Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 January 2018 to 16 Mar 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Version / remarks:
Adopted 28 July 2015
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
CAS 1629579-82-3
Molecular formula C18H32N2O11
EC 818-033-1
Specific details on test material used for the study:
Identification: Bis-Aminopropyl Diglycol Dimaleate
Appearance: Clear to transparent yellow liquid
Purity/Composition: Not indicated
Test item storage: At room temperature
Stable under storage conditions until: 13 November 2019 (expiry date)

Additional information
Test Facility test item number: 207396/C
Purity/Composition correction factor: Contains 74% water
Test item handling: No specific handling conditions required
Stability at higher temperatures: Yes, maximum temperature: 40°C, maximum duration: 60 minutes
Chemical name (IUPAC, synonym or trade name: Bis-Aminopropyl Diglycol Dimaleate
CAS number: 11629579-82-3

Molecular structure:
Molecular formula: C18H32N2O11
Molecular weight: 452.46

Test animals / tissue source

Species:
human

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
The liquid test item was applied undiluted (50 µL) directly on top of the tissue.
Two tissues were treated with 50 µL Milli-Q water (negative control) and 2 tissues with
50 µL Methyl Acetate (positive control) respectively.
Duration of treatment / exposure:
30 minutes ±2 minutes at 37.0 ± 1.0°C
Duration of post- treatment incubation (in vitro):
After rinsing the cell culture inserts were each dried carefully and immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre-labeled 12-well plate for a 12 ± 2 minute immersion incubation at room temperature (Post-Soak). After the Post-Soak period cell culture inserts were each dried carefully and transferred to the 6-well plate containing 1.0 mL of warm Assay Medium and were incubated for 120 ± 15 minutes at 37°C.
Number of animals or in vitro replicates:
The test was performed on a total of 2 tissues per test item together with a negative control and positive control.
Details on study design:
Test System
Test System
EpiOcular™ (OCL-200-EIT MatTek Corporation, Lot: 27420, kit A and B, Appendix 4).
The EpiOcular tissue construct is a non-keratinized epithelium (0.6 cm2) prepared from normal human keratinocytes (MatTek). It models the cornea epithelium with progressively stratified, but not cornified cells. These cells are not transformed or transfected with genes to induce an extended life span in culture. The “tissue” is prepared in inserts with a porous membrane through which the nutrients pass to the cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test material to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo.

Rationale
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro eye irritation tests is the EpiOcular test, which is recommended in international guidelines and scientific publications (e.g. OECD).

EXPERIMENTAL DESIGN

Test for the Interference of the Test Item with the MTT Endpoint
A test item may interfere with the MTT endpoint if it is colored and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test item is present on the tissues when the MTT viability test is performed.

Test for Color Interference by the Test Item
Bis-Aminopropyl Diglycol Dimaleate was checked for possible color interference before the study was started. Some non-colored test items may change into colored items in aqueous conditions and thus stain the tissues during the exposure. To assess the color interference, 50 µL of Bis-Aminopropyl Diglycol Dimaleate or 50 µL sterile Milli-Q water as a negative control was added to 1.0 mL Milli-Q water. The mixture was incubated for at least 1 hour at 37.0 ± 1.0°C in the dark. Furthermore, 50 µL of Bis-Aminopropyl Diglycol Dimaleate or 50 µL sterile Milli-Q water as a negative control was added to 2.0 mL isopropanol. The mixture was incubated for 2 - 3 hours at room temperature with gentle shaking.
At the end of the exposure time, the absorbance of the solutions was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader. Centrifugation was not considered necessary. If after subtraction of the negative control, the OD for the test item solution is >0.08, the test item is considered as possibly interacting with the MTT measurement.

Test for Reduction of MTT by the Test Item
Bis-Aminopropyl Diglycol Dimaleate was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, 50 µL of Bis-Aminopropyl Diglycol Dimaleate was added to 1 mL MTT solution (1 mg/mL MTT in phosphate buffered saline). The mixture was incubated for approximately 3 hours at 37.0 ± 1.0°C in the dark. A negative control, 50 µL sterile Milli-Q water was tested concurrently. If the MTT solution color turned blue / purple or if a blue / purple precipitate was observed the test item interacts with MTT. Only test items which bind to the tissue after rinsing can interact with MTT in the main assay.

Test System Set Up
On the day of receipt the tissues were equilibrated (in its 24-well shipping container) to room temperature. Subsequently, tissues were transferred to 6-well plates and incubated for 20 ± 4 hours at 37°C in 1.0 mL fresh pre-warmed Assay Medium.
DMEM (Dulbecco’s Modified Eagle’s Medium)
Supplemented DMEM medium, serum-free supplied by MatTek Corporation.
MTT medium
MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent.

Environmental conditions
All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 78 - 86%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.8 - 37.0°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Test Item Preparation
No correction was made for the purity/composition of the test item.
The liquid test item was applied undiluted (50 µL) directly on top of the tissue.
Any residual volumes were discarded.

Application/Treatment of the Test Item
The test was performed on a total of 2 tissues per test item together with a negative control and positive control.
Before the assay was started the entire tissues were pre-wetted with 20 μL of Ca2+Mg2+-Free-DPBS. The tissues were incubated at standard culture conditions for 30 ± 2 minutes.
Two tissues were treated with 50 µL Milli-Q water (negative control) and 2 tissues with 50 µL Methyl Acetate (positive control) respectively.
Fifty µL of the undiluted test item was added into the 6-well plates on top of the tissues.
After the exposure period with Bis-Aminopropyl Diglycol Dimaleate (30 ± 2 minutes at 37.0 ± 1.0°C), the tissues were thoroughly rinsed with Ca2+Mg2+-free D-PBS (brought to room temperature) to remove residual test item.
After rinsing the cell culture inserts were each dried carefully and immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre-labeled 12-well plate for a 12 ± 2 minute immersion incubation at room temperature (Post-Soak). After the Post-Soak period cell culture inserts were each dried carefully and transferred to the 6-well plate containing 1.0 mL of warm Assay Medium and were incubated for 120 ± 15 minutes at 37°C.

Cell Viability Measurement
After incubation, cell culture inserts were dried carefully to remove excess medium. The cell culture inserts were transferred into a 24-wells plate prefilled with 0.3 mL MTT-medium (1.0 mg/mL). The tissues were incubated for 180 ± 10 minutes at 37°C.
After incubation with MTT-medium the tissues were placed on blotting paper to dry the tissues and then transferred to a pre-labelled 24-well plate containing 2.0 mL of isopropanol in each designated well so that isopropanol is flowing into the insert on the tissue surface. Formazan was extracted with 2 mL isopropanol refrigerated for 18 ± 2 hours in the dark. At the end of the extraction period, the liquid within each insert was decanted/pipetted into the well from which it was taken.
The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Eye hazard potential of the test item was classified according to remaining cell viability following exposure of the test item.

ACCEPTABILITY CRITERIA
The in vitro eye irritation test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be > 0.8 and < 2.5.
b) The mean relative tissue viability of the positive control should be <50% relative to the negative control.
c) The difference between the % tissue viabilities of the two identically treated replicates should be <20.
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

INTERPRETATION
The test chemical is identified as not requiring classification and labelling according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60%. In this case no further testing in other test methods is required.
The test chemical is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and postexposure incubation is less than or equal (≤) to 60%.

ANALYSIS
Calculation of Cell Viability
Optical Density readings were transferred into Microsoft Excel to allow further calculations to be performed.
The corrected OD (ODc) for each sample or control was calculated by subtracting the value of the blank mean (ODbl) from each reading (ODraw).

ODc = ODraw – ODbl

The OD value representing 100% cell viability is the average OD of the negative controls (ODlt_u+MTT).
The %Viability for each sample and positive control is calculated as follows:
%Viability = (ODc/mean ODlt_u+MTT) * 100

Results and discussion

In vitro

Results
Irritation parameter:
other: Optical density score
Run / experiment:
OD570
Value:
ca. 1.308
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
RESULTS

Interference of the Test Item with the MTT Endpoint
The test item was checked for color interference in aqueous conditions. Addition of the test item to Milli-Q and isopropanol resulted after subtraction of the blank in an OD of -0.0012 and 0.0005, respectively. Therefore it was concluded that the test item did not induce color interference.
In addition, because no color change was observed in the presence of MTT it was concluded that Bis-Aminopropyl Diglycol Dimaleate did not interact with the MTT endpoint.

Main Assay
The mean absorption at 570 nm measured after treatment with the test item and controls are presented in "Any other information" section.
The individual OD570 measurements are presented in "Any other information" section.
The mean tissue viability obtained ("Any other information" section) after 30 ± 2 minutes treatment with the test item compared to the negative control tissues. Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 30 ± 2 minutes treatment the test item compared to the negative control tissues was 81%. Since the mean relative tissue viability for the test item was above 60% Bis-Aminopropyl Diglycol Dimaleate is considered to be non-irritant.
The positive control had a mean cell viability after 30 ± 2 minutes exposure of 33%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range ("Any other information" section). The difference between the percentages of viability of two tissues treated identically was less than 8%, indicating that the test system functioned properly.

Any other information on results incl. tables

Table1          
Mean Absorption in the EpiOcular™ Test with Bis-Aminopropyl Diglycol Dimaleate

 

A

(OD570)

B

(OD570)

Mean

(OD570)

 

SD

Negative control

1.674

1.552

1.613

±

0.086

Bis-Aminopropyl Diglycol Dimaleate

1.285

1.331

1.308

±

0.033

Positive control

0.594

0.483

0.539

±

0.078

OD = optical density

SD = Standard deviation

Duplicate exposures are indicated by A and B.

In this table the values are corrected for background absorption (0.041). Isopropanol was used to measure the background absorption.

 

Table2          
Mean Tissue Viability in the EpiOcular™ Test with Bis-Aminopropyl Diglycol Dimaleate

 

Mean tissue viability (percentage of control)

Difference between two tissues
 (percentage)

Negative control

100

7.6

Bis-Aminopropyl Diglycol Dimaleate

81

2.9

Positive control

33

0.5


  Individual OD Measurements at 570 Nm

 

A

(OD570)

B

(OD570)

Negative control

 

 

OD570 measurement 1

1.6411

1.5925

OD570 measurement 2

1.7893

1.5936

Bis-Aminopropyl Diglycol Dimaleate

 

 

OD570 measurement 1

1.2703

1.3803

OD570 measurement 2

1.3820

1.3645

Positive control

 

 

OD570 measurement 1

0.6109

0.5155

OD570 measurement 2

0.6595

0.5338

OD = Optical density

Duplicate exposures are indicated by A and B.


 

Historical Control Data for EpiOcular™ Studies

Liquids

 

Negative control

(absorption; OD570)

Positive control

(absorption; OD570)

Positive control

(viability; %)

Range

1.077 – 1.805

0.029 – 0.793

2.11 – 48.25

Mean

1.52

0.42

26.86

SD

0.21

0.23

14.11

n

16

16

16

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of January 2013 to May 2017.

Applicant's summary and conclusion

Conclusions:
Bis-Aminopropyl Diglycol Dimaleate, the test substance, was found to be non-irritant in the EpiOcular™ test under the experimental conditions described in this report (OECD 492).
Executive summary:

The objective of this study was to evaluate the eye hazard potential of Bis-Aminopropyl Diglycol Dimaleate. For this purpose Bis-Aminopropyl Diglycol Dimaleate was topically applied on the Reconstructed Human EpiOcular™ Model.

The possible eye hazard potential of Bis-Aminopropyl Diglycol Dimaleate was tested through topical application for 30 minutes.

The study procedures described in this report were based on the most recent OECD guideline.

The test item was a clear to transparent yellow liquid. The test item was applied undiluted (50 µL) directly on top of the tissue for 30±2 minutes. 

After exposure the cornea epithelial construct was thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 2 hours at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect.

The positive control had a mean cell viability of 33% after 30±2 minutes exposure. The absolute mean OD570(optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The difference between the percentages of viability of two tissues treated identically was less than 8%, indicating that the test system functioned properly.

Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 30±2 minutes treatment with the test item compared to the negative control tissues was 81%. Since the mean relative tissue viability for the test item was above 60% after 30±2 minutes treatment the test item is considered to be non-irritant.

In conclusion, Bis-Aminopropyl Diglycol Dimaleate is non-irritant in the EpiOcular™ test under the experimental conditions described in this report.