IPEMA

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-03-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

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:

2020-06-26

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

impurity 1

Reference substance name:

3-methylbuten-2-yl methacrylate

EC Number:

286-559-3

EC Name:

3-methylbuten-2-yl methacrylate

Cas Number:

85269-36-9

Molecular formula:

C9H14O2

IUPAC Name:

2-methyl-1-methylenepropyl methacrylate

impurity 2

Reference substance name:

3-methylbut-3-en-1-ol

EC Number:

212-110-8

EC Name:

3-methylbut-3-en-1-ol

Cas Number:

763-32-6

Molecular formula:

C5H10O

IUPAC Name:

3-methylbut-3-en-1-ol

impurity 3

Reference substance name:

Unknown

Molecular formula:

Unknown

IUPAC Name:

Unknown

Constituent 1

Reference substance name:

2-Propenoic acid, 2-methyl-, 3-methyl-3-buten-1-yl ester

Cas Number:

156291-88-2

Molecular formula:

C9H14O2

IUPAC Name:

2-Propenoic acid, 2-methyl-, 3-methyl-3-buten-1-yl ester

Test material form:

liquid

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Abattoir Vion Beef B.V., Buchloe, Germany (isolated corneas obtained as by-product from animals freshly slaughered)
- Storage, temperature and transport conditions of ocular tissue: transported in HBSS containing Pen/Strep on ice to the laboratory.
- Time interval prior to initiating testing: Immediately after arrival of the eyes, cornea preparation was initiated.
- Selection and preparation of corneas: The eyes were carefully examined for defects and any defective eyes were discarded. The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (Duratec GmbH) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI 1640 medium (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI 1640 medium). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 +-1 °C.

Test system

Vehicle:

physiological saline

Remarks:

0.9 % Sodium Chloride Solution (aq)

Controls:

yes

Amount / concentration applied:

750 µg/L of the test substance or the control substance was introduced into the anterior chamber.

Duration of treatment / exposure:

After 10 minutes incubation at 32 ± 1 °C either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI 1640 medium (without phenol red).

Duration of post- treatment incubation (in vitro):

After the illuminance measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI 1640 medium. 1 mL of a 4 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 ± 1 °C.

Number of animals or in vitro replicates:

3 corneas for the test item
3 corneas as negative controls treated with physiological saline 0.9% NaCl
3 corneas as positive control treated with ethanol 100%

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated. The eyes were carefully examined for defects and any defective eyes were discarded. The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (Duratec GmbH) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 ± 1 °C.

QUALITY CHECK OF THE ISOLATED CORNEAS:
Only corneas that had an initial illuminance reading I > I0/1.1651 lux were used for the assay. Eyes that were noted to have defects were discarded and not used on the study.

NUMBER OF REPLICATES: 3
VEHICLE CONTROL USED: yes
POSITIVE CONTROL USED: yes
APPLICATION DOSE AND EXPOSURE TIME: 750 µg/L and 10 minutes
REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: The epithelium was washed at least three times with MEM (containing phenol red). Once the medium was free of test item, the cornea was finally rinsed with complete RPMI (without phenol red).
- POST-EXPOSURE INCUBATION: 2 hours at 32 ± 1 °C; 90 minutes at 32 +- 1°C with 1mL of 4 mg/mL sodium fluorescein solution

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacimeter (BASF-OP3.0, Duratec GmbH)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry
- Others: Pertinent observations
SCORING SYSTEM: In Vitro Irritancy Score (IVIS).
This was detailed in the study report as follows:
= 3 = no category
> 3; = 55 = no stand-alone prediction can be made
>55 = Category 1.
An identification of test substances that should be classified as irritating to eyes (UN GHS Category 2 or Category 2A) or test substances that should be classified as mildly irritating to eyes (UN GHS Category 2B) cannot be made.

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used: Yes.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

0.16

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

The eye irritancy potential of 3-Methyl-3-butenyl 2-methyl-2-propenoate was investigated in the bovine corneal opacity and permeability assay.
The test item was tested as provided by the sponsor.
None of the corneas treated with 3-Methyl-3-butenyl 2-methyl-2-propenoate showed any opacity of the tissue.
The following mean in vitro irritation score was calculated:
0.16
Therefore, the test item was classified into UN GHS No Category.
The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.
The negative control responses should result in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

Any other information on results incl. tables

Opacity

Cornea No.	Test Item	Initial Opacity	Final Opacity	Change of Opacity Value	Corrected Opacity Value
1		2.66	4.52	1.86
2	Negative	3.03	5.41	2.38
3	Control	2.88	3.89	1.02
MV		2.85	4.61	1.75
4		5.20	27.15	21.94	20.19
5	Positive	1.42	26.07	24.65	22.90
6	Control	1.53	21.34	19.81	18.06
MV		2.72	24.85	22.13	20.38
7		2.41	4.88	2.47	0.72
8	Test Item	2.55	5.41	2.86	1.11
9		3.59	4.76	1.17	-0.58
MV		2.85	5.01	2.17	0.41

MV = mean value

 

Permeability

Cornea No.	Test Item	OD490	Corrected OD490 Value
1		0.021
2	Negative	0.028
3	Control	0.036
MV		0.028
4		0.696	0.668
5	Positive	1.173	1.145
6	Control	1.292	1.264
MV		1.054	1.025
7		0.008	-0.020
8	Test Item	0.010	-0.018
9		0.017	-0.011
MV		0.012	-0.017

MV = mean value

 

In Vitro Irritation Score

Cornea No.	Test Item	Corrected Opacity	Corrected OD490 Value	IVIS
1		1.86	0.021
2	Negative	2.38	0.028
3	Control	1.02	0.036
MV		1.75	0.028	2.18
4		20.19	0.668
5	Positive	22.90	1.145
6	Control	18.06	1.264
MV		20.38	1.025	35.76
7		0.72	-0.020
8	Test Item	1.11	-0.018
9		-0.58	-0.011
MV		0.41	-0.017	0.16

MV = mean value

 

Historical Mean In Vitro Irritation Score of the Positive Control

	IVIS Positive Control - Ethanol 100 %
Mean Value (MV)	47.24
Standard Deviation (SD)	8.86
MV- 2xSD	29.52
MV+2xSD	64.95
Number of Replicates providing Historical Mean: 74

Positive controls are updated after every single experiment or at least every 3 months

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

According to the evaluation criteria the test item 3-Methyl-3-butenyl 2-methyl-2-propenoate is classified into UN GHS No Category.

Conclusions:

According to the evaluation criteria the test item 3-Methyl-3-butenyl 2-methyl-2-propenoate is classified into UN GHS No Category.

Executive summary:

The eye irritancy potential of 3-Methyl-3-butenyl 2-methyl-2-propenoate was investigated in the bovine corneal opacity and permeability assay. None of the corneas treated with 3-Methyl-3-butenyl 2-methyl-2-propenoate showed any opacity of the tissue. The mean in vitro irritation score was 0.16. Therefore IPEMA was not classified as eye irritant