Reaction mass of 4,4'-Isopropylidenediphenol, 1-chloro-2,3-epoxypropane and ethylenediamine

Endpoint summary

Description of key information

According to the read across approach used, BADGE-EDA should be considered corrosive to the skin (Category. 1) according to EU CLP and UN GHS.

Under the experimental conditions reported, 4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with ethylenediamine should be considered seriously eye damaging (CLP/EPA/GHS (Cat 1).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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

Justification for type of information:

Information is used in a read across approach, for information on justification please see document enclosed in Chapter 13.

Reason / purpose for cross-reference:

read-across: supporting information

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)

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

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

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%)

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 EpiDerm^TMwere 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.

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.

Reference 2

Endpoint:

skin irritation / corrosion, other

Type of information:

read-across from supporting substance (structural analogue or surrogate)

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

Justification for type of information:

Information is generated using a read across approach, for information on justification please see document enclosed in Chapter 13.

Reason / purpose for cross-reference:

read-across source

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)

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

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

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%)

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 EpiDerm^TMwere 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.

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 ethylenediamine 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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

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:

This study was conducted on 21 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

July 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with ethylenediamine
- CAS No. 72480-18-3
- Appearance: Colorless solidified liquid - as determined by Envigo CRS GmbH laboratory staff-
- Analytical purity: Not supplied
- Impurities (identity and concentrations): Not supplied
- Composition of test material, percentage of components: Not supplied
- Purity test date: Not supplied
- Lot/batch No.: BBF01102V1
- Expiration date of the lot/batch: 01 January 2021
- Isomers composition: Not applicable
- Other: Storage conditions: Room tremperature

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Schlachthof Aschaffenburg, 63739 Aschaffenburg, Germany
- Number of animals: Multiple
- Characteristics of donor animals (e.g. age, sex, weight): least 9 month old
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The isolated eyes were transported to the laboratory in HBSS supplemented with streptomycin / penicillin at ambient temperature.
- Time interval prior to initiating testing: The corneae were isolated on the same day after delivery of the eyes.
- indication of any existing defects or lesions in ocular tissue samples: none
- Indication of any antibiotics used: None reported

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.75 mL per cornea - 20% solution (w/v) in saline

Duration of treatment / exposure:

240 Minutes

Duration of post- treatment incubation (in vitro):

90 minutes

Number of animals or in vitro replicates:

3

Details on study design:

Experimental Design and Study Conduct

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 transported to the laboratory in HBSS supplemented with streptomycin / penicillin at ambient temperature. The corneae were isolated on the same day after delivery of the eyes.

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 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 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 the negative and positive controls.

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 solution or negative or positive control at a volume of 0.75 mL each on the 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 control items, respectively, were rinsed off from the application side with saline, and fresh incubation medium was added into the anterior compartment and opacity was measured (t240). The opacity measurement is described below.
In the second step of the assay, permeability of the cornea was determined. The permeability measurement is described below.

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 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 ± 10 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 and the optical density at 490 nm (OD490) was determined with a spectrophotometer.
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)

Irritation parameter:

in vitro irritation score

Value:

> 133.34 - < 145.91

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

cornea opacity score

Value:

> 119.67 - < 134.67

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

This in vitro study was performed to assess the corneal damage potential of 4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with ethylenediamine 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) solution in saline of the test item 4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with ethylenediamine, the positive, and the negative controls were applied to corneae and incubated for 240 minutes at 32 ± 1 °C. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae and opacity 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 (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 1.17).
The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae (mean IVIS = 105.73) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)).
Relative to the negative control, the test item caused a distinct increase of the corneal opacity and permeability. The calculated mean IVIS was 140.99 (threshold for serious eye damage: IVIS ≥ 55). According to OECD 437 the test item is classified as serious eye damaging

Other effects:

None

Results after 240 minutes incubation

Test Group	Opacity value = Difference (t240-t0) of Opacity	Permeability at 490 nm (OD490)	IVIS	Proposed in vitro Irritancy Score
Negative Control	0	0.055	0.83	Not categorized
	0	0.057	0.86	Not categorized
	1	0.055	1.83	Not categorized
Positive Control	104.67	0.106	106.26	Category 1
	95.67	0.168	98.19	Category 1
	109.67	0.204	112.73	Category 1
Test Item	134.67	0.749	145.91	Category 1
	119.67	0.911	133.34	Category 1
	127.67	1.070	143.72	Category 1

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

According to the current study and under the experimental conditions reported, 4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with ethylenediamine is serious eye damaging (CLP/EPA/GHS (Cat 1).

Executive summary:

This in vitro study was performed to assess the corneal damage potential of 4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with ethylenediamine 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) solution in saline (0.9% (w/v) NaCl in deionised water) of the test item 4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with ethylenediamine, the positive, and the negative controls were applied to 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 and opacity 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’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with ethylenediamine caused a strong increase of the corneal opacity and permeability compared with the values caused by the negative control. The calculated mean in vitro irritancy score was 140.99. According to OECD 437 (see table in chapter 3.8.3) the test item is classified as serious eye damaging (CLP/EPA/GHS (Cat 1).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Justification for classification or non-classification

According to the read across approach used, BADGE-EDA, BADGE-DETA and BADGE-TETA should be considered corrosive to the skin (Category. 1) according to EU CLP and UN GHS.

Under the experimental conditions reported, BADGE-EDA, BADGE-DETA and BADGE-TETA should be considered seriously eye damaging (CLP/EPA/GHS (Cat 1)