure: The test material percentage viability was 0.4.  The negative and positive controls were 100 and 3.1 % respectively.

The criteria required for acceptance of results in the test were satisfied.

Under the conditions of the study the test material was classified as corrosive, Category 1A.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the material does require classification with respect to skin and eye corrosion category 1A (H314 Causes severe skin burns and eye damage).