Reaction mass of dieuropium tricarbonate and digadolinium tricarbonate and disamarium tricarbonate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 September 2017 to 15 September 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU Method B.40. bis.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of dieuropium tricarbonate and digadolinium tricarbonate and disamarium tricarbonate

EC Number:

945-733-5

Molecular formula:

See remarks

IUPAC Name:

Reaction mass of dieuropium tricarbonate and digadolinium tricarbonate and disamarium tricarbonate

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: Natural white powder
- Storage conditions: Room temperature (15-25 °C), protected from light and humidity

In vitro test system

Test system:

human skin model

Remarks:

EPISKIN™(SM) model

Source species:

other: EPISKIN™(SM) model

Cell type:

other: EPISKIN™(SM) three-dimensional human skin model comprising a reconstructed epidermis with a functional stratum corneum.

Cell source:

other: Not specified

Source strain:

not specified

Details on animal used as source of test system:

SOURCE
EPISKIN™(SM) (Manufacturer: SkinEthic, France, Batch No.: 17-EKIN-037, Expiry Date: 18 September 2017) is a three-dimensional human epidermis model. 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 comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al., 1994). Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

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)

Remarks:

No formulation was required.

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™(SM) model
- Tissue batch number(s): Batch No.: 17-EKIN-037
- Expiry date: 18 September 2017
- Date of initiation of testing: 14 September 2017

- Pre-incubation (Day [-1]): The Maintenance Medium was pre-warmed to 37°C. The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight at 37°C in an incubator with 5% CO2 in a > 95% humidified atmosphere.

- Application (Day 0): The Assay Medium was pre-warmed to 37°C. The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, whereby each epidermis was in contact with the medium in the corresponding well underneath. Two epidermis units were used for each test or control materials.
- 20 mg of test material was applied evenly to the epidermal surface of each of two test material treated skin units and each additional control skin units and then 100 μL physiological saline was added to the test material to ensure good contact with the epidermis.
- 50 μL of physiological saline was added to each of the two negative control skin units.
- 50 μL of glacial acetic acid was added to each of the two positive control skin units.
The plates with the treated epidermis units were incubated for 4 hours (± 10 min) at room temperature (23.6 - 25.2°C) covered with the plate lids.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 23.6 - 25.2°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: After the incubation time (4 hours), all test material treated tissues and also the positive control tissues were removed and rinsed thoroughly with PBS solution to remove all the remaining test or positive control material from the epidermal surface. Likewise, negative control tissues were processed accordingly.
The rest of the PBS was removed from the epidermal surface using a pipette (without touching the epidermis).

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1] was diluted in phosphate buffered saline (PBS) at a final concentration of 3 mg/mL (MTT stock solution). The obtained stock solution (prepared on 13 September 2017) was stored in refrigerator (2-8°C) protected from light. It was diluted with pre-warmed (37°C) Assay Medium to a final concentration of 0.3 mg/mL (MTT working solution) immediately before use.
MTT solution (2 mL of 0.3 mg/mL MTT working solution) was added to each well below the skin units (except of the two living colour control units). The lid was replaced.
- Incubation time: The plate was incubated at 37°C in an incubator with 5% CO2 in a > 95% humidified atmosphere for 3 hours (± 15 minutes), protected from light.
- Spectrophotometer: The OD (optical density or absorbance) of the samples was measured using a plate reader
- Wavelength: 570 nm

- Formazan extraction (Day 0): At the end of incubation with MTT a formazan extraction was undertaken. A disk of epidermis was cut from each skin unit (this procedure involved the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube containing 500 μL acidified isopropanol (one tube corresponded to one well of the assay plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material and the acidified isopropanol, and then incubated overnight at room temperature protected from light with gentle agitation (~ 150 rpm) for formazan extraction.
A blank sample containing 2 mL of acidified isopropanol was processed in parallel.
Isopropanol was acidified with HCl acid to achieve a final concentration of 0.04N HCl (1.8 mL of 12N HCl acid was diluted in 500 mL isopropanol, or similar ratio was applied). The solution was prepared on the day of use.

- Cell viability measurements: Following the formazan extraction, 2 × 200 μL sample from each tube were placed into the wells of a 96-well plate (labelled appropriately).
The mean of 6 wells of acidified isopropanol solution (200 μL/well) was used as blank.

- Indicator for potential false viability
Chemical action by the test material on MTT may mimic that of cellular metabolism leading to a false estimate of viability. This may occur when the test material is not completely removed from the tissue by rinsing or when it penetrates the epidermis. If the test material directly acts on MTT (MTT-reducer), is naturally coloured, or becomes coloured during tissue treatment, additional controls should be used to detect and correct for test item interference with the viability measurement. Methods of how to correct direct MTT reduction and interferences by colouring agents are detailed in the following paragraphs.

- Check-method for possible direct MTT reduction with test material:
20 mg of test material was added to 2 mL MTT working solution and mixed. The mixture was incubated at 37°C in an incubator with 5% CO2, in a > 95% humidified atmosphere for 3 hours and then any colour change was observed:
- Test material which do not react with MTT: Yellow
- Test material reacting with MTT: Blue or purple
After three hours of incubation, yellow colour of the mixture was detected; therefore additional controls were not used in the experiment.

- Check-method to detect the colouring potential of test material:
Prior to treatment, the test material was evaluated for its intrinsic colour or ability to become coloured in contact with water and/or isopropanol* (simulating a tissue humid environment). As the test material had an intrinsic colour, thus further evaluation to detect colouring potential was necessary. Non Specific Colour % (NSCliving %) was determined in order to evaluate the ability of test material to stain the epidermis by using additional control tissues.
* Note: Water is the environment during exposure, isopropanol is the extracting solution.

Therefore, in addition to the normal procedure, two additional test material-treated living tissues were used for the non-specific OD evaluation. These tissues followed the same test material application and all steps as for the other tissues, except for the MTT step: MTT incubation was replaced by incubation with fresh Assay Medium to mimic the amount of colour from the test material that may be present in the test disks. OD readings were conducted following the same conditions as for the other tissues.


NUMBER OF REPLICATE TISSUES:
- In this assay, two replicates per test material were used. Two negative controls and two positive controls were also run in this assay. Furthermore, as the test material was coloured, two additional test material-treated living tissues were used for the non-specific OD evaluation.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues
- N. of replicates: Duplicate

CALCULATION OF VAIBILITY PERCENTAGES
The data calculation using two replicates is shown below. Results are calculated in a similar way when more replicates are used.

Blank: The mean of the 6 blank 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 material: Individual test material 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 material values was calculated
- The % viability for each test material 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 material was calculated:
Mean TT % = (TT1 % + TT2 %) / 2
- The variability for 2 disks was calculated as:
(Disk1-Disk2)/((Disk1+Disk2)/2) x 100 %

Data calculation for test items having MTT-interacting potential
Test materials 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 material treated killed tissues OD
ODNC: negative control OD
If NSMTT% is ≤ 50 %, then true MTT metabolic conversion (TODTT) has to be undertaken as follows:
TODTT = [ODTT – (ODKT – ODKNC)]
ODTT: test material treated viable tissues
– The % relative viability (RV%) for each test material 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 material 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.

- Data calculation for test material having colouring potential:
For test materials detected as able to stain the tissues the non-specific OD was 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 % (NSCliving %):
NSCliving % = (mean ODCTV / mean ODNC)×100
ODCTV: test material treated viable tissue (not incubated with MTT)
ODNC: negative control OD (incubated with MTT)
If NSC living % is ≤ 5 % then the normal calculation mode was used.
If NSC living % is > 5 % and ≤ 5 0 %, then additional correction (TODTT) has to be undertaken as follows:

TODTT = [ODTV - ODCTV]
ODTT: test material treated viable tissue (incubated with MTT)
ODCTV: test material treated viable tissue (not incubated with MTT)
– The % relative viability (RV% %) for each test material 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 material 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 material must be considered as incompatible with the test.

- Data calculation for test materials having both MTT-interacting and colouring potential:
For test materials detected as able to both stain the tissues and interfere with MTT 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 materisl 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]
ODTT: test material treated viable tissues (incubated with MTT)
ODKT: test material treated killed tissues OD
ODKNC: negative control killed tissues OD
ODCTV: test material treated viable tissues (not incubated with MTT)
ODCTK: test material treated killed tissues (not incubated with MTT)
The % relative viability (% RV) for each test material 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 material is calculated:
Mean Relative Viability % = (RV1 % + RV2 %) / 2

PREDICTION MODEL / DECISION CRITERIA
The prediction model below corresponds to the methods agreed by EU regulatory agencies in line with OECD No. 431 (OECD, 2016).
The cut-off value of 35% and classification method was validated in an international validation study of this kit (Fentem, 1998).
For 2 disks:
If both disks have mean viability of ≥ 35% = Non Corrosive
If both disks have mean viability of < 35 = Corrosive (at the corresponding incubation period)

For more than 2 disks:
If the mean value is ≥ 35% and the variability is less than 50% = Non Corrosive
If the mean value is < 35% and the variability is less than 50% = Corrosive

Otherwise:
If the classification is not made with these criteria, retest with 2 more disks. Take the mean of the 4 disks to classify as above or below 35%. Outlier values may be excluded where there are scientific reasons, such as where application or rinsing is difficult and that the Study Director considers that a result is not representative.

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 test material

NEGATIVE CONTROL
- Amount applied: 50 µL physiological saline

POSITIVE CONTROL
- Amount(s) applied: 50 µL glacial acetic acid

Duration of treatment / exposure:

The plates with the treated epidermis units were incubated for 4 hours (± 10 min) at room temperature (23.6 - 25.2°C) covered with the plate lids.

Number of replicates:

The test material was applied to two skin units and each additional skin unit.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of two runs

Value:

104.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

ADDITIONAL CONTROLS
As the test material was coloured, two additional test material-treated living tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of these tissues was determined as 0.007, Non Specific Colour% (NSCliving%) was calculated as 0.9%. 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 material 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.

Any other information on results incl. tables

Validity of the Test

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.

The mean OD value of the two negative control tissues was in the recommended range (0.788).

The two positive control treated tissues showed 0.6% viability demonstrating the proper performance of the assay.

The OD data of one replicate tissue at the positive control in this study was lower (0.001) than the minimum OD of the historical control range (0.005). This fact has no impact on the results or integrity of the study since the positive control material showed severe effect.

The difference of viability between the two test material-treated tissue samples in the MTT assay was 6.1%.

The difference of viability between the two negative control tissue samples in the MTT assay was 1.5%.

The mean OD value of the blank samples (acidified isopropanol) was 0.046. All these parameters were within acceptable limits and therefore the study was considered to be valid.

Applicant's summary and conclusion

Interpretation of results:

other: Not classified according to EU criteria.

Conclusions:

Under the conditions of the test the test material was not corrosive to the skin.

Executive summary:

The corrosivity of the test material was evaluated according to the standardised giudlines OECD 431.

During the study, disks of EPISKIN™ (SM) (two units) were treated with the test material 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 (NSCliving%) from the test material. 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 material is considered to be corrosive to skin.

Following exposure with the test material, the mean cell viability was 104.2% compared to the negative control. This is above the threshold of 35%, therefore the test material 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™ (SM) model test with the test material, the results indicate that the test material is non-corrosive to the skin.