1,3-Propanediaminium, 2-hydroxy-N1,N1,N1,N3,N3-pentamethyl-N3-[3-[(2-methyl-1-oxo-2-propen-1-yl)amino]propyl]-, chloride (1:2)

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 08 October to 27 November 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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:

Methacryloamidopropyl-pentamethyl-1,3-propylene-2-ol-ammonium dichloride

EC Number:

617-909-9

Cas Number:

86702-10-5

Molecular formula:

C15 H33 N3 O2 .2Cl

IUPAC Name:

Methacryloamidopropyl-pentamethyl-1,3-propylene-2-ol-ammonium dichloride

Test material form:

solid - liquid: aqueous solution

Details on test material:

- State of aggregation: N/A
- Particle size distribution: N/A
- Mass median aerodynamic diameter (MMAD): N/A
- Geometric standard deviation (GSD): N/A
- Shape of particles: N/A
- Surface area of particles: N/A
- Crystal structure: N/A
- Coating: N/A
- Surface properties: N/A
- Density: 1.135
- Moisture content: N/A
- Residual solvent: N/A
- Activation: N/A
- Stabilisation: N/A
- Other:

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: CY50561001
- Expiration date of the batch: 24/04/2016
- Purity test date: 65.4%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature 15-25°C, below 70 RH%
- Solubility and stability of the test substance in the solvent/vehicle: soluble in water. Stability not checked

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none (The test item was applied in its original form.)
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

In vitro test system

Test system:

human skin model

Remarks:

EPISKIN-SM is a three-dimensional human epidermis model.

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Adult donors

Source strain:

not specified

Details on animal used as source of test system:

Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period.

Justification for test system used:

The EPISKIN-SM model has been validated for corrosivity testing in an international trial (Fentem, 1998) and its use is recommended by the relevant OECD guideline for corrosivity testing (OECD No. 431); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN-SM model
- Tissue batch number(s): 15-EKIN-040
- Production date: Unknown
- Shipping date: Unknown
- Delivery date: Unknown
- Expiry date: 12 October 2015
- Date of initiation of testing: 08 October 2015

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature
- Temperature of post-treatment incubation (if applicable): not applicable

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: one, with approximately 25 mL PBS solution.
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT stock solution= 3 mg/mL ; MTT final concentration (working solution) = 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Thermo Fisher Scientific, Catalogue Number: 240 72800, Serial Number: 0920-14
- Wavelength: 570 nm.
- Filter: unknown
- Filter bandwidth: unknown
- Linear OD range of spectrophotometer: unknown

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
The quality of the final product is assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecylsulphate (SDS). These quality control experiments were conducted at SkinEthic laboratories (supplier of the EpiSkin-SM test kits used in the present study).

NUMBER OF REPLICATE TISSUES: Two epidermis units were used for each test or control materials.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE : As the test item was coloured, two additional test item-treated tissues were used for the non specific OD evaluation.
- Fresh tissues
- Procedure used to prepare the killed tissues (if applicable): N/A
- N. of replicates : two
- Method of calculation used: The mean optical density (measured at 570 nm) of these tissues was determined as 0.009, Non Specific Colour % was calculated as 1.0% (see Table 1). This is below the threshold of 5%, therefore correction due to colouring potential was not necessary.

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

PREDICTION MODEL / DECISION CRITERIA:
If both disks have mean viability of ≥35% = Non Corrosive
If both disks have mean viability of <35% = Corrosive (at the corresponding incubation period)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 μL
- Concentration (if solution): N/A (The test item was applied in its original form (it was supplied in a form of 65.4% solution), no formulation was required.)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration (if solution): N/A

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration (if solution): N/A

Duration of treatment / exposure:

4 hours (±10 min)

Duration of post-treatment incubation (if applicable):

N/A

Number of replicates:

Two epidermis units were used for each test or control materials.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

ca. 89.3

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: After receipt, the two indicators of the delivered kit were checked in each case. Based on the observed colours, the epidermis units were in proper conditions.
- Direct-MTT reduction: no
- Colour interference with MTT: As the test item was coloured, two additional test item-treated tissues were used for the non specific OD evaluation. The mean optical density (measured at 570 nm) of these tissues was determined as 0.009, Non Specific Colour % was calculated as 1.0% (see Table 1). This is below the threshold of 5%, therefore correction due to colouring potential was not necessary. As no colour change was observed after three hours of incubation of the test item in MTT solution, thus the test material did not interact with MTT. Therefore, additional controls and data calculations were not necessary to exclude the false estimation of viability.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Negative and positive controls as well as variability between replicate measurements were within acceptable limits and therefore the study was considered to be valid.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD value of the two negative control tissues was in the recommended range (0.889).
- Acceptance criteria met for positive control: The positive control treated tissues showed 0.9% viability demonstrating the proper performance of the assay.
- Acceptance criteria met for variability between replicate measurements: The difference of viability between the two test item-treated tissue samples in the MTT assay was 9.3%.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Following exposure with Diquat, the mean cell viability was 89.3% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.
In conclusion, in this in vitro EPISKIN model test with Diquat, the results indicate that the test item (tested in a form of 65.4% solution) is not corrosive to the skin.

Executive summary:

An in vitro skin corrosivity test of Diquat test item was performed in a reconstructed human epidermis model. EPISKIN-SM is designed to predict and classify the corrosive potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (detailed in 3.6. section). The corrosivity of the test item was evaluated according to the OECD No. 431 guideline.

Disks of EPISKIN (two units) were treated with Diquat test item and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with Phosphate Buffered Saline solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

Physiological saline (0.9% (w/v) NaCl solution) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively (two units / control). Two additional disks were used to provide an estimate of colour contribution from the test item. For each treated tissue viability was expressed as a % relative to the negative control. If the mean relative viability after 4 hours of exposure is below 35% of the negative control, the test item is considered to be corrosive to skin.

Following exposure with Diquat, the mean cell viability was 89.3% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKIN model test with Diquat, the results indicate that the test item (tested in a form of 65.4% solution) is not corrosive to the skin.