Dipotassium methanedisulphonate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 December 2017 - 16 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Dipotassium methanedisulphonate

EC Number:

228-543-0

EC Name:

Dipotassium methanedisulphonate

Cas Number:

6291-65-2

Molecular formula:

CH4O6S2.2K

IUPAC Name:

dipotassium methanedisulphonate

Test material form:

solid

In vitro test system

Test system:

human skin model

Remarks:

SkinEthic RHE® model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: not specified

Justification for test system used:

This study makes use of reconstructed human epidermis (RhE) (obtained from human derived non-transformed epidermal keratinocytes) which closely mimics the histological, morphological, biochemical and physiological properties of the upper parts of the human skin, i.e., the epidermis. Use of reconstructed human epidermis (RhE) is also recommended by OECD and other regulatory authorities. SkinEthicTM RHE model has been validated and is part of OECD validated reference methods (VRMs) and is also a recommended model for conducting in vitro skin irritation studies.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic RHE® model
- Tissue batch number(s): 17-RHE-127
- Production date:
- Shipping date:
- Delivery date:
- Date of initiation of testing: 12/12/2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature for 3 min exposure and 37°C ± 1°C for 1 hour exposure.
- Temperature of post-treatment incubation (if applicable): 37ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 20 x 1 mL of DPBS
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 hours at 37°C, 5% CO2
- Spectrophotometer: Synergy™ microplate reader (BioTek)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD =1.1 (CV = 4.5%) specification OD > 0.7. Historical negative control mean OD range = 1.610-2.594 (3 min exposure) and 1.806-2.693 (60 min exposure) (acceptability criteria, 0.8≤OD≤3).
- Barrier function: 4.5 h (Specification 4.0h < ET50< 10h)
- Morphology: 6 Cell layers, absence of significant histological abnormalities, well differentiated epidermis, specification > 4
- Contamination: no

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE:
- Freeze killed tissues (for positive control)
- Procedure used to prepare the killed tissues (if applicable): Killed tissues were prepared by incubating the viable tissues at -20 ± 5°C for 48 hours.
- N. of replicates : 2
- Method of calculation used: The true tissue viability is calculated as the percent tissue viability obtained with living tissues exposed to the positive minus the percent non-specific MTT reduction obtained with the killed tissues exposed to the same, calculated relative to the negative control run concurrently (%NSMTT). NSMTT was < 0% relative to the negative control, therefore true tissue viability was not determined.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20 mg (40 mg/cm2)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 40 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 40 µL
- Concentration (if solution): N/A

Duration of treatment / exposure:

3 minutes and 60 minutes

Duration of post-treatment incubation (if applicable):

3 hours (incubation in MTT solution)

Number of replicates:

3

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean after 3 min exposure

Value:

108

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

(distilled water)

Positive controls validity:

not applicable

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean after 1 hour exposure

Value:

95

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

(distilled water)

Positive controls validity:

valid

Remarks:

5.31% viability (8N KOH)

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: For adapted positive control treated tissues, NSMTT was < 0% relative to the negative control, therefore true MTT metabolic conversion of treated tissue was not determined.
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes. A demonstration of proficiency was performed for the SkinEthic RHE® model. Adequate results were obtained for the evaluated chemicals.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes. The mean OD of negative control tissues for treatment time 3 min and 1 hour were 2.328 and 2.324 (acceptability criteria, 0.8≤OD≤3 for the SkinEthic RHE® model).
- Acceptance criteria met for positive control: yes. Mean viability of the tissue replicates exposed for 1 hour, expressed as % of the negative control, is 5.31% < 15%.
- Acceptance criteria met for variability between replicate measurements: yes. 1.53% (for 3 min exposure) and 1.00% (for 60 min exposure) < 30%.

Any other information on results incl. tables

Table 1: Data Summary of Percent Viability

Treatment	Exposure Time	Tissue Replicate	O.D.	Corrected O.D.	Mean of Corrected O.D.	Mean O.D. of Replicate tissues	% Viability/ Tissue	Mean % Viability	S.D. of % Viability	% C.V. of % Viability	Corrosivity Class
Negative Control (Sterile Distilled water)	3 Minutes	1	2.352	2.309	2.294	2.328	100	100	NA	NA	NA
			2.331	2.288
			2.328	2.285
		2	2.411	2.368	2.353
			2.434	2.391
			2.343	2.3
		3	2.329	2.286	2.336
			2.433	2.39
			2.374	2.331
	60 Minutes	1	2.408	2.365	2.306	2.324	100	100	NA	NA
			2.324	2.281
			2.314	2.271
		2	2.335	2.292	2.299
			2.332	2.289
			2.36	2.317
		3	2.447	2.404	2.368
			2.342	2.299
			2.444	2.401
Positive Control (8N KOH)	60 Minutes	1	0.172	0.129	0.125	0.123	5.38	5.31	0.07	1.32	Corrosive
			0.165	0.122
			0.166	0.123
		2	0.172	0.129	0.123		5.29
			0.167	0.124
			0.16	0.117
		3	0.15	0.107	0.122		5.25
			0.179	0.136
			0.167	0.124

Treatment	Exposure Time	Tissue Replicate	O.D.	Corrected O.D.	Mean of Corrected O.D.	Mean O.D. of Replicate tissues	% Viability/ Tissue	Mean % Viability	S.D. of % Viability	% C.V. of % Viability	Corrosivity Class
Dipotassium Methanedisulphonate	3 Minutes	1	2.636	2.593	2.506	2.522	108	108	1.53	1.42	Non-corrosive
			2.499	2.456
			2.512	2.469
		2	2.572	2.529	2.570		110
			2.651	2.608
			2.617	2.574
		3	2.491	2.448	2.489		107
			2.518	2.475
			2.588	2.545
	60 Minutes	1	2.318	2.275	2.21	2.204	95	95	1.00	1.05
			2.199	2.156
			2.242	2.199
		2	2.381	2.338	2.225		96
			2.208	2.165
			2.216	2.173
		3	2.2	2.157	2.177		94
			2.237	2.194
			2.224	2.181

Key: O.D. = Optical density, S.D. = Standard deviation, C.V. = Coefficient variation

Note: Mean O.D. of Blank at 03 minutes and 60 minutes exposure was 0.042. Mean ODNC at 3 minutes exposure was 2.328 and Mean ODNC at 60 minutes exposure was 2.324.

Table 2: Data Summary of Adapted Control for Non-Specific MTT Reduction (NSMTT)

Treatment	Exposure Time	Tissue Replicate	O.D.	Corrected O.D.	Mean of Corrected O.D.	Mean OD of Replicate tissues	% NSMTT	Mean % NSMTT	TODTT	% Relative Viability
Negative Control (Untreated killed tissues)	60 minutes	1	0.17	0.128	0.127	0.129	NA	NA	NA	NA
			0.17	0.128
			0.168	0.126
		2	0.175	0.133	0.130
			0.168	0.126
			0.172	0.13
Positive Control (Positive control treated killed tissues)	60 minutes	1	0.046	0.004	0.005	0.005	-5.0	-5.0	NA	NA
			0.047	0.005
			0.047	0.005
		2	0.046	0.004	0.004		-5.0
			0.045	0.003
			0.046	0.004

Keys: O.D. = Optical Density, NSMTT = Non-specific MTT reduction calculation, NA = Not applicable, TODTT = Treated tissue true metabolic conversion,

Note: 1) For positive control (exposure period of 60 minutes) and Dipotassium Methanedisulphonate  (exposure period of 03 and 60 minutes), NSMTT was < 0% relative to the negative control, hence true MTT metabolic conversion of treated tissue was not determined; 2) Mean O.D. Blank was 0.042;  3) Mean ODNC for 60 minutes exposure was 2.324 and mean ODNC for 03 minutes exposure was 2.328; 4) Distilled water was used for Negative control tissues and 8N KOH was used for positive control tissues

Applicant's summary and conclusion

Interpretation of results:

other: No category (CLP Regulation EC no. 1272/2008)

Conclusions:

Under RHE test method performed in SkinEthic RHE® model the test item does not have to be classified as skin corrosive (cat. 1).

Executive summary:

An in vitro skin corrosion test for the test item was performed in a reconstructed human epidermis SkinEthic RHE® model, according to OECD TG 431 (GLP study). Three epidermis units were treated with 20 mg test item for 3 minutes at room temperature and for 1 hour, at 37°C, 5% CO2. Exposure of the test item was terminated by rinsing with 20 x 1 mL of DPBS. The viability of each disk was assessed by incubating the tissues with MTT, extracting the precipitated formazan crystals using isopropanol and incubating the tissues for overnight in a refrigerator protected from light, and finally measuring the concentration of formazan by determining the OD at 570 nm. Under the test conditions, the mean percent viabilities of the treated tissues were 108% (for 3 min exposure) and 95% (for 60 min exposure) versus 5.31% (for 60 min exposure) of the positive control item (potassium hydroxide 8N). Therefore, the test item must be considered as non-corrosive.