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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 14 March 2017 and 06 April 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

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 2016
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:
1,1'-[isopropylidenebis(p-phenyleneoxy)]bis[3-[[2-[(2-aminoethyl)amino]ethyl]amino]-2-propanol
Cas Number:
108248-15-3
Molecular formula:
C29H50N6O4
IUPAC Name:
1,1'-[isopropylidenebis(p-phenyleneoxy)]bis[3-[[2-[(2-aminoethyl)amino]ethyl]amino]-2-propanol
Constituent 2
Chemical structure
Reference substance name:
3,3'-[iminobis(ethane-2,1-diylimino)]bis{1-[4-(2-{4-[3-({2-[(2-aminoethyl)amino]ethyl}amino)-2-hydroxypropoxy]phenyl}propan-2-yl)phenoxy]propan-2-ol}
Molecular formula:
C54H87N9O8
IUPAC Name:
3,3'-[iminobis(ethane-2,1-diylimino)]bis{1-[4-(2-{4-[3-({2-[(2-aminoethyl)amino]ethyl}amino)-2-hydroxypropoxy]phenyl}propan-2-yl)phenoxy]propan-2-ol}
Constituent 3
Chemical structure
Reference substance name:
3-[4-(2-{4-[3-({2-[(2-aminoethyl)amino]ethyl}amino)-2-hydroxypropoxy]phenyl}propan-2-yl)phenoxy]propane-1,2-diol
Molecular formula:
C25H39N3O5
IUPAC Name:
3-[4-(2-{4-[3-({2-[(2-aminoethyl)amino]ethyl}amino)-2-hydroxypropoxy]phenyl}propan-2-yl)phenoxy]propane-1,2-diol
Constituent 4
Reference substance name:
Polymeric Adducts of 2,​2'-​[(1-​methylethylidene)​bis(4,​1-​phenyleneoxymethylen​e)​]​bis-oxirane and N1-​(2-​aminoethyl)​-1,​2-ethanediamine
IUPAC Name:
Polymeric Adducts of 2,​2'-​[(1-​methylethylidene)​bis(4,​1-​phenyleneoxymethylen​e)​]​bis-oxirane and N1-​(2-​aminoethyl)​-1,​2-ethanediamine
Constituent 5
Reference substance name:
Unknown components
IUPAC Name:
Unknown components
Specific details on test material used for the study:
Identification : 4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with diethylenetriamine
CAS No : 31326-29-1
Batch : Ei 3041
Purity : 94% (dose calculation will not be adjusted to purity)
Appearance : Yellow/brown viscous liquid
Colourless solidified liquid*
Expiry Date : 31 May 2021
Storage Conditions: At room temperature
Stability in Solvent: Not indicated by the Sponsor
Purpose of Use : Industrial chemical

*determined by Envigo CRS GmbH laboratory staff

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: 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
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.: 25803
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
3.3.2 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

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.

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 mmdiam.).
EpiDerm™ tissues were shipped at 4 °C on medium-supplemented agarose gels in a 24-well plate and reached Envigo CRS GmbH on 04 April 2017. On day of receipt the pre-incubation phase of the EpiDerm™ tissues started.

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
25 ± 2 mg 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) should be further evaluated for their potential to interfere with MTT. To test if an item directly reduces MTT, 25 ± 2 mg 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 proved to be a MTT reducer, an additional functional check (step 4) had to be performed.

Step 4:
The procedure employed freeze-killed tissues that possess no metabolic activity but absorb and bind the test item extract similar to viable tissues.
Each MTT reducing chemical was applied to two freeze-killed tissues. In addition, two freeze killed tissues were left untreated. (Note: The untreated killed controls show a small amount of MTT reduction due to residual reducing enzymes within the killed tissue).The entire assay protocol was performed on the frozen tissues in parallel to the assay performed with the live EpiDerm tissues.

Data were then corrected as follows:
Data correction procedure
True viability = Viability of treated tissue – Interference from test chemical =
ODtvt – ODkt where ODkt = (mean ODtkt – mean ODukt)
tvt = treated viable tissue kt = killed tissues
tkt = treated killed tissue ukt = untreated killed tissue (NC treated tissue)
Since the interference by the test item is < 30% of the negative control value, the net OD of the test item treated killed control was subtracted from the mean OD of the test item extract treated viable tissues to obtain the true amount of MTT reduction that reflects metabolic conversion only.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
Due to the test item’s extreme viscous property, it was used as a solid item. 25 ± 2 mg (39.7 mg/cm2 according to guideline) of the test item were applied onto the surface of duplicate EpiDermTM tissue. It was taken care, that the tissue surface was covered with the test item as evenly as possible. The test item was wetted with 25 µL of deionised water.

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
Duration of post-treatment incubation (if applicable):
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).
Number of replicates:
2

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 Minute Exposure
Value:
ca. 39.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
22.5
Remarks on result:
other: corrosive to skin
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 Minute Exposure
Value:
ca. 18.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100
Positive controls validity:
valid
Remarks:
3.4
Remarks on result:
other: corrosive to skin
Other effects / acceptance of results:
Quality Criteria
The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not led to a change in colour.
Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent showed blue colour.
The test item is considered to be corrosive to skin:
• since the viability after 3 minutes exposure is lower than 50% (with and without taking the correction factor derived from the additional test with freeze-killed tissues into account)

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: 1.557 to 1.681)
• the mean viability of the tissue replicates treated with the positive control for 1 hour, is <15% compared to the negative control (3.4%)
• the Coefficient of Variation (CV) in the range 20 – 100% viability between tissue replicates is ≤ 30% (range: 1.2% to 13.0%)

Any other information on results incl. tables

Results after treatment with 4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with diethylenetriamine and the controls:

Dose Group

Ex-posure Interval
[min]

Ab-sorbance (OD) 570 nm
Well 1

Ab-sorbance (OD) 570 nm
Well 2

Ab-sorbance (OD) 570 nm
Well 3

Mean Ab-sorbance (OD) of 3 Wells

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

Mean Ab-sorbance (OD) of 2 Tissues

Rel. Absorbance
[% of Negative Control]*

Mean Rel. Absorbance
[% of Negative Control]

CV [%]

Corrected Rel. Absorbance [% of Negative Control]**

Blank

 

0.036

0.037

0.037

0.037

0.000

 

 

Negative Control Tissue 1

3

1.603

1.545

1.551

1.566

1.530

1.553

98.5

100.0

2.1

 

Negative Control Tissue 2

1.635

1.572

1.630

1.612

1.576

101.5

Positive Control Tissue 1

0.392

0.391

0.384

0.389

0.352

0.349

22.7

22.5

1.2

Positive Control Tissue 2

0.385

0.385

0.380

0.383

0.346

22.3

Test Item Tissue 1

0.684

0.708

0.698

0.697

0.660

0.635

42.5

40.9

5.6

39.2

Test Item Tissue 2

0.645

0.647

0.647

0.646

0.609

39.3

Negative ControlFreeze Killed TissueTissue 1

0.126

0.137

0.139

0.134

0.098

0.098

6.3

6.3

1.4

 

Negative ControlFreeze Killed TissueTissue 2

0.136

0.138

0.134

0.136

0.099

6.4

Test ItemFreeze Killed TissueTissue 1

0.168

0.167

0.168

0.168

0.131

0.125

8.4

8.0

6.9

Test ItemFreeze Killed TissueTissue 2

0.155

0.155

0.157

0.156

0.119

7.7

Blank

 

0.036

0.036

0.036

0.036

0.000

 

 

Negative Control Tissue 1

60

1.674

1.674

1.696

1.681

1.645

1.583

103.9

100.0

5.6

 

Negative Control Tissue 2

1.573

1.543

1.555

1.557

1.520

96.1

Positive Control Tissue 1

0.092

0.089

0.091

0.091

0.054

0.054

3.4

3.4

0.2

Positive Control Tissue 2

0.090

0.091

0.090

0.090

0.054

3.4

Test Item Tissue 1

0.356

0.371

0.384

0.370

0.334

0.368

21.1

23.3

13.0

18.9

Test Item Tissue 2

0.440

0.438

0.437

0.438

0.402

25.4

Negative ControlFreeze Killed TissueTissue 1

0.119

0.117

0.114

0.117

0.080

0.085

5.1

5.3

7.3

 

Negative ControlFreeze Killed TissueTissue 2

0.126

0.125

0.126

0.125

0.089

5.6

Test ItemFreeze Killed TissueTissue 1

0.185

0.189

0.187

0.187

0.151

0.153

9.5

9.7

2.3

Test ItemFreeze Killed TissueTissue 2

0.192

0.193

0.190

0.192

0.155

9.8

*         Relative absorbance [rounded values]

**       Corrected Relative Viability (%)     

 Discussion

Thisin vitrostudy was performed to assess the corrosive potential of 4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with diethylenetriamine by means of the Human Skin Model Test with EpiDerm™ tissues models.

The test item passed the colour interference pre-test. Due to its MTT reducing capacity, an additional test with freeze-killed tissues was performed.

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

Afterwards, the test and the control items were rinsed off the tissues, and a 3 hour incubation period (37 ± 1 °C, 5 ± 0.5 % CO2) with MTT solution followed. MTT solution was then aspirated from the wells and the wells were rinsed with DPBS. Inserts were transferred into new 24 well plates. The formazan salt was extracted for 17 hours at room temperature.

The required acceptability criteria were met.

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 (22.5%) and for the 1 hour exposure period (3.4%) thus confirming the validity of the test system and the specific batch of tissue models.

After exposure to the test item 4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with diethylenetriamine the relative absorbance value decreased to 40.9% after 3 minutes exposure (corrected value: 39.2%). After 1 hour exposure the relative absorbance value was reduced to 23.3% (corrected value: 18.9%). Only the value of the 1 hour exposure period did not exceed the threshold for corrosivity which is defined to be 15%. After the 3 minutes exposure period the threshold for corrosivity of< 50%was exceeded. Therefore, the test item is considered to be corrosive.

 

Applicant's summary and conclusion

Interpretation of results:
other: 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’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with diethylenetriamine is corrosive to skin according to EU CLP and UN GHS.
Executive summary:

Thisin vitrostudy was performed to assess the corrosive potential of 4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with diethylenetriamine by means of the Human Skin Model Test with EpiDerm™ tissues models.

The test item did not dye water in the pre-test for colour interference, but it reduced MTT in the pre-test for direct MTT reduction. Consequently, an additional test with viable tissues (without MTT addition) was not necessary, but an additional test with freeze-killed tissues had to be performed to determine a correction factor for calculating the true viability in the main experiment.

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 40.9% after 3 minutes exposure (corrected value: 39.2%). After 1 hour exposure the relative absorbance value was reduced to 23.3% (threshold for corrosivity:15%) without taking the correction factor derived from the additional test with freeze-killed tissues into consideration (with correction: 18.9%). Since the 3-minutes value exceeded the threshold for corrosivity, which is defined to be 50%, the test item is 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’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with diethylenetriamine iscorrosiveto skin according to EU CLP and UN GHS.

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