(8α,9R,8'''α,9'''R)-1,1'-[(2,3,5,6-tetrafluoro-1,4-phenylene)bis(methylene)]bis(6'-methoxycinchonan-1-ium-9-ol) dibromide

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(8α,9R,8'''α,9'''R)-1,1'-[(2,3,5,6-tetrafluoro-1,4-phenylene)bis(methylene)]bis(6'-methoxycinchonan-1-ium-9-ol) dibromide

EC Number:

857-673-6

Cas Number:

1879067-61-4

Molecular formula:

C48H52F4N4O4.2Br

IUPAC Name:

(8α,9R,8'''α,9'''R)-1,1'-[(2,3,5,6-tetrafluoro-1,4-phenylene)bis(methylene)]bis(6'-methoxycinchonan-1-ium-9-ol) dibromide

Test material form:

solid: particulate/powder

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from multiple donors

Vehicle:

physiological saline

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN
- Tissue batch number(s): Batch No.: 19-EKIN-030
- Production date: 23 July 2019
- Expiry date: 29 July 2019
- Date of initiation of testing: 25 July 2019

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C
- Temperature of post-treatment incubation: 23.7 to 24.2 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: washing was done with phosphate buffered saline, volume and number of washes not reported
- Observable damage in the tissue due to washing: not reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL, prepared from 3 mg/mL stock solution
- Incubation time: 3 hours
- Spectrophotometer: not reported
- Wavelength: 570 nm
- Filter: not reported
- Filter bandwidth: not reported
- Linear OD range of spectrophotometer: not reported

NUMBER OF REPLICATE TISSUES: two

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues
- Procedure used to prepare the killed tissues: living epidermis units (Manufacturer: SkinEthic, France, Batch No.:19-EKIN-016, Expiry Date: 22 April 2019) on 16 April 2019 were placed in a 12 well plate with 2 mL of distilled water, then incubated at 37 °C in an incubator with 5% CO2, in a >95% humidified atmosphere for 48 hours (±1 hour). At the end of the incubation the water was discarded and the epidermis units were frozen on 18 April 2019
- N. of replicates : two per control
- Method of calculation used:
Blank:
– The mean of the 6 blank (as in 3.8.5) OD values was calculated
Negative control:
– Individual negative control OD values (NCraw) were corrected with the mean blank OD:
OD Negative Control (ODNC) = ODNCraw – ODblank mean
– The corrected mean OD of the 2 negative control values was also calculated: this corresponds to 100% viability
Positive control:
– Individual positive control OD values (PCraw) were corrected with the mean blank OD:
OD Positive Control (ODPC) = ODPCraw – ODblank mean
– The corrected mean OD of the 2 positive control values was calculated
– The % viability for each positive control replicate was calculated relative to the mean negative control:
Positive Control1 % = (ODPC1 / mean ODNC) ×100
Positive Control2 % = (ODPC2 / mean ODNC) ×100
– The mean value of the 2 individual viability % for positive control was calculated:
Mean PC % = (PC1 % + PC2 % ) / 2
Test item:
– Individual test item OD values (TTraw) were corrected with the mean blank OD:
OD Treated Tissue (ODTT) = ODTTraw – ODblank mean
– The corrected mean OD of the 2 test item values was calculated
– The % viability for each test item replicate was calculated relative to the mean negative control:
Treated Tissue1 % = (ODTT1 / mean ODNC) ×100
Treated Tissue2 % = (ODTT2 / mean ODNC) ×100
– The mean value of the 2 individual viability % for test item was calculated:
Mean TT % = (TT1 % + TT2 %) / 2
– The variability for 2 disks is calculated as:
(Disk1-Disk2)/((Disk1+Disk2)/2) x 100%
Test items that interfere with MTT can produce non-specific reduction of the MTT. In this case, additional control samples are used to determine the OD value derived from non-specific reduction of the MTT. The measured OD value is corrected by the result of the additional controls before calculation of viability % as follows:
Non-specific MTT reduction calculation (NSMTT%):
NSMTT% = [(ODKT- ODKNC) / ODNC] × 100
ODKNC: negative control treated killed tissues OD
ODKT: test item treated killed tissues OD
ODNC: negative control OD
If NSMTT% is ≤ 50%, then true MTT metabolic conversion (TODTT) is undertaken as follows:
TODTT = [ODTT – (ODKT – ODKNC)]
ODTT: test item treated viable tissues
– The % relative viability (RV%) for each test item replicate is calculated relative to the mean negative control:
RV1 % = [TODTT1 / mean ODNC] × 100
RV2 % = [TODTT2 / mean ODNC] × 100
– The mean value of the 2 individual relative viability % for test item is calculated:
Mean Relative Viability % = (RV1 % + RV2 %) / 2
If NSMTT% is > 50% relative to the negative control: additional steps must be undertaken if possible, or the test item must be considered as incompatible with the test.
For test items detected as able to stain the tissues the non-specific OD is evaluated due to the residual chemical colour (unrelated to mitochondrial activity) and subtracted before calculation of the “true” viability % as detailed below:
Non-Specific Colour % (NSC living %):
NSC living % = (mean ODCTV / mean ODNC)×100
ODCTV: test item treated viable tissue (not incubated with MTT)
ODNC: negative control OD (incubated with MTT)
If NSC living % is ≤ 5 % then the normal calculation mode is used.
If NSC living % is > 5 % and ≤ 50 %, then additional correction (TODTT) is undertaken as follows:
TODTT = [ODTT - ODCTV]
ODTT: test item treated viable tissue (incubated with MTT)
ODCTV: test item treated viable tissue (not incubated with MTT)
– The % relative viability (RV%) for each test item replicate is calculated relative to the mean negative control:
RV1 % = [TODTT1 / mean ODNC] × 100
RV2 % = [TODTT2 / mean ODNC] × 100
– The mean value of the 2 individual relative viability % for test item is calculated:
Mean Relative Viability % = (RV1 % + RV2 %) / 2
If NSC living % is > 50 % relative to the negative control, additional steps must be undertaken if possible, or the test item must be considered as incompatible with the test.
For test items detected as able to both stain the tissues (3.9.3) and interfere with MTT (3.9.2) may also require a third set of controls before calculation of the “true” viability %.
Non-Specific Colour % with killed tissues (NSCkilled %):
NSCkilled % = (mean ODCTK / mean ODNC)×100
ODCTK: test item treated killed tissues (not incubated with MTT)
ODNC: negative control OD (incubated with MTT)
In that case the true MTT metabolic conversion (TODTT) is undertaken as follows:
TODTT = [ODTT – (ODKT – ODKNC) – mean ODCTV +mean ODCTK]
Report Number: 19/181-039B Page 24 of 38
ODTT: test item treated viable tissues (incubated with MTT)
ODKT: test item treated killed tissues OD
ODKNC: negative control killed tissues OD
ODCTV: test item treated viable tissues (not incubated with MTT)
ODCTK: test item treated killed tissues (not incubated with MTT)
The % relative viability (RV %) for each test item replicate is calculated relative to the mean negative control:
RV1 % = [TODTT1 / mean ODNC] × 100
RV2% = [TODTT2 / mean ODNC] × 100
The mean value of the 2 individual relative viability % for test item is calculated:
Mean Relative Viability % = (RV1 % + RV2 %) / 2

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The cut-off value of 35% and classification method was validated in an international validation of this kit (Fentem, 1998).
- If both disks have a mean viability of ≥35% = Non Corrosive; If both disks have a 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: 20 mg
- 100 μL of physiological saline was added to ensure good contact with the epidermis

NEGATIVE CONTROL
- Amount(s) applied: 50 μL

POSITIVE CONTROL
- Amount(s) applied: 50 μL

Duration of treatment / exposure:

4 hours

Duration of post-treatment incubation (if applicable):

3 hours incubation with MTT solution

Number of replicates:

Two per treatment, negative control, positive control
Two additional with killed epidermis units for testing MTT interaction potential of the test substance
Two additional with killed epidermis units for negative control
Two additional with killed epidermis units for non-specific colour

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of two disks

Value:

111.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

After receipt, the two indicators of fitness for the delivered kit (reflecting the storage temperature history and the pH) were checked. Based on the observed colours, the epidermis units were in suitable condition for use in the assay.
The mean OD value of the two negative control tissues was 0.768, which satisfied the acceptability criterion of being 0.6-1.5.
The positive control treated tissues showed 0.4% cell viability demonstrating the proper performance of the assay.
The difference of cell viability between the two test item-treated tissue samples in the MTT assay was 2.3%.
The difference of cell viability between the two negative control treated tissue samples in the MTT assay was 15.1%.
The mean OD value of the blank samples (acidified isopropanol) was 0.047.
All these parameters met the acceptability criteria, therefore the study was considered to be valid.
High viability results (>100%) do regularly occur in cases where the test item causes metabolic stimulation in the exposed cells, so the study result is not considered to be invalid.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Following exposure of the test model to the substance, the mean cell viability was 111.5% compared to the negative control. This is clearly above the threshold of 35%, and the test substance is considered to be non-corrosive to skin.

Executive summary:

The skin corrosion potential of the substance was studied under GLP to OECD TG 431, in an in vitro study using a reconstructed human epidermis model, EpiSkin. After receipt, the two indicators of fitness for the delivered test model kit (reflecting the storage temperature history and the pH of the agar medium) were checked. Based on the observed colours, the epidermis units were in suitable condition for use in the assay. All cell units were maintained under proper conditions before their use. On the day prior to the test, the epidermis units were placed in the appropriate number of wells in an assay plate, which were filled with pre-warmed maintenance medium, and incubated overnight at 37 °C in an incubator with 5% carbon dioxide in a >95% humidified atmosphere. On the day of the experiments, two epidermis disks were treated with 20 mg of the powdered test substance for a period of 4 hours at room temperature. Exposure was terminated by rinsing the units with phosphate buffered saline solution. The cell viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution (at a concentration of 0.5 mg/mL). The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically at 570 nm. The two negative control epidermis units were treated with physiological saline (0.9% w/v NaCl solution in purified water). The two positive control epidermis units were treated with glacial acetic acid. Two additional disks were used to provide an estimate of colour distribution from the test substance. The possible interaction of the test substance with MTT was examined using two killed epidermis units treated with the substance and two additional killed epidermis units treated with physiological saline. To avoid a possible double correction for colour interference, a third set of two control epidermis units for non-specific colounr in killed tissues were used. For each treated tissue, the viability was expressed as a percentage relative to the negative control. The experiment met all the validity criteria, e.g. with regards to cell tissue suitability, negative and positive controls. Following exposure to the test substance over a period of four hours, the mean cell viability in the treated epidermis units was 111.5% compared to the negative control. This is above the threshold of 35%, and the test substance was therefore considered to be not corrosive to skin.