Reaction products of diethylenetriamine and 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

Endpoint summary

Description of key information

An In vitro skin irritation: reconstructed human epidermis test (EPISKIN) was carried out according to OECD guideline 439 and EU method B. 46. Under the conditions of this test, BADGE-DETA is irritant to skin according to UN GHS and EU CLP regulation.

An in vitro skin corrosion, Reconstructed human epidermis test was carried out according to OECD guideline 431 and EU Method B 40. The study results showed BADGE-DETA is corrosive to skin according to EU CLP and UN GHS.

An In vitro eye irritation (Bovine Corneal Opacity and Permeability test) was carried out according to OECD guideline 437. According to this study, BADGE-DETA is not seriously eye damaging (CLP/EPA/GHS (Cat 1) but a prediction for the damage hazard cannot be made (GHS).

An In vivo eye irritation study was carried out according to OECD guideline 405 and EU Method B. 5. In this study the test item produced irreversible ocular damage and was considered to be corrosive to the rabbit eye. BADGE-DETA was classified as Category 1 (irreversible effects on the eye) according to the Globally Harmonized System of Classification and Labelling of Chemicals.

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

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: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between24 November 2016 and 13 January 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:

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 was not adjusted to purity)
Appearance: Yellow/brown viscous liquid
Colourless solidified liquid*
Expiry Date: 01 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:

non-transformed keratinocytes

Cell source:

other: The EpiSkin™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis.

Source strain:

not specified

Details on test system:

EpiSkin™ Kit Components Needed for the Assay
EpiSkin™ Kit Lot No.: 17-EKIN-002
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

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Test Item Preparation
5 µL of deionised water were topically applied to the epidermal surface in order to
improve further contact between the solid and the epidermis. Each approximately 10 mg of the test item were applied to the wetted tissues and spread to match the surface of the tissue

Duration of treatment / exposure:

15 Minutes

Duration of post-treatment incubation (if applicable):

42 hours post-exposure incubation

Number of replicates:

3. Triplicate tissues were treated with: test substance, positive control or negative control.

Details on study design:

Study Design
MTT-Solution
MTT Formazan salt was dissolved in DMEM or assay medium to reach a final concentration of 0.3 mg/mL.

Cell Culture
EpiSkin™ kits are purchased from SkinEthic Laboratories (69007 Lyon, France). The EpiSkin™ tissue consists of normal, human-derived epidermal keratinocytes 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 10 January 2017. On the day of receipt 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 test item’s colour interference potential had to be evaluated. For this purpose the test item (about 10 mg) was mixed with 90 µL of deionised water in a pre-experiment. The test item/water mixture was gently shaken for 15 minutes at room temperature.
The test item did not dye water or did not change colour 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 about 10 mg 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 with 10 µL of DMEM was used as the control. Since the MTT solution colour turned purple, the test item was presumed to have reduced the MTT. Therefore, it was necessary to perform an additional test with two freeze-killed tissues, which possess no metabolic activity but absorb and bind the test item like viable tissues. Killed tissues are prepared e. g. by adding 2 mL of deionised water to the living tissue and incubating at 37 ± 1.5 °C (5 ± 0.5% CO2) for 48 ± 1 hour. After discarding the water the tissues were placed in the freezer (< -18 °C). Tissues should be de-frozen before use at room temperature (1 hour in 2 mL of maintenance medium). Once killed, the tissue may be stored up to a half of year in the freezer. In addition to the normal test procedure described in chapter 3.6.3, the functional check will employ two killed tissues treated with the MTT reducing test item and two untreated killed tissues that will show the small amount of MTT reduction due to residual NADH and associated enzymes within the killed tissue.
If the direct reduction by the test item is > 30% of the negative control value, the test item is considered incompatible with this endpoint. If the direct reduction of MTT by the test item is ≤ 30% of the negative control the net OD of the test item treated killed tissues will be subtracted from the net OD of the test item treated viable tissues to obtain the true amount of MTT reduction that reflects metabolic conversion only.

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 about 23 hours.

Treatment
The test item tissues were wetted with 5 µL of deionised water. The negative control, positive control and the test item were added into the insert atop the concerning EpiSkin™ triplicate tissues. The 12-well plates were treated 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. The test item could not be removed completetly. 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.

MTT Assay
A 12-well plate was filled with 2 mL assay medium containing 0.3 mg/mL MTT per well.
After the treatment procedure 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 aspirated from the wells and the wells were rinsed three times with PBS. 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 vials were sealed to inhibit isopropanol evaporation. The formazan salt was extracted for nearly 3 hours while shaking at room temperature.
Per tissue sample 2  200 µL aliquots of the formazan blue solution 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 are presented in tabular form, including experimental groups with the test item, negative and positive controls.

Data Evaluation
The mean OD of the three 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 testitem or positive control) / mean OD negative control) x 100

Data correction procedure:

True ODtest item = mean ODtest item - (mean ODfreeze-killed tissue test item – mean ODfreeze-killed tissue negative control)

For the test item and the positive control, the mean relative viability  rel. standard deviation of the three individual tissues was calculated and used for classification according to the following prediction model:
For the current test, an irritation potential of a test item according to H315 classification of EU (according to directive 67/548/EEC and according to regulation (EC) 1272/2008), and GHS category 2 according to UN GHS (published 2003, last (3rd) revision 2009) is recommended if the mean relative tissue viability of three individual tissues is reduced by < 50% of the negative control.

In Vitro Result In Vivo Prediction
Mean tissue viability < 50% Irritant (I), H315 (category 2)
Mean tissue viability > 50% Non-irritant (NI)

Acceptability of the Assay
The absolute OD 570 nm of the negative control tissues in the MTT test is an indicator of tissue viability obtained after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is meeting the acceptance criterion if the mean OD of the three tissues is  0.6 till ≤ 1.5.
The rel. standard deviations in between tissues of the same treatment group should be ≤ 18%.
An assay is meeting the acceptance criterion if mean relative tissue viability of the positive control is  40%.
The data of the quality control (determined by SkinEthic Laboratories, 69007 Lyon, France) of the respective EpiSkin™ lot is mentioned in the present report (the acceptance limit of the IC50 should be between 1.0 and 3.0 mg/mL after 18 hours treatment with SLS).

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 Minute exposure/42h observation

Value:

17.5

Negative controls validity:

valid

Remarks:

Set to 100%

Positive controls validity:

valid

Remarks:

8.1

Remarks on result:

positive indication of irritation

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

Item	Absorbance 570nm of tissues*	Mean Absorbance of triplicate tissues	Relative Absorbance (%)**	Relative SD (%)	Mean Relative Absorbance (% Negative control)***	OD correction	Corrected Mean Relative Absorbance (% Negative control)***
Negative Control	1.111		105.5
	1.094	1.053	103.9	8.2	100
	0.954		90.6
Positive Control	0.074		7.0
	0.082	0.086	7.8	15.9	8.1
	0.101		9.6
Test Item	0.071		6.7
	0.059	0.061	5.6	14.0	5.8	0.061 -(0.096 -0.219)	17.5
	0.051		5.1
Negative Control freeze-killed	0.232	0.219
	0.206
Test item freeze-killed	0.105	0.096
	0.088

*       Mean of two replicate wells after blank correction

**       relative absorbance per tissue [rounded values]: 100 x Absorbance tissue / mean absorbance negative control

***       relative absorbance per treatment group [rounded values]: 100 x mean absorbance test item / mean absorbance negative control

 

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 3 hour incubation with MTT-reagent showed blue colour.

The corrected mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 17.5% (threshold for irritancy: ≤ 50%), consequently the test item was irritant to skin.

 

Discussion

This in vitro study was performed to assess the irritation 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.

The test item passed the pre-test for colour interference, but it reduced MTT (pre-test for direct MTT reduction). Consequently, an additional test with freeze-killed tissues to determine a correction factor for calculating the true viability in the main experiment was necessary.

Each three tissues of the human skin model EpiSkin™ were treated with the test item, the negative or the positive control for 15 minutes.

The test item and the positive and negative controls were washed off the skin tissues after 15 minutes treatment. After further incubation for about 42 hours the tissues were treated with the MTT solution for 3 hours following nearly 3 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD ³ 0.6 till ≤ 1.5 for the 15 minutes treatment interval thus showing the quality of the tissues.

Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 8.1% thus ensuring the validity of the test system.

The rel. standard deviations between the % variability values of the test item, the positive and negative controls were below 16% (threshold of the "OECD TG 439 Guideline for the Testing of Chemicals 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”: 18%), thus ensuring the validity of the study.

After treatment with the test item the corrected mean relative absorbance value was reduced to 17.5%. This value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Remarks:

Criteria used for interpretation of results: EU CLP and UN GHS

Conclusions:

it can be stated that in this study and under the experimental conditions reported, 4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with diethylenetriamine is 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’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with diethylenetriamine by means of the Human Skin Model Test according to OECD TG 439.

The test item did not dye water or change colour when mixed with deionised water (test for colour interference), but it reduced MTT (test for direct MTT reduction. Consequently, an additional test with freeze-killed tissues had to be performed to determine a correction factor for calculating the true viability.

Each three tissues of the human skin model EpiSkin™ were treated with the test item, the negative control (deionised water) 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 OD  0.6 till ≤ 1.5 for the 15 minutes treatment interval 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 for the 15 minutes treatment interval thus ensuring the validity of the test system.

After treatment with the test item the corrected mean relative absorbance value decreased to 17.5%. This value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential.

In conclusion, it can be stated that in this study and under the experimental conditions reported, the 4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with diethylenetriamine is irritant to skin according to UN GHS and EU CLP regulation.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

Eye irritation

Link to relevant study records

Reference

Reference 1

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 diethylenetriamine
- CAS No. 31326-29-1
- Analytical purity: 94%
- Lot/batch No.: Ei 3041
- Expiration date of the lot/batch: 01 May 2021
- Isomers composition: Not applicable
- Other: Storage conditions: Room tremperature
- Appearance: Colorless solidified liquid as determined by Envigo CRS GmbH laboratory staff

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: not specified - multiple
- Characteristics of donor animals (e.g. age, sex, weight): 9 month old donor cattle
- 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: Not 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 replicates

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:

> 41.17 - < 47.99

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Not categorized

Positive controls validity:

valid

Remarks:

Category 1

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Value:

> 36.67 - < 40.67

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Not categorized

Positive controls validity:

valid

Remarks:

Category 1

Remarks on result:

no 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 diethylenetriamine 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 diethylenetriamine, 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 an increase of the corneal opacity and permeability. The calculated mean IVIS was 43.76 (threshold for serious eye damage: IVIS ≥ 55). According to OECD 437 no prediction for the damage hazard of the test item to the eye can be made.

Results after 240 minutes incubation time:

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	36.67	0.300	41.17	No hazard prediction can be made
	40.67	0.488	47.99	No hazard prediction can be made
	37.67	0.297	42.13	No hazard prediction can be made

 

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, 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 diethylenetriamine is not serious eye damaging (CLP/EPA/GHS (Cat 1) but a prediction for the damage hazard cannot be made (GHS).

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 diethylenetriamine 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 diethylenetriamine, 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 diethylenetriamine caused an increase of the corneal opacity and permeability compared with the values caused by the negative control. The calculated mean in vitro irritancy score was 43.76. According to OECD 437 (see table in chapter 3.8.3) the test item the test item is not classified as serious eye damaging (CLP/EPA/GHS (Cat 1) but the test item’s hazard for eye damaging cannot be predicted.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 06 March 2017 and 11 April 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 405 and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

02 October 2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Version / remarks:

EC 440/2008

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
Batch: Ei3041
Purity: 94%
Physical state/Appearance: yellow/brown extremely viscous liquid
Expiry Date: 01 May 2021
Storage Conditions: room temperature in the dark

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Two New Zealand White (Hsdlf:NZW) strain rabbits were supplied by Envigo RMS (UK) Limited, Leicestershire, UK. At the start of the study the animals weighed 2.84 or 3.42 kg and were 12 to 52 weeks old. After an acclimatization period of at least 5 days each animal was given a number unique within the study which was written with a black indelible marker pen on the inner surface of the ear and on the cage label.

Animal Care and Husbandry
The animals were individually housed in suspended cages. Free access to mains drinking water and food (2930C Teklad Global Rabbit diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study. The diet and drinking water were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
The temperature and relative humidity were set to achieve limits of 17 to 23 °C and 30 to 70% respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.1 mL test item as supplied

Observation period (in vivo):

21 days

Number of animals or in vitro replicates:

2

Details on study design:

Immediately before the start of the test, both eyes of the provisionally selected test rabbits were examined for evidence of ocular irritation or defect with the aid of a light source from a standard ophthalmoscope. Only animals free of ocular damage were used.
Initially, a single rabbit was treated. A subcutaneous injection of buprenorphine 0.01 mg/kg was administered 60 minutes prior to test item application to provide a therapeutic level of systemic analgesia. Five minutes prior to test item application, a pre dose anesthesia of ocular anesthetic (two drops of 0.5% proxymetacaine hydrochloride) was applied to each eye.
A volume of 0.1 mL of the test item was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test item, and then released. The left eye remained untreated and was used for control purposes. Immediately after administration of the test item, an assessment of the initial pain reaction was made according to the six point scale shown in Table1.

Eight hours after test item application, a subcutaneous injection of post dose analgesia, buprenorphine 0.01 mg/kg and meloxicam 0.5 mg/kg, was administered to provide a continued therapeutic level of systemic analgesia. The treated animal was checked for signs of pain and suffering approximately 12 hours later. No further analgesia was required.
After consideration of the ocular responses produced in the first treated animal, a second animal was similarly treated.
Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the numerical evaluation (Draize, J.H, 1977) given in Table 2.
Any other ocular effects were also noted. Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.
Any clinical signs of toxicity, if present, were also recorded.
Additional observations were made on Days 7, 14 and 21 to assess the reversibility of the ocular effects.
Individual body weights were recorded on Day 0 (the day of dosing) and at the end of the observation period.

Data Evaluation
If evidence of irreversible ocular damage is noted, the test item will be classified as corrosive to the eye.
The results were also evaluated according to the Globally Harmonized System of Classification and Labelling of Chemicals.

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

14 d

Score:

0

Max. score:

1

Reversibility:

not reversible

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

21 d

Score:

1

Max. score:

1

Reversibility:

not reversible

Irritation parameter:

iris score

Basis:

animal #1

Time point:

14 d

Score:

0

Max. score:

1

Reversibility:

not reversible

Irritation parameter:

iris score

Basis:

animal #2

Time point:

21 d

Score:

0

Max. score:

1

Reversibility:

not reversible

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

14 d

Score:

0

Max. score:

2

Reversibility:

not reversible

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

21 d

Score:

0

Max. score:

2

Reversibility:

not reversible

Irritant / corrosive response data:

Diffuse corneal opacity was noted in both treated eyes 1 hour after treatment and at the 24, 48, 72 Hour and 7 Day observations. Diffuse corneal opacity persisted in the second treated eye at the 14 and 21 Day observations. A small area of vascularisation, measuring approximately 2 mm x 5 mm, on the upper edge of the cornea, was noted in the second treated eye at the 7 Day observation. The area of vascularisation noted in the second treated eye had increased in size to approximately 5 mm x 7 mm and extended towards the center of the cornea at the 14 and 21 Day observations. An additional area of vascularisation, measuring approximately 3 mm x 4 mm on the lower right section of the cornea, was also noted in the second treated eye at the 14 and 21 Day observations.
Iridial inflammation was noted in both treated eyes 1 hour after treatment and at the 24, 48 and 72 Hour observations and persisted in the second treated eye at the 7 Day observation.
Moderate conjunctival irritation was noted in both treated eyes 1 hour after treatment and at the 24, 48 and 72 Hour observations. Moderate conjunctival irritation persisted in the second treated eye with minimal conjunctival irritation noted in the first treated eye at the 7 Day observation. Minimal conjunctival irritation was noted in the second treated eye at the 14 Day observation.
The reactions noted at the 21 Day observation in the second treated eye were considered to be indicative of irreversible effects.

Other effects:

Body Weight
Both animals showed expected gain in body weight during the study.

Individual Scores for Ocular Irritation:

	Rabbit 75702 Male  IPR = ^						Rabbit 75706 Female    IPR = 0
Time after treatment	1h	24h	48h	72h	7 days	14 days	1h	24h	48h	72h	7 days	14 days	21 days
CORNEA
Degree of opacity	1	1	1	1	1	0	1	1	1	1	1	1	1
Area of Cornea Involved	1	2	2	1	1	0	1	2	2	2	1 V	1 V+*	1 V+*
IRIS	1	1	1	1	0	0	1	1	1	1	1	0	0
CONJUNCTIVAE
Redness	2	2	2	2	1	0	2	2	2	2	2	1	0
Chemosis	3	2	2	2	1	0	2	2	2	1	1	0	0
Discharge	2	2	1	1	0	0	2	2	2	1	1	0	0

 

IPR =       Initial pain reaction

^ =           Due to technician error initial pain reaction was not recorded

V =          Small area of vascularisation, measuring approximately 2 mm x 5 mm, on the upper edge of the cornea

V+ =        Small area of vascularisation, measuring approximately 5 mm x 7 mm and extending towards the center of the cornea

* =           Additional area of vascularisation, measuring approximately 3 mm x 4 mm on the lower right section of the cornea

 

Body Weight

Both animals showed expected gain in body weight during the study.

Individual Body Weights and Body Weight Change

Rabbit Number and Sex	Individual Body Weight (kg)		Body Weight Change (kg)
	Day 0	Day 14 (Day 21*)
75702 Male	3.42	3.50	0.08
75706 Female	2.84	2.98*	0.14

 

Interpretation of results:

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

Conclusions:

The test item produced irreversible ocular damage and was considered to be CORROSIVE to the rabbit eye.
The test item was classified as Category 1 (irreversible effects on the eye) according to the Globally Harmonized System of Classification and Labelling of Chemicals.

Executive summary:

The study was performed to assess the irritancy potential of the test item to the eye of the New Zealand White rabbit.

Results

A single application of the test item to the non-irrigated eye of two rabbits produced diffuse corneal opacity, iridial inflammation and moderate conjunctival irritation.  Vascularisation of the cornea, up to 5mm x 7mm in length and extending towards the centre of the cornea, was also noted in the second treated eye.  The first treated eye appeared normal at the 7 Day observation.

The reactions noted at the 21 Day observation in the second treated eye were considered to be indicative of irreversible effects.

Conclusion

The test item produced irreversible ocular damage and was considered to be CORROSIVE to the rabbit eye.

The test item was classified as Category 1 (irreversible effects on the eye) according to the Globally Harmonized System of Classification and Labelling of Chemicals.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

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)