Fatty acids, tall-oil, reaction products with linseed oil, maleic anhydride and methanol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Guideline study under GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

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:

yes

Remarks:

minor. The test item was stored at 25.9 deg C for 8.5 h, rather than at 20 deg C as stated in the protocol. Also,the measurement of opacity was done in a photometer rather than an opacitometer, but an appropriate calculation can be made for correction.

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Addition product of maleic anhydride, tall oil fatty acids, linseed oil and methanol

Molecular formula:

not available for the UVCB substance

IUPAC Name:

Addition product of maleic anhydride, tall oil fatty acids, linseed oil and methanol

Test material form:

liquid

Details on test material:

Name ZWA 5496/100
Batch no. 210150084
Appearance brown liquid
Composition Reaction products of maleic anhydride (MA), disproportionated tall oil fatty acid, linseed oil andmethanol
Molecular weight 380-1400 g/mol
Purity 100% (UVCB

Test animals / tissue source

Species:

cattle

Strain:

other: Bos primagenius Taurus

Details on test animals or tissues and environmental conditions:

Bovine corneas were used from previously slaughtered cattle which were between ages 12-60 months. SOURCE OF COLLECTED EYES
- Source:
- Number of animals:
- Characteristics of donor animals (e.g. age, sex, weight): age 12-60 months, M/F,
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):
- Time interval prior to initiating testing:
- indication of any existing defects or lesions in ocular tissue samples: eyes checked for defects as part of the selection criteria
- Indication of any antibiotics used:
TEST ANIMALS
- Source: Muller Fleisch GmbH,Birkenfeld, Germany. Isolated fresh.
- Age at study initiation:12-60 months
- Weight at study initiation: No data


ENVIRONMENTAL CONDITIONS
- Temperature (°C): After receipt, incubated 1 h at 32 deg C in Hank's Balanced Salt Solution in exposure chambers. Vessels were sterilized glass or sterilizable plastic.
- Humidity (%):
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light):

IN-LIFE DATES: 2 Sept. 2015

Test system

Vehicle:

other: Minimal Essential Medium (MEM) without penol but supplemented with sodium bicarbinate, L-glutamine and 10% fetal calf serum (FCS).

Controls:

yes, concurrent positive control

yes, concurrent negative control

no

Amount / concentration applied:

750 μL

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

2 hours

Number of animals or in vitro replicates:

Bovine eyes ex vivo were used in the study.

Details on study design:

Light transmission was recorded through each cornea in a spectrophotomer at 570 nm, prior to exposure. This comprised the baseline opacity.

Three replicate measurements were obtained.

The closed chamber method was used. The respective substance (negative control solution, test item or positive control) was applied by pipetting 750 μL of the appropriate liquid through the refill hole in the holder of the cornea. The test item was applied to the epithelium in such a manner that as much as possible of the cornea was covered with the test item.

Exposure time was 10 min at 32 ± 1 °C. After rinsing with cMEM with phenol red and final rinsing with cMEM without phenol red, the anterior chamber was filled with cMEM without phenol red, and the corneas were stored for additional 2 h at 32 ± 1 °C (post-incubation). The cMEM without phenol red was renewed in both chambers of the apparatus. Then, the final opacity value of each cornea was recorded (again by measurement at 570 nm).

The cMEM without phenol red was removed from the front chamber, and 1 mL sodium fluorescein solution (concentration: 4 mg/mL) was added to the front chamber. The chambers were then closed and incubated for 90 min at 32 ± 1 °C. After incubation, the content of the posterior chamber was thoroughly mixed. Then, the permeability of the cornea was measured by reading the optical density at 490 nm of the liquid in the posterior chamber.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

1.83

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Mean opacity difference of the negative control is 0.1220.
For the test item, the calculated IVIS (In vitro irritancy scoe) is 1.83. The experiment is considered as sufficient for the classification of the test item because two of the three replicates of the test item lead to the same assessment for the test item.

Any other information on results incl. tables

Optical Density of the test substance:

Replicate	Negative Control			Test Item ZWA 5496/100			Positive Control
Measured values	-0.0004	0.0007	0.0031	0.0060	0.0413	0.0240	0.1997	0.2629	0.2637
*Corrected values	-0.0020	0.0035	0.0155	0.0300	0.2065	0.1200	0.9985	1.3145	1.3185
Mean	0.0057

Absorbance and Opacity Values of the Negative Control:

Parameter	Negative Control
Absorbance before exposition	0.1888	0.1907	0.1792
Absorbance after exposition	0.2395	0.2033	0.2148
Opacity before exposition	1.5445	1.5513	1.5108
Opacity after exposition	1.7358	1.5970	1.6398
Opacity Difference	0.1913	0.0457	0.1291

Absorbance and Opacity Values for the Test Item and the Positive Control:

Parameter	Test Item ZWA 5496/100			Positive Control
Absorbance before exposition	0.1297	0.2813	0.1462	0.1425	0.1296	0.1766
Absorbance af- ter exposition	0.1560	0.3495	0.2432	1.9991	1.8398	1.9957
Opacity before exposition	1.3480	1.9112	1.4002	1.3884	1.3477	1.5018
Opacity after exposition	1.4322	2.2361	1.7507	99.7930	69.1512	99.0148
Opacity Difference	0.0842	0.3250	0.3504	98.4046	67.8035	97.5130

In Vitro Irritancy Scores:

Test Group	IVIS	Mean IVIS	Relative Standard Deviation IVIS
Negative Control 0.9% NaCl	0.16	0.21	66.4%
	0.10
	0.36
Test Item ZWA5496/100	0.33	1.83	79.2%
	3.22
	1.94
Positive Control DMF undiluted	113.18	105.86	15.3%
	87.31
	117.08

Note: the high relative standard deviations of the IVIS of test item and negative control are due to mathematical reasons, as the respective means are very small.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The calculated IVIS (in vitro irritancy score) of the undiluted test substance in the BCOP is 1.83. The criteria for a non-irritant (IVIS of 3 or less) is met and the substance is evaluated as not irritating to the eye.

Executive summary:

less