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Diss Factsheets

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 January 2018 - 20 January 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
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
2,6-dimethyloct-7-en-2-yl formate
EC Number:
246-788-1
EC Name:
2,6-dimethyloct-7-en-2-yl formate
Cas Number:
25279-09-8
Molecular formula:
C11H20O2
IUPAC Name:
2,6-dimethyloct-7-en-2-ol
Test material form:
liquid

Test animals / tissue source

Species:
other: Cow
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Deonar Abattoir slaughter house, Mumbai, Maharashtra
- Characteristics of donor animals (e.g. age, sex, weight): Between 1 to 5 years
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Transported (in a sealed plastic container) under cold condition in Hanks’ Balanced Salt Solution containing antibiotics [e.g., penicillin at 100 IU/mL and streptomycin at 100 μg/mL]
- Time interval prior to initiating testing: The eyes were used within 24 hours from slaughter
- indication of any existing defects or lesions in ocular tissue samples: Corneas from eyes free of visible defects were used.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL

Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
2 h
Number of animals or in vitro replicates:
3 replicates
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
Eyes were examined prior to use. Corneas from eyes free of visible defects were used. Corneas that have opacity lesser than seven opacity units or equivalent for the opacitometer were used in the study.

QUALITY CHECK OF THE ISOLATED CORNEAS

NUMBER OF REPLICATES : 3

NEGATIVE CONTROL USED : Yes (nomral saline)

POSITIVE CONTROL USED : Yes (dimethylformamide)

APPLICATION DOSE AND EXPOSURE TIME : 750 µL of test item; corneas were exposed for approx. 10 min.

TREATMENT METHOD: Open chamber
A volume of 750 μL was introduced into the anterior chamber through the dosing holes on the top surface of the chamber. The anterior compartment was then plugged. Post application of the test item the holders were turned to a horizontal position and slightly rotated to ensure uniform covering of the test item over the cornea.

POST-INCUBATION PERIOD: yes (2 h)
Once the medium was free of test item, the corneas were given a final rinse with EMEM (without phenol red). Media in the anterior and the posterior chamber was removed and fresh EMEM (without phenol red) was filled. The compartments were plugged and post treatment opacity of each cornea was recorded. Once the medium was free of test item, the corneas were given a final rinse with EMEM (without phenol red). Anterior chamber was then refilled with fresh EMEM without phenol red. After rinsing, the corneas were incubated for an additional period of approximately 2 hours ± 10 minutes at 32 ± 1ºC. At the end of post-exposure incubation period the media in the anterior and the posterior chamber were removed and fresh EMEM (without phenol red) was filled. The compartments were plugged and post treatment opacity of each cornea recorded.

After opacity measurement, the medium was removed from the anterior chamber and was filled with 1 mL of 4 mg/mL fluorescein solution whilst the posterior chamber was filled with fresh EMEM (without phenol red). The holders were then incubated in a horizontal position for approximately 90 ± 5 min at 32 ± 1 ºC. After the incubation the medium in the posterior chamber was transferred into labelled tubes. An aliquot of the medium from the posterior chamber was transferred to a 96-well plate. The plate reader was set to read at OD490. A reading of EMEM (without phenol red) was also recorded which served as blank.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Once, until no visual evidence of test item.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacitometer
- Corneal permeability: passage of sodium fluorescein dye (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
> 55 Category 1

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Run / experiment:
1
Value:
0.34
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
2
Value:
1.31
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
3
Value:
0.96
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The mean IVIS score for the corneas treated with test item was found to be 0.87.

OTHER EFFECTS:
- Visible damage on test system: No.

DEMONSTRATION OF TECHNICAL PROFICIENCY: JRF Study nº 530-01-01-10123 (2015)

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes (mean IVIS = -0.49)
- Acceptance criteria met for positive control: Yes (mean IVIS = 160.97)
- Range of historical values if different from the ones specified in the test guideline:
Dimethylformide (positive control): Mean ± SD = 120.37 ± 30.51 (min 58.94, max 165.18)

Any other information on results incl. tables

Corneal Opacity

The mean final opacity values for dihydromyrcenyl formate treated eyes (0.68) did not show any observable increase in comparison to the control group (-0.78). An observable marked increase in final mean opacity was observed in the corneas treated with N,N-dimethylformamide (124.96).

Corneal Permeability

The mean final corneal permeability values for dihydromyrcenyl formate treated eyes (0.012) did not show any observable increase in comparison to the control group (0.019). An observable marked increase in mean final corneal permeability was observed in the corneas treated with N,N- dimethylformamide (2.400).

In vitro Irritancy Score (IVIS)

The mean In-Vitro Irritancy Score (IVIS) of normal saline (control) and N,N-dimethylformamide (positive control) treated corneas were found to be -0.49 and 160.97 which confirmed the reliability of the test procedure.

The mean IVIS score for the corneas treated with dihydromyrcenyl formate was found to be 0.87.

Dihydromyrcenyl Formate (0.75 mL)

Cornea holder N°

Io

 (LUX)

I (Initial)

(LUX)

Initial Opacity Value

I

(Post

Treatment)

(LUX)

Post Treatment Opacity Value

Corr. Opacity Value

Final Opacity Value

OD490

Value

Corr. OD490Value

Final OD490

Value

IVIS

20

1083

1019

2.92

1029

2.51

-0.41

0.37

0.065

0.017

-0.002

0.34

21

1103

1011

4.05

1011

4.05

0.00

0.78

0.102

0.054

0.035

1.31

22

1112

1061

2.34

1058

2.46

0.12

0.90

0.071

0.023

0.004

0.96

Mean

0.68

-

0.031

0.012

0.87

SD

0.28

-

0.020

0.020

0.49

Normal Saline (0.75 mL)

Cornea Holder N°

Io

 (LUX)

I (Initial)

(LUX)

Initial Opacity Value

I

(Post Treatment)

(LUX)

Post Treatment Opacity

Value

Corr. Opacity

Value

OD490

Value

Corr. OD490

Value

IVIS

13

1112

1054

2.61

1053

2.65

0.04

0.060

0.012

0.22

14

1067

961

4.82

1021

2.22

-2.60

0.091

0.043

-1.96

15

1052

990

2.92

985

3.13

0.21

0.051

0.003

0.26

Mean

-0.78

-

0.019

-0.49

SD

1.58

-

0.021

1.27

N,N-Dimethylformamide (0.75 mL)

Cornea holder N°

Io

 (LUX)

I (Initial)

(LUX)

Initial Opacity Value

I

(Post

Treatment)

(LUX)

Post Treatment Opacity

Value

Corr. Opacity

Value

Final Opacity

Value

OD490

Value

Corr. OD490

Value

Final OD490

Value

IVIS

17

1088

1016

3.25

233

146.62

143.37

144.15

1.908

1.860

1.841

171.77

18

1094

969

5.56

287

112.45

106.89

107.67

3.385

3.337

3.318

157.44

19

1103

1024

3.50

266

125.79

122.29

123.07

2.108

2.060

2.041

153.69

Mean

124.96

-

2.419

2.400

160.97

SD

18.31

-

0.801

0.801

9.54

Keys: IVIS = In Vitro Irritation Score, Io =Baseline Reading (With medium but without cornea), I = LUX Reading with Medium and Cornea, OD490= Optical Density at 490 Wave Length, - = Not Applicable, Corr. = Corrected. Blank OD490value = 0.048.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was determined to be not irritating to the eye.
Executive summary:

An in-vitro eye irritation bovine corneal opacity and permeability test was performed according to OECD Guideline 437 (GLP study). Three sets, each consisting of three corneas were tested. The first set served as control and was treated with 750 μL normal saline. The second set served as positive control and was treated with 750 μL dimethylformamide with the third set treated with 750 μL test item. Post application the corneas were incubated for approximately 10 minutes after which the test item was washed-off the cornea and the corneas kept in incubation for approximately 2 h at 32 ± 1 ºC. At the end of the incubation period opacity readings were taken. Post opacity permeability reading was measured by applying 1 mL of fluorescein sodium solution (4 mg/mL) on to the anterior surface of the cornea and was incubated for approximately 90 min at 32 ºC. At the end of the incubation period the Optical Density (OD) was measured at 490 nm for the fluid collected from the posterior chamber. The mean In-Vitro Irritancy Score (IVIS) of normal saline (control) and dimethylformamide (positive control) treated corneas were found to be -0.49 and 160.97 which confirmed the reliability of the test procedure. The IVIS score for the corneas treated with test item was found to be 0.87 (No category according to OECD 437). Thus, the test item was determined to be not irritating to the eye.