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Skin irritation / corrosion

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
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)

Test material

Constituent 1
Chemical structure
Reference substance name:
1,3-Bis[2-(4-cyanatophenyl)-2-propyl]benzene
Cas Number:
127667-44-1
Molecular formula:
C26 H24 N2 O2
IUPAC Name:
1,3-Bis[2-(4-cyanatophenyl)-2-propyl]benzene
Constituent 2
Chemical structure
Reference substance name:
1,3,5-triazine-2,4,6-triyltris(oxybenzene-4,1-diylpropane-2,2-diylbenzene-3,1-diylpropane-2,2-diylbenzene-4,1-diyl) tricyanate
Molecular formula:
C78H72N6O6
IUPAC Name:
1,3,5-triazine-2,4,6-triyltris(oxybenzene-4,1-diylpropane-2,2-diylbenzene-3,1-diylpropane-2,2-diylbenzene-4,1-diyl) tricyanate
Constituent 3
Chemical structure
Reference substance name:
4-{2-[3-(prop-1-en-2-yl)phenyl]propan-2-yl}phenyl cyanate
Molecular formula:
C19H19NO
IUPAC Name:
4-{2-[3-(prop-1-en-2-yl)phenyl]propan-2-yl}phenyl cyanate
Constituent 4
Chemical structure
Reference substance name:
4-(2-{3-[2-(4-cyanatophenyl)propan-2-yl]phenyl}propan-2-yl)phenyl N-{(E)-[4-(2-{3-[2-(4-cyanatophenyl)propan-2-yl]phenyl}propan-2-yl)phenoxy]methylidene}carbamimidate
Molecular formula:
C52H50N4O4
IUPAC Name:
4-(2-{3-[2-(4-cyanatophenyl)propan-2-yl]phenyl}propan-2-yl)phenyl N-{(E)-[4-(2-{3-[2-(4-cyanatophenyl)propan-2-yl]phenyl}propan-2-yl)phenoxy]methylidene}carbamimidate
Constituent 5
Chemical structure
Reference substance name:
4-{2-[3-(prop-1-en-2-yl)phenyl]propan-2-yl}phenyl N-{(Z)-[4-(2-{3-[2-(4-cyanatophenyl)propan-2-yl]phenyl}propan-2-yl)phenoxy]methylidene}carbamimidate
Molecular formula:
C45H45N3O3
IUPAC Name:
4-{2-[3-(prop-1-en-2-yl)phenyl]propan-2-yl}phenyl N-{(Z)-[4-(2-{3-[2-(4-cyanatophenyl)propan-2-yl]phenyl}propan-2-yl)phenoxy]methylidene}carbamimidate
Constituent 6
Chemical structure
Reference substance name:
4-{2-[3-(prop-1-en-2-yl)phenyl]propan-2-yl}phenyl N-[(E)-(4-{2-[3-(prop-1-en-2-yl)phenyl]propan-2-yl}phenoxy)methylidene]carbamimidate
Molecular formula:
C38H40N2O2
IUPAC Name:
4-{2-[3-(prop-1-en-2-yl)phenyl]propan-2-yl}phenyl N-[(E)-(4-{2-[3-(prop-1-en-2-yl)phenyl]propan-2-yl}phenoxy)methylidene]carbamimidate
Constituent 7
Chemical structure
Reference substance name:
2,4,6-tris(4-{2-[3-(prop-1-en-2-yl)phenyl]propan-2-yl}phenoxy)-1,3,5-triazine
Molecular formula:
C57H57N3O3
IUPAC Name:
2,4,6-tris(4-{2-[3-(prop-1-en-2-yl)phenyl]propan-2-yl}phenoxy)-1,3,5-triazine
Constituent 8
Chemical structure
Reference substance name:
4-[2-(3-{2-[4-({4,6-bis[4-(2-{3-[2-(4-cyanatophenyl)propan-2-yl]phenyl}propan-2-yl)phenoxy]-1,3,5-triazin-2-yl}oxy)phenyl]propan-2-yl}phenyl)propan-2-yl]phenyl N-{(E)-[4-(2-{3-[2-(4-cyanatophenyl)propan-2-yl]phenyl}propan-2-yl)phenoxy]methylidene}carbamimidate
Molecular formula:
C104H98N8O8
IUPAC Name:
4-[2-(3-{2-[4-({4,6-bis[4-(2-{3-[2-(4-cyanatophenyl)propan-2-yl]phenyl}propan-2-yl)phenoxy]-1,3,5-triazin-2-yl}oxy)phenyl]propan-2-yl}phenyl)propan-2-yl]phenyl N-{(E)-[4-(2-{3-[2-(4-cyanatophenyl)propan-2-yl]phenyl}propan-2-yl)phenoxy]methylidene}carbamimidate
Constituent 9
Reference substance name:
Polymeric impurites: reaction product of Bishenol M with cyanogen bromide
Molecular formula:
not available
IUPAC Name:
Polymeric impurites: reaction product of Bishenol M with cyanogen bromide
Details on test material:
Identification: Sodium benzoate
Purity: >99.5%
Physical state/Appearance: white granular solid
Expiry Date: 09 May 2022
Storage Conditions: room temperature over silica gel
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

In vitro test system

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

Results and discussion

In vitro

Resultsopen allclose all
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%)

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.