4,4'-bis(chloromethyl)-1,1'-biphenyl

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8 December 2016 to 9 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 July 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU Method B.40.Bis (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-bis(chloromethyl)-1,1'-biphenyl

EC Number:

216-784-4

EC Name:

4,4'-bis(chloromethyl)-1,1'-biphenyl

Cas Number:

1667-10-3

Molecular formula:

C14H12Cl2

IUPAC Name:

4,4'-bis(chloromethyl)-1,1'-biphenyl

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: White crystalline powder
- Storage conditions: Controlled room temperature (15-25°C, below 70% RH%), protected from humidity, under inert gas.

Specific details on test material used for the study:

The test material was applied as supplied, no formulation was required (although the test material was ground to a fine powder).
No correction for purity of the test material was applied.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The EPISKIN™(SM) model has been validated for corrosivity testing in an international trial 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) three-dimensional human epidermis model
- Source: SkinEthic
- Tissue lot number(s): 16-EKIN-049
- Expiry date: 12 December 2016
- 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.

TEST FOR DIRECT MTT REDUCTION
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.
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.

ASSESSMENT OF 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. As the test material had an intrinsic colour, further evaluation was necessary to detect colouring potential. Non Specific Colour % (NSCliving %) was determined in order to evaluate the ability of test material to stain the epidermis by using additional control tissues.
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.

MAIN TEST
PRE-INCUBATION
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
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 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 (22.9 - 24.2°C) covered with the plate lids.

NUMBER OF REPLICATE TISSUES:
- Test material: 2
- Negative controls: 2
- Positive controls: 2
Furthermore, as the test item was coloured, two additional test material-treated tissues were used for the non specific OD evaluation.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 22.9 - 24.2°C

REMOVAL OF TEST MATERIAL AND CONTROLS
After the incubation time (4 hours), all test material treated tissues or 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 TEST
MTT solution (2 mL of 0.3 mg/mL MTT working solution) was added to each well below the skin units (except for the two living colour control units). The lid was replaced and the plate incubated at 37°C in an incubator with 5% CO2 for 3 hours (± 15 min), protected from light.

FORMAZAN EXTRACTION
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.

CELL VIABILITY MEASUREMENTS
Following the formazan extraction, 2×200 μL samples from each tube were placed into the wells of a labelled 96-well plate. The OD (optical density or absorbance) of the samples was measured using a plate reader at 570 nm. The mean of 6 wells of acidified isopropanol solution (200 μL/well) was used as blank.
The proper status of the instrument was verified by measuring a Verification plate at the required wavelength on each day before use.

DATA EVALUATION
- The relative mean viabilities were calculated in the following way:
Relative mean viability (%) = (mean OD570 of the test material / mean OD570 of negative control) x 100

INTERPRETATION OF RESULTS
If both disks have mean viability of ≥ 35% = Non Corrosive
If both disks have mean viability of < 35% = Corrosive (at the corresponding incubation period)

Otherwise:
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.

VALIDITY OF THE TEST
The mean OD value of the two negative control tissues should be ≥ 0.6 and ≤ 1.5 and negative control OD values should not be below historically established boundaries.
The acceptable mean viability % range for positive controls is ≤ 20%.
The difference of viability between the two tissue replicates should not exceed 30%.
The mean OD value of the blank samples (acidified isopropanol) should be <0.1.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 20 mg

NEGATIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration (if solution): 0.9% (w/v) NaCl solution

POSITIVE CONTROL
- Amount(s) applied: 50 µL

Duration of post-treatment incubation (if applicable):

4 hours

Number of replicates:

2

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

test material

Value:

92

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

positive control

Value:

0.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

DIRECT MTT REDUCTION
The MTT solution containing the test material did not turn blue/purple. This was taken to indicate the test material did not reduce MTT.

ADDITIONAL CONTROLS
As the test material was coloured, two additional test material-treated tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of these tissues was determined as 0.002, Non Specific Colour % was calculated as 0.3%. This is below the threshold of 5%, therefore correction due to colouring potential was not necessary.

VIABILITY RESULTS
The mean OD value for the test material treated skin samples showed a 92.0% relative viability.
The mean OD value for the positive control treated skin samples showed a 0.8% relative viability.

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 condition.
- The mean OD value of the two negative control tissues was in the recommended range (0.836).
- The two positive control treated tissues showed 0.8% viability demonstrating the proper performance of the assay.
- The difference of viability between the two test material-treated tissue samples in the MTT assay was 13.9%.
- The difference of viability between the two negative control tissue samples in the MTT assay was 15.0%.
- 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.

Any other information on results incl. tables

Table 1: Optical Density (OD) and the calculated relative viability % of the samples

Substance	Optical density (OD)			Viability (%)
	Measured		Blank corrected
Negative control	1	0.819	0.773	92.5
	2	0.944	0.898	107.5
	mean	-	0.836	100.0
Positive control	1	0.052	0.006	0.8
	2	0.053	0.007	0.8
	mean	-	0.007	0.8
Test material	1	0.868	0.822	98.4
	2	0.761	0.715	85.5
	mean	-	0.768	92.0

 

Applicant's summary and conclusion

Interpretation of results:

other: Not corrosive according to EU criteria

Conclusions:

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

Executive summary:

The skin corrosion potential of the test material was investigated in accordance with the standardised guidelines OECD 431 and EU Method B.40.bis, under GLP conditions.

During the study, disks of EPISKIN™(SM) were treated with 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 from the test material. For each treated tissue viability was expressed as a % relative to the negative control.

Following exposure with the test material, the mean cell viability was 92.0% 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, the results indicate that the test item is non corrosive to the skin.