Registration Dossier

Administrative data

Description of key information

Under the experimental conditions reported, 4,4’-(1,3-Phenylenediisopropylidene) diphenylcyanate is non-irritant to skin or eyes according to UN GHS and EU CLP regulation.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation / corrosion, other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
THis study was conducted between 01 June 2017 and 06 July 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was assigned Reliability 1 as it was conducted to OECD TG 431 and in compliance with GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
29 July 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Version / remarks:
EC No. 440/2008 30 May 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Identification: 4,4’-(1,3-Phenylenediisopropylidene) diphenylcyanate
CAS No.: 127667-44-1
Batch: FAR366085A
Purity: Not indicated by the Sponsor
Appearance: Highly viscous yellowish liquid
Expiry Date: 29 September 2019
Storage Conditions: In the refrigerator
Stability in Solvent: Not indicated by the Sponsor
Purpose of Use: Industrial chemical
Test system:
human skin model
Source species:
human
Cell type:
other: The epidermis model (e.g. EpiDermTM) is derived from human keratinocytes and consists of normal, human-derived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis.
Cell source:
other: Not specified as study used an EpiDerm™ Reconstructed Human Epidermis Model Kit
Source strain:
not specified
Details on animal used as source of test system:
Not applicable
Justification for test system used:
Recognised in vitro test for corrosivity
Vehicle:
unchanged (no vehicle)
Details on test system:
Test System

Epi-200 Kit Components Needed for the Assay
EpiDerm™ Kit Lot No.: 25828
1 Sealed 24-well plate Contains 24 inserts with EpiDerm™ tissues on agarose
2 24-well plates For MTT viability assay
4 6-well plates For storing inserts, or for topically applying test agents
1 bottle Serum-Free Assay Medium DMEM-based medium
1 bottle DPBS Rinse Solution For rinsing the inserts in MTT assay

MTT-100 Assay Kit Components
1 vial, 2 mL MTT concentrate
1 vial, 8 mL MTT diluent (supplemented DMEM) For diluting MTT concentrate prior to use in the MTT assay
1 bottle, 60 mL Extractant Solution (Isopropanol) For extraction of formazan crystals

3.3.3 MTT-Solution
The MTT-solution was prepared freshly on day of use (resulting: 1 mg/mL).
For use in the pre-test (step 3): MTT from Sigma, Germany, DMEM from Gibco, Germany
For use in the main experiment: MTT concentrate from MatTek, MTT diluent from MatTek.

3.3.4 Cell Culture
Epi-200 kits and MTT-100 assays were purchased from MatTek Corporation (Bratislava, Slovakia). The EpiDerm™ tissue consisted of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consisted of organised basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm™ tissues (surface 0.63 cm²) are cultured on specially prepared cell culture inserts (MILLICELL, 10 mm ).
EpiDerm™ tissues were shipped on cool packs and on medium-supplemented agarose gels in a 24-well plate and reached Envigo CRS GmbH on 04 July 2017. On day of receipt the pre-incubation phase of the EpiDerm™ tissues started.
3.4 Test for Direct MTT Reduction and Colour Interference
A test item may interfere with the MTT endpoint if: a) it is coloured and/or b) able to directly reduce MTT. The MTT assay is affected only if the test item is present in the tissues when the MTT viability test is performed.
Some non-coloured test items may change into coloured test items in wet or aqueous conditions and thus stain tissues during the 60 min exposure. Therefore, before exposure, a functional check for this possibility should be performed (step 1).
Step 1
50 µL of the test item were added to 0.3 ml of deionised water (transparent glass test-tube). The mixture was incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5 % CO2) for 60 min. At the end of the exposure time, the mixture was shaken and the presence and intensity of the staining (if any) was evaluated. If the solution changed colour significantly, the test item is presumed to have the potential to stain the tissue. An additional test on viable tissues (without MTT addition) should be performed (step 2).
Since the test item did not dye water when mixed with it, step 2 did not have to be performed.
Step 3
All test items (including those already evaluated in step 1 and step 2) should be further evaluated for their potential to interfere with MTT. To test if an item directly reduces MTT, 50 µL of the test item were added to 1 ml of a MTT/DMEM solution (1 mg/mL) and were incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5 CO2) for 60 minutes. Untreated MTT/DMEM solution (1 mg/mL) medium was used as control. If the MTT/DMEM solution (1 mg/mL) turns blue/purple, the test item reduces MTT and an additional test on freeze-killed tissues (step 4) must be performed.
Since the test item did not prove to be a MTT reducer, step 4 did not have to be performed.

EXPERIMENTAL PERFORMANCE
Pre-warming of EpiDerm™ Tissues
19 to 20 hours before dosing, EpiDerm™ tissues were removed from the refrigerator / unpacked, and the inserts were transferred into 6-well plates containing the pre-warmed assay medium under sterile conditions using sterile forceps. A 24-well plate was prepared as holding plate containing 300 µL assay medium. The holding plate was pre warmed in an incubator (37 ± 1.5 °C, 5 ± 0.5 % CO2) until use.

Treatment
Duplicate EpiDermTM tissues were treated with the test item, positive control or negative control for the following exposure times:
• Test Item: 3 ± 0.5 minutes, 60 ± 5 minutes
• Negative Control: 3 ± 0.5 minutes, 60 ± 5 minutes
• Positive Control: 3 ± 0.5 minutes, 60 ± 5 minutes
After the pre-incubation of the EpiDermTM tissues was completed the DMEM-based medium in each well was replaced with 0.9 mL fresh assay medium. The 6-well plates for the 3 ± 0.5 minutes exposure periods stayed at room temperature in the sterile bench, the 6-well plates for the 60 ± 5 minutes exposure period were placed into an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2).
At the end of the exposure period the tissues were removed from the 6-well plate and gently rinsed with DPBS to remove any residual test material (20 times). Excess DPBS was removed by gently shaking the tissue insert and blotting the lower surface with blotting paper. The tissues were placed in the prepared holding plate until all tissues were rinsed.

MTT Assay
Two 24-well plates were prepared prior to the end of the tissue pre-warming period. MTT solution (300 µL) was added to each well and the plates were kept in an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2) until required.
Following rinsing, the tissues were transferred from the holding plates to the MTT-plates. After a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2) the tissues were rinsed three times with DPBS and carefully dried with blotting paper. The inserts were transferred into new 24-well plates. The tissues were each immersed in 2 mL of extractant solution (isopropanol) pipetted in each well ensuring that the tissues were completely covered. The 24-well plates were sealed to minimise isopropanol evaporation. The formazan salt was extracted for 16.5 hours without shaking in the refrigerator.
After the extraction period the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken, and the insert was discarded. 24-well plates were then placed on a shaker for 15 minutes until the solution was homogeneous in colour.
3 x 200 µL aliquots of the blue formazan solution were transferred from each tissue into a 96-well flat bottom microtiter plate. The OD was determined in a microplate reader (Versamax®, Molecular Devices, SoftMax Pro Enterprise (version 4.7.1)) at 570 nm (OD570) without reference filter. The mean values were calculated for each set of 3 wells per tissue.

Data Evaluation
The mean OD of the duplicate negative control tissues was calculated after blank correction. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula:

Relative viability (%) = (Mean OD test item or positive control / Mean OD562 negative control) x 100

Interpretation of Results
For the test item and the positive control the mean relative viability  rel. standard deviation of the two individual tissues for both exposure periods was calculated and used for classification according to the following prediction model:
Viability measured after exposure time points Prediction to be considered
< 50% after 3 minutes exposure Corrosive
≥ 50% after 3 minutes exposure AND
< 15% after 60 minutes exposure Corrosive
≥ 50% after 3 minutes exposure AND
≥ 15% after 60 minutes exposure Non-corrosive
Test Item Identified as Corrosive
< 25% after 3 minutes exposure Optional Sub-category 1A*
≥ 25% after 3 minutes exposure A combination of optional Sub-categories 1B and 1C

* According to the data generated in view of assessing the usefulness of the RhE test methods for supporting sub-categorisation, it was shown that around 29%, 31% and 33% of the Sub-category 1A results of the EpiDERMTM test method may actually constitute Sub-category 1B or Sub-category 1C substances/mixtures (i.e. over-classifications).

Acceptability of the Assay
An assay met the acceptance criteria if
• the mean OD of the tissue replicates treated with the negative control is ≥ 0.8 and ≤ 2.8 for every exposure time
• the mean viability of the tissue replicates treated with the positive control for 1 hour, is <15% compared to the negative control
• the Coefficient of Variation (CV) in the range 20 – 100% viability between tissue replicates is ≤ 30%
The quality certificate of the supplier of the test kit demonstrating its robustness (treatment with 1% Triton X-100: 4.77 hours ≤ ET50 ≤ 8.72 hours) is annexed to the report.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Prior to use an aliquot of the test item was pre-warmed in a water bath (37 °C) to improve the test item’s liquidity. 50 µL (79.4 µL/cm2 according to guideline) of the test item were dispensed directly onto duplicate EpiDermTM tissue surface or used in the colour and MTT interference pre-tests

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL Sterile distilled water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL Potassium Hydroxide
- Concentration (if solution): 8.0N
Duration of treatment / exposure:
Duplicate EpiDermTM tissues were treated with the test item, positive control or negative control for the following exposure times:
• Test Item: 3 ± 0.5 minutes, 60 ± 5 minutes
• Negative Control: 3 ± 0.5 minutes, 60 ± 5 minutes
• Positive Control: 3 ± 0.5 minutes, 60 ± 5 minutes
Number of replicates:
2
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 Minute Exposure
Value:
94.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
25.9
Remarks on result:
other: Not corrosive to skin
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 Minute Exposure
Value:
ca. 93.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100
Positive controls validity:
valid
Remarks:
5.5
Remarks on result:
other: Not corrosive to skin
Other effects / acceptance of results:
The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not lead to a change in colour.
Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

The test item is considered to be non-corrosive to skin:
• since the viability after 3 minutes exposure is greater than 50% and
• the viability after 1 hour exposure is greater than 15%.

The acceptance criteria are met:
• the mean OD of the tissue replicates treated with the negative control is ≥ 0.8 and ≤ 2.8 for every exposure time (range: 1.581 to 1.746)
• the mean viability of the tissue replicates treated with the positive control for 1 hour, is <15% compared to the negative control (5.5%)
• the Coefficient of Variation (CV) in the range 20 – 100% viability between tissue replicates is ≤ 30% (range: 0.6% to 2.8%)

Mean OD562 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Dose Group

Ex-posure Inter-val

Absor-bance
Well 1
(Tissue 1/2)

Absor-bance
Well 2 (Tissue 1/2)

Absor-bance
Well 3 (Tissue 1/2)

Mean Absor-bance (Tissue 1/2)

Mean Ab-sorbance (OD) of 3 Wells minus Blank

Mean Ab-sorbance (OD) of 2 Tissues

Rel. Absor-bance [%]

CV
[%]

Mean Rel. Absorbance [%]

Blank

 

0.037

0.037

0.037

0.037

0.000

 

Negative Control

3
minutes

1.746

1.675

1.716

1.712

1.675

1.658

101.0

1.5

100.0

1.683

1.673

1.677

1.678

1.641

99.0

Positive Control

0.477

0.465

0.453

0.465

0.428

0.430

25.8

0.6

25.9

0.474

0.464

0.467

0.468

0.431

26.0

Test Item

1.598

1.592

1.599

1.597

1.560

1.566

94.1

0.6

94.4

1.619

1.596

1.613

1.609

1.572

94.8

Blank

 

0.036

0.036

0.036

0.036

0.000

 

Negative Control

1
hour

1.672

1.581

1.633

1.629

1.593

1.586

100.4

0.6

100.0

1.603

1.607

1.638

1.616

1.580

99.6

Positive Control

0.109

0.111

0.111

0.111

0.075

0.087

4.7

20.6

5.5

0.147

0.136

0.125

0.136

0.100

6.3

Test Item

1.563

1.559

1.522

1.548

1.512

1.483

95.3

2.8

93.5

1.496

1.488

1.485

1.490

1.454

91.6

OD =  Optical density

Interpretation of results:
other: Not Corrosive to skin
Remarks:
according to EU CLP and UN GHS criteria
Conclusions:
In conclusion, it can be stated that in this study and under the reported experimental conditions, 4,4’-(1,3-Phenylenediisopropylidene) diphenylcyanate is non corrosive to skin according to EU CLP and UN GHS.
Executive summary:

Thisin vitrostudy was performed to assess the corrosive potential of 4,4’-(1,3-Phenylenediisopropylidene) diphenylcyanate by means of the Human Skin Model Test with EpiDerm™ tissues models.

The test item did not reduce MTT (pre-test for direct MTT reduction), and it did not dye deionised water or changed its colour when mixed with it (pre-test for colour interference). Consequently, additional tests with freeze-killed or viable tissues to determine correction factors for calculating the true viability in the main experiment were not necessary.

Independent duplicate tissues of EpiDerm™ were exposed to either the test item, the negative control (deionised water) or the positive control (8.0 N KOH) for 3 minutes and 1 hour, respectively.

After exposure to the negative control the absorbance values met the required acceptability criterion of a mean OD570≥ 0.8 and ≤ 2.8 for both treatment intervals thereby confirming the acceptable quality of the tissues.

Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period and for the 1 hour exposure period. The 1 hour exposure caused a decrease of the cell viability < 15% of the negative control. The CV in the range 20 – 100% viability between the tissue replicates is ≤ 30%, thus the validity of the test system and the specific batch of the tissue models is confirmed.

After exposure of the tissues to the test item the relative absorbance value decreased to 94.4% after 3 minutes exposure. After 1 hour exposure the relative absorbance value was reduced to 93.5%. Both values did not exceed the threshold for corrosivity which is defined to be 50% after the 3 minutes exposure and 15% after the 1 hour exposure. Therefore, the test item is not considered to be corrosive.

In conclusion, it can be stated that in this study and under the reported experimental conditions, the test item 4,4’-(1,3-Phenylenediisopropylidene) diphenylcyanate isnon corrosiveto skin according to EU CLP and UN GHS.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
This study was conducted between 01 June 2017 and 09 June 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is considered to be reliability 1 as it has been conducted according to OECD Test Guideline 439 using the EPISKIN™ Reconstructed Human Epidermis Model and in compliance with GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
23 July 2009
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
The test item was supplied by or on behalf of the Sponsor including the following information.
Identification: 4,4’-(1,3-Phenylenediisopropylidene) diphenylcyanate
CAS No.: 127667-44-1
Batch: FAR366085A
Purity: Not indicated by the Sponsor
Appearance: Highly viscous yellowish liquid
Expiry Date: 29 September 2019
Storage Conditions: In the refrigerator at 2 to 8° C
Stability in Solvent: Not indicated by the Sponsor
Purpose of Use: Industrial chemical
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
Dermal irritation is defined as "the production of reversible inflammatory changes in the skin". The potential for chemical induced skin irritation is an important consideration in establishing procedures for the safe handling, packing and transport of chemicals. It is usually determined in vivo using the Draize rabbit skin irritation test as described in OECD guideline 404. Because systemic reactions play a minor role in modulating local skin toxicity potential of chemicals, skin irritation potential may be predicted by in vitro systems, provided they are sufficiently complex to mimic human skin barrier and cell reactivity. In an international validation study performed by ECVAM, the in vitro skin irritation test using the human skin models EpiSkin™ and EpiDerm™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential .
The test consists of a topical exposure of the neat test item to a human reconstructed epidermis model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazoliumbromide], in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison with untreated negative controls is used to predict skin irritation potential (see OECD TG 439) and is used for the purpose of classification as irritating or non-irritating according to chemicals law (EU CLP, UN GHS). Depending on the regulatory framework and applicability of the test guideline, chemicals that produce cell viabilities above the defined threshold level, are considered non-irritants. The test chemical is considered to be irritant to skin in accordance with UN GHS and EU CLP Category 2 if the tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50%.
Vehicle:
unchanged (no vehicle)
Details on test system:
EpiSkin™ Kit Components Needed for the Assay
EpiSkin™ Kit Lot No.: 17-EKIN-023
1 Sealed 12-well plate Contains 12 inserts with EpiSkin™ tissues on agarose
1 12-well plate For MTT viability assay
1 bottle Assay Medium Basic medium for use in MTT assays
1 bottle EpiSkin™ Maintenance Medium Basic medium for incubations

MTT-Solution
The MTT-solution was prepared freshly on day of use with assay medium
(concentration: 0.3 mg/mL). 

Cell Culture
EpiSkin™ kits are purchased from SkinEthic Laboratories (69007 Lyon, France). The EpiSkin™ tissue consists of NHEK, which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiSkin™ tissues (surface 0.38 cm²) are cultured on specially prepared cell culture inserts.
EpiSkin™ tissues were shipped at ambient temperature on medium-supplemented agarose gels in a 12-well plate and reached Envigo CRS GmbH on 07 June 2017. On the day of experiment the pre-incubation phase of the EpiSkin™ tissues started.

Test for Direct MTT Reduction and Colour Interference
Prior to the start of the test, the colour interference potential of the test item had to be evaluated. For this purpose 10 µL of the test item was mixed with 90 µL of deionised water in a pre-experiment. The mixture was gently shaken for 15 minutes at room temperature.
The colour of the mixture did not change during the incubation period compared with the colour of the pure test item. Therefore, the measurement of the OD of the test item in water at 570 nm was not required and consequently not performed.
For correct interpretation of results it is necessary to assess the ability of the test item to directly reduce MTT. To test for this ability 10 µL of the test item was added to 2 mL of MTT solution (0.3 mg/mL) and the mixture was incubated in the dark at
37 ± 1.5 °C (5 ± 0.5% CO2) for 3 hours. MTT solution was used as the control. If the MTT solution colour turns blue/purple, the test item is presumed to have reduced the MTT.
Since the colour did not turn blue/purple, the test item was not considered to be a MTT reducer.

EXPERIMENTAL PERFORMANCE
Pre-warming of EpiSkin™ Tissues
Under sterile conditions using sterile forceps, the inserts were transferred into 12-well plates containing the pre-warmed (37 ± 1.5 °C) maintenance medium. The EpiSkin™ tissues were incubated for approximately 3 hours.

Treatment
The negative control, positive control and the test item were added into the insert atop the concerning EpiSkin™ triplicate tissues for 15 minutes.
After the end of the treatment interval the inserts were immediately removed from the 12-well plate. The tissues were gently rinsed with PBS to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper. The inserts were placed in the plates with 2 mL maintenance medium. The tissues were incubated for approximately 42 hours at 37 ± 1.5 °C, 5 ± 0.5% CO2.

IL-1 α Immunoassay
Samples of all treatment groups were taken from the wells. The plates were shaken for approximately 15 minutes to homogenise the released mediators in the medium before sampling. At least 1.6 mL medium from each well was taken and was stored in the freezer at –20 °C. Since the results derived from the MTT assay were not unclear or borderline, the IL-1 α concentration in the medium was not determined and the taken samples were discarded after report finalization.

MTT Assay
A 12-well plate was filled with 2 mL assay medium containing 0.3 mg/mL MTT per well.
After the 42 ± 1 hour incubation period was completed for all tissues the cell culture inserts were transferred from the holding plates to the MTT-plates. After a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2) MTT solution was rinsed three times with PBS and the tissues were plotted. Tissue samples were cut out of the inserts with a biopsy punch and transferred into plastic vials. The tissue samples were immersed into extractant solution by gently pipetting 0.5 mL extractant solution (isopropanol containing 0.04 N HCl) into each vial. The tissue samples were completely covered by isopropanol. The formazan salt was extracted for about 2.5 hours at room temperature with gentle agitation.
Per tissue sample 2 x 200 µL aliquots of the formazan extract were transferred into a 96-well flat bottom microtiter plate. OD was read in a microplate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, version 4.7.1) with 570 nm filter. Mean values were calculated from the 2 wells per tissue sample.

Data Recording
The data generated were recorded in the laboratory protocol. The results were presented in tabular form, including experimental groups with the test item, negative and positive controls.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
10 µL (26.3 µL/cm2) of the undiluted test item were applied to each of the triplicate tissues. Due to the highly viscous property of the test item, each an aliquod for the pre-tests and for the main experiment was warmed in a water bath at 37 °C to change the physical state to liquid enabling the technicians to use pipettes for application

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL,

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): as a 5% solution in deionised water
Duration of treatment / exposure:
15 Minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Details on study design:
Study Design
Pre-Test Procedure
Assessment of Direct Test Item Reduction of MTT
MTT Salt Metabolism, Cell Viability Assay
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 formazan salt by mitochondrial succinate dehydrogenase in viable cells.
One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified by using killed tissues to act as controls.

Test for Direct MTT Reduction
As specified, a test item may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, the test item is checked for the ability to directly reduce MTT according to the following procedure:
10 µL of the test item was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37”C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control.
If the MTT solution containing the test item turns blue or purple, the test item is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and water killed tissues for quantitative correction of the results.

Assessment of Color Interference with the MTT endpoint
A test item may interfere with the MTT endpoint if it is colored. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed.
10 µL of test item was added to 90 µL of sterile water. After mixing for 15 minutes on a plate shaker a visual assessment of the color was made.

Pre-incubation (Day 0: Tissue Arrival)
Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert:
Tissues Satisfactory : Yes
Temperature Indicator Color Satisfactory : Yes
Agar Medium Color Satisfactory : Yes

2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre labeled 12 well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37 °C, 5% CO2 in air overnight.

Main Test
Application of Test Item and Rinsing (Day 1)
2 mL of maintenance medium, warmed to approximately 37”C, was pipetted into the second column of 3 wells of the 12 well plate.
Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. 10 µL (26.3 µL/cm2) of the test item was applied to the epidermis surface. Triplicate tissues treated with 10 µL of DPBS served as the negative controls and triplicate tissues treated with 10 µL of SDS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the center). After a 7 Minute contact time the SDS solution was re spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period (re-spreading is not required for the negative control or test item). The plates were kept in the biological safety cabinet at room temperature for 15 minutes.
At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37”C, 5% CO2 in air for 42 hours.

MTT Loading/Formazan Extraction (Day 3)
Following the 42 Hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre labeled micro tubes and stored in a freezer at 14 to 30 ºC for possible inflammatory mediator determination.
2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5% CO2 in air. At the end of the 3 Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labeled 1.5 mL micro tubes containing 500 µL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.

Absorbance/Optical Density Measurements (Day 6)
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous colored solution.
For each tissue, duplicate 200 µL samples were transferred to the appropriate wells of a pre labeled 96 well plate. 200 µL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 562 nm (without a reference filter) using the Anthos 2001 microplate reader.

Data Evaluation
Quantitative MTT Assessment (Percentage Tissue Viability)
For the test item the relative mean tissue viabilities obtained after the 15 Minute exposure period followed by the 42 Hour post exposure incubation period were compared to the mean of the negative control treated tissues (n=3). The relative mean viabilities were calculated in the following way:

Relative mean viability (%) = (Mean OD562 of test item / Mean OD562 of negative control) x 100

Classification of irritation potential is based upon relative mean tissue viability following the 15 Minute exposure period followed by the 42 Hour post exposure incubation period according toTable 1 below.
Irritation / corrosion parameter:
other: other: relative mean viability
Run / experiment:
15 Minute exposure/42h observation
Value:
> 85.5 - < 94.8
Negative controls validity:
valid
Remarks:
Set to 100%
Positive controls validity:
valid
Remarks:
1.2
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The mean OD of the three negative control exposed tissues is  0.6 till ≤ 1.5 (range: 1.384 to 1.468).
The rel. standard deviations between tissues of the same treatment group was ≤ 18% (3.2% (negative control) and 5.4% (test item)). For the positive control, the acceptance criteria were not met (28.7%). Reason: extremely low absorbance values of 0.014, 0.014 and 0.022, where small differences cause high rel. standard deviations. The OECD guideline 439 and the SkinEthic protocol request only the simple standard deviation being ≤ 18% as acceptance criteria between tissues of the same treatment group. If the simple standard deviation is considered (0.3%), the acceptance criteria are met. This deviation from the acceptance criteria is not considered to have an impact on the outcome of the study.
The mean relative tissue viability of the positive control was ≤ 40% (1.2%).
The acceptance limit of the IC50 of the respective EpiSkin™ lot was between 1.5 and 3.0 mg/mL after 18 hours treatment with SLS (1.8 mg/mL).

Results after treatment with the test item and controls

Test Group

Absor-bance 570 nm
Tissue Well 1

Absor-bance 570 nm
Tissue Well 2

Mean Absor-bance 570 nm

Mean Absor-bance 570 nm*

Mean Absor-bance of 3 Tissues

Standard
Deviation of
 Tissue Absorbance

Relative Absor-bance [%] Tissue 1, 2 + 3**

Standard Deviation [%]

Rel. Standard Deviation [%]****

Rel. Absorbance

[% of Negative Control]***

Blank

0.050

0.048

0.049

0.000

 

Negative Control

1.480

1.457

1.468

1.420

1.387

0.045

102.4

3.2

3.2

100.0

1.469

1.438

1.453

1.405

101.3

1.432

1.337

1.384

1.336

96.3

Positive Control

0.063

0.062

0.062

0.014

0.017

0.005

1.0

0.3

28.7

1.2

0.061

0.065

0.063

0.014

1.0

0.076

0.066

0.071

0.022

1.6

Test Item

1.363

1.363

1.363

1.314

1.238

0.067

94.8

4.9

5.4

89.3

1.267

1.203

1.235

1.186

85.5

1.197

1.325

1.261

1.213

87.5

The optical pre-experiment (colour interference pre-experiment) to investigate the colour change potential of the test item in water did not lead to a change in colour.

Optical evaluation of the MTT-reducing capacity of the test item after 3 hour incubation with MTT-reagent did not show blue colour.

The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 89.3% (threshold for irritancy: ≤ 50%), consequently the test item was not irritant to skin.

The mean OD of the three negative control exposed tissues is > 0.6 till ≤ 1.5 (range: 1.384 to 1.468).

The rel. standard deviations between tissues of the same treatment group was ≤ 18% (3.2% (negative control) and 5.4% (test item)). For the positive control, the acceptance criteria were not met (28.7%). Reason: extremely low absorbance values of 0.014, 0.014 and 0.022, where small differences cause high rel. standard deviations. The OECD guideline 439 and the SkinEthic protocol request only the simple standard deviation being ≤ 18% as acceptance criteria between tissues of the same treatment group. If the simple standard deviation is considered (0.3%), the acceptance criteria are met. This deviation from the acceptance criteria is not considered to have an impact on the outcome of the study.

The mean relative tissue viability of the positive control was ≤ 40% (1.2%).

The acceptance limit of the IC50 of the respective EpiSkin™ lot was between 1.5 and 3.0 mg/mL after 18 hours treatment with SLS (1.8 mg/mL). Historical Data for the negative control are not available. Reason: Envigo CRS GmbH changed the negative control for the test system “In vitroSkin Irritation Assay using RHE supplied by SkinEthic” from deionized water to PBS. The test is performed at Envigo CRS GmbH a long time prior to issue of OECD 439 guideline, where deionized water or PBS are suggested as negative control. Originally, SkinEthic requested deionized water to be used as negative control in their protocol. All of Envigo CRS GmbH’s historical data are based on deionized water. In the current SkinEthic protocol, PBS is suggested as negative control. Therefore, Envigo CRS GmbH decided to change the negative control to be in accordance with the SkinEthic protocol, but the number of performed assays using PBS as negative control is still too low to issue historical data.
Historical Data for the positive control are available (see annex 1). But the determined viability value of 1.2% is not within the historical data (viability range: 3.90% - 32.73%).
Since the historical data at Envigo CRS GmbH are updated at least once a year, after next update the present positive control viability of 1.2% will be within the historical range. Since neither the OECD guideline nor SkinEthic requests historical data for the positive control, the outcome of the study is not impacted by this.

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, it can be stated that in this study and under the experimental conditions reported, 4,4’-(1,3-Phenylenediisopropylidene) diphenylcyanate is non-irritant to skin according to UN GHS and EU CLP regulation
Executive summary:

This in vitro study was performed to assess the irritation potential of 4,4’-(1,3-Phenylenediisopropylidene) diphenylcyanate by means of the Human Skin Model Test according to OECD TG 439.

The test item did not reduce MTT (pre-test for direct MTT reduction), and it did not dye water, when mixed with it (pre-test for colour interference). Also its intrinsic colour was nto intensive. Consequently, additional tests with freeze-killed or viable tissues were not necessary.

Three tissues of the human skin model EpiSkin™ were treated with the test item, the negative control (PBS) or the positive control (5% sodium lauryl sulfate) for 15 minutes.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD³0.6 till ≤ 1.5 thus showing the quality of the tissues.

Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control thus ensuring the validity of the test system.

After treatment with the test item the mean relative absorbance value decreased to 89.3%. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

In conclusion, it can be stated that in this study and under the experimental conditions reported, 4,4’-(1,3-Phenylenediisopropylidene) diphenylcyanate is non-irritant to skin according to UN GHS and EU CLP regulation
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted on 12 June 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is considered to be reliability 1 as it has been conducted according to OECD Test Guideline 437 using the Bovine Corneal Opacity and Permeability Assay method and in compliance with GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
26 July 2013
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
EC No. 440/2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
The test item was supplied by or on behalf of the Sponsor including the following information:
Identification: 4,4’-(1,3-Phenylenediisopropylidene) diphenylcyanate
CAS No.: 127667-44-1
Batch: FAR366085A
Purity: Not indicated by the Sponsor
Appearance: Highly viscous yellowish liquid
Expiry Date: 29 September 2019
Storage Conditions: In the refrigerator
Stability in Solvent: Not indicated by the Sponsor
Purpose of Use: Industrial chemical
Species:
other: Eyes from >9 month old cattle
Strain:
other: Not applicable
Details on test animals or tissues and environmental conditions:
Test System: Freshly isolated bovine cornea (at least 9 month old donor cattle)
Rationale: OECD 437
Source: AB Schlachthof GmbH & Co. KG, 63739 Aschaffenburg, Germany

Collection of Bovine Eyes
Freshly isolated bovine eyes of at least 9 month old donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were stored in HBSS containing 1% (v/v) Penicillin/Streptomycin (100 units/mL penicillin and 100 µg/mL streptomycin) in the cooled slaughter-house until transportation on the same morning to the laboratory using a Styrofoam box. The corneae were isolated on the same day after delivery of the eyes.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
0.75 mL of the test item or control items were applied to the cornea.
Duration of treatment / exposure:
240 minutes
Number of animals or in vitro replicates:
3 corneas per treatment
Details on study design:
Preparation of Corneae
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. The corneae were directly used in the BCOP test on the same day.
Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OECD guideline 437, which consists of anterior and posterior compartments, which interface with the epithelial and the endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure that no air bubbles were present within the compartments.
For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath.
At the end of the incubation period, the basal opacity was determined (t0).
The basal opacity of all corneae was recorded. Each cornea with a value of the basal opacity > 7 was discarded. Sets of three corneae were used for treatment with the test item and for the negative and positive controls, respectively.

Exposure of the Corneae to the Test Groups
The corneae were distributed as follows:
Groups Number of Corneae
1 Negative Control 3
2 Positive Control 3
3 Test Item 3
The anterior compartment received the test item suspension or the negative or positive controls at a volume of 0.75 mL ensuring sufficient coverage of the epithelial surface of the corneae. The corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath.
The incubation time lasted 240 minutes.
Afterwards, the test item or the control items, respectively, were each rinsed off from the according application sides with saline, and fresh incubation medium was added into the anterior compartment and opacity was measured (t240). The opacity measurement is described in chapter 3.5.
In the second step of the assay, permeability of the cornea was determined. The permeability measurement is described in chapter 3.6.

Opacity Measurement
The opacitometer determines changes in the light transmission passing through the corneae, and displays a numerical opacity value. This value was recorded in a table. The opacitometer (OP_KiT opacitometer (Electro Design, 63-Riom, France)) was calibrated as described in the manual and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
After exposure of the corneae to the different test groups, and after rinsing the opacity value was determined again (t240).

Permeability Determination
Following to the opacity readings, the permeability was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the incubation medium was removed from the anterior compartment and replaced by 1 mL of a 0.5% (w/v) sodium fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 minutes in a water-bath at 32 ± 1 °C. Incubation medium from the posterior compartment were removed, well mixed and transferred into a 96 well plate.
The optical density was measured with a microplate reader (Versamax® Molecular Devices) at 490 nm (OD490). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

Data Recording
The data generated were recorded in the raw data file. The results are presented in tabular form, including experimental groups with the test item as well as negative and positive controls.

Data Evaluation
pacity
The change of the opacity value of each treated cornea or of the positive and negative control corneae is calculated by subtracting the initial basal opacity from the post treatment opacity reading (t240 – t0), for each individual cornea.
The average change in opacity of the negative control corneae is calculated and this value is subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.
Permeability
The corrected OD490 value of each cornea treated with positive control or test item is calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.
IVIS Calculation
The following formula is used to determine the IVIS of the negative control:

IVIS = opacity value + (15 x OD490 value)

The following formula is used to determine the IVIS of the positive control and the test item:

IVIS = (opacity value – corrected opacity value mean negative control) + (15 x corrected OD490 value)

The mean IVIS value of each treated group is calculated from the IVIS values.
Depending on the IVIS score obtained, the test item is classified into the following Category according to OECD guideline 437:

IVIS UN GHS
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
> 55 Category 1

Criteria for Determination of a Valid Test
The test will be acceptable if
• the positive control gives an IVIS that falls within two standard deviations of the current historical mean (updated every three months), and if
• the negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.


Irritation parameter:
in vitro irritation score
Value:
> 0.08 - < 1.56
Negative controls validity:
valid
Remarks:
1.36
Positive controls validity:
valid
Remarks:
126.28
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
Corneal epithelium condition
The corneas treated with the test item were cloudy post treatment and post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation

In vitro irritancy scores:
The in vitro irritancy score for the test item was 0.94
The in vitro irritancy score for the negative control was 1.36
The in vitro irritancy score for the positive control was 126.28
Other effects:
No other effects were observed

Results after 240 Minutes Treatment Time


Test Group

Opacity value = Difference (t240-t0) of Opacity

Permeability at 490 nm (OD490)

IVIS

Mean IVIS

Proposedin vitroIrritancy Score

 

 

Mean

 

Mean

 

 

 

Negative Control

0

0.33

0.062

0.068

0.93

1.36

Not categorized

0

0.080

1.20

1

0.063

1.95

Positive Control

121.67*

0.187*

124.47

126.28

Category 1

116.67*

0.133*

118.66

131,67*

0.271*

135.73

Test Item

0.67*

0.060*

1.56

0.94

Not categorized

-0.33*

0.100*

1.16

-0.33*

0.028*

0.08

*corrected values

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, according to the current study and under the experimental conditions reported, 4,4’-(1,3-Phenylenediisopropylidene) diphenylcyanate is not categorized (GHS).
Executive summary:

This in vitro study was performed to assess thecorneal damage potentialof4,4’-(1,3-Phenylenediisopropylidene) diphenylcyanate by means of the BCOP assay using fresh bovine corneae.

After a first opacity measurement of the fresh bovine corneae (t0), the 20% (w/v) suspensionin saline (0.9% (w/v) NaCl in deionised water) of the test item 4,4’-(1,3-Phenylenediisopropylidene) diphenylcyanate as well as the positive and the negative controls were each applied to different corneae fixed in an incubation chamber in horizontal position and incubated for 240 minutes at 32 ± 1 °C. The posterior chamber contained incubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae andopacity was measured again (t240).

After the opacity measurements permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.

With the negative control (physiological saline) neither an increase of opacity nor permeability of the corneae could be observed.

The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as serious eye damage (CLP/EPA/GHS (Cat 1)).

 

Relative to the negative control, the test item 4,4’-(1,3-Phenylenediisopropylidene) diphenylcyanate did not cause an increase of the corneal opacity and permeability. The calculated meanin vitroirritancy score was 0.94.According to OECD 437 the test item is not categorized (GHS).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

Justification for classification or non-classification