Glycerides, tall-oil mono-

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23. Jan. 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

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)

Version / remarks:

OECD Guideline for the Testing of Chemicals, Method No. 437, adopted 09. Oct. 2017: “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

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EU) 2017/735 amending Regulation (EC) No. 440/2008, EU Method B.47 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, adopted 14. Feb. 2017

Deviations:

no

Principles of method if other than guideline:

OECD Guideline for the Testing of Chemicals, Series on Testing and Assessment No. 160: “GUIDANCE DOCUMENT ON “THE BOVINE CORNEAL OPACITY AND PERMEABILITY (BCOP) AND ISOLATED CHICKEN EYE (ICE) TEST METHODS: COLLECTION OF TISSUES FOR HISTOLOGICAL EVALUATION AND COLLECTION OF DATA ON NON-SEVERE IRRITANTS”; 25. Oct. 2011

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Glycerides, tall-oil mono-

EC Number:

263-034-7

EC Name:

Glycerides, tall-oil mono-

Cas Number:

61789-12-6

Molecular formula:

C19H38O4 to C21H42O4 * (mono-glycerides) # C37H66O5 to C39H76O4 * (di-glycerides) # C55H96O6 to C57H108O4 * (tri-glycerides) # * in H2 increments (i,e, decreasing # of double bonds)

IUPAC Name:

Glycerides, tall-oil mono-

Test material form:

liquid

Details on test material:

Name: LUMULSE GMT-K
Batch no.: 385932
Appearance: opaque amber liquid
Purity: 100%
Homogeneity: homogeneous
Expiry date: 26. Oct. 2019
Storage: Room Temperature: (20 ± 5°C)

Specific details on test material used for the study:

No further details specified in the study report.

Test animals / tissue source

Species:

cattle

Strain:

other: Bos primigenius Taurus

Details on test animals or tissues and environmental conditions:

Species: Bos primigenius Taurus (fresh bovine corneas)
Origin: Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany, on the day of the test. The cattle were between 12 and 60 months old. The eyes were transported to the test facility in Hanks’ Balanced Salt Solution with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 μg/mL) in a suitable cooled container within 1 hour.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

750 μL negative control solution, test item and positive control were applied to each replicate.

Duration of treatment / exposure:

Exposure time of the test item on the corneas was 10 minutes at 32 ± 1 °C.

Duration of post- treatment incubation (in vitro):

2 hours at 32 ± 1 °C (post-incubation).

Number of animals or in vitro replicates:

For each treatment group (negative control solution, test item and positive control), three replicates were used.

Details on study design:

Preparations
After having carefully cleaned and sterilised the cornea holders, they were kept in the incubation chamber at 32 ± 1 °C.
On the day of the assay, the MEM without phenol red was supplemented with sodium bicarbonate, L-glutamine and 1% fetal calf serum (= complete MEM) and stored in a water bath at 32 ± 1 °C.
The same was performed with the MEM with phenol red, but without addition of sodium bicarbonate.
After the arrival of the corneas, they were examined and only corneas which were free from damages were used. The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM (32 ± 1 °C) without phenol red was filled. The holders were then incubated for 1 hour in the incubation chamber at 32 ± 1 °C.

Experimental Parameters
Date of treatment: 23. Jan. 2018
Incubation time: 10 min.
Negative control: HBSS
Positive control: Dimethylformamide (undiluted)

Method Description
After the initial incubation, the medium was changed and the baseline opacity for each cornea was recorded. None of the corneas showed tissue damage; therefore, all corneas were used. For each treatment group (negative control solution, test item and positive control), three replicates were used. After removal of the pre-incubation medium (cMEM without phenol red), 750 μL negative control solution, test item and positive control were applied to each replicate.
According to the characteristics of the test item, the following treatment procedure was performed:

Closed Chamber Method
The “closed chamber method” is used for liquid substances.
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 on the cornea. The test item was given on the epithelium in such a manner that as much as possible of the cornea was covered with the test item.
Exposure time of the test item on the corneas was 10 minutes at 32 ± 1 °C. After thorough 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 hours at 32 ± 1 °C (post-incubation).
After post-incubation time, the cMEM without phenol red was renewed in both chambers.
Then, the final opacity value of each cornea was recorded. 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.

Permeability Test
After the recording of the final opacity value, the cMEM without phenol red was removed from the front chamber, and 1 mL sodium fluorescein solution was added to the front chamber for the detection of permeability of the corneas.
For the closed chamber method, a sodium fluorescein solution with a concentration of 4 mg/mL was used.
The chambers were then closed again and incubated for 90 minutes at 32 ± 1 °C. After incubation, the content of the posterior chamber was thoroughly mixed. Then, its optical density at 492 nm was measured with the microtiter plate photometer.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean IVIS

Value:

1.35

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

In the negative control, no signs of eye irritation were observed.
The positive control induced serious eye damage, which would be classified as GHS category I.
The test item LUMULSE GMT-K showed no effects on the cornea of the bovine eye. The calculated IVIS is 1.35.
The experiment is considered as sufficient for the classification of the test item, because all three replicates of the test item lead to the same assessment for the test item.

Any other information on results incl. tables

Opacity and Permeability Values

The illuminance (unit: LUX) values which were measured before and after exposure are given in the following table:

Illuminance Values

Parameter	Negative Control			Test Item			Positive Control
	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.
(I) Measured values before exposure	983	990	990	1008	998	957	956	986	977
(I) Measured values after exposure	957	968	962	975	912	913	268	286	362

Rep. = Replicate

 

The values in the following tables present the calculated opacity values, according to evaluation:

Opacity Values negative Control

Parameter	Negative Control
	1. Rep.	2. Rep.	3. Rep.
Opacity before exposure	3.82	3.51	3.51
Opacity after exposure	4.99	4.49	4.76
Opacity Difference	1.17	0.98	1.25
Mean Opacity Difference	1.13

Rep. = Replicate

 

Opacity Values Test Item and Positive Control

Parameter	Test item			Positive Control
	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.
Opacity before exposure	2.74	3.60	4.99	5.04	3.68	4.08
Opacity after exposure	4.17	7.18	7.13	119.16	109.18	77.98
Opacity Difference	1.43	3.58	2.14	114.12	105.50	73.90
Opacity Difference corrected	0.29	2.45	1.01	112.99	104.36	72.77
Mean Opacity Difference corrected	1.25			96.71

Rep. = Replicate

 

For the permeability measurement, three replicates for each treatment group were measured three times. cMEM without phenol red was measured as blank value as well. The optical density values at 492 nm are given in the following tables:

Optical density at 492 nm of Blank

Parameter	cMEM without phenol red
1. Measurement	0.036
2. Measurement	0.038
3. Measurement	0.036
Mean	0.037

 

Optical density at 492 nm of Negative Control, Test Item and Positive Control

Parameter	Negative Control			Test Item			Positive Control
	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.
1. Measurement	0.061	0.050	0.036	0.037	0.043	0.086	1.367	0.630	0.852
2. Measurement	0.061	0.048	0.031	0.037	0.040	0.084	1.411	0.647	0.881
3. Measurement	0.057	0.051	0.036	0.038	0.040	0.085	1.390	0.648	0.871
1. Measurement – blank	0.0243	0.0133	- 0.0007	0.0003	0.0063	0.0493	1.3303	0.5933	0.8153
2. Measurement – blank	0.0243	0.0113	- 0.0057	0.0003	0.0033	0.0473	1.3743	0.6103	0.8443
3. Measurement – blank	0.0203	0.0143	- 0.0007	0.0013	0.0033	0.0483	1.3533	0.6113	0.8343
Mean of each replicate	0.0230	0.0130	- 0.0023	0.0007	0.0043	0.0483	1.3527	0.6050	0.8313
Mean of the 3 replicates	0.0112			--			--
Corrected	--	--	--	-0.0106	-0.0069	0.0371	1.3414	0.5938	0.8201
Corrected mean of the 3 replicates	--			0.0066			0.9484

Rep. = Replicates

 

IVIS Values

The calculated IVIS for each replicate and the corresponding means are presented in the following table:

IVIS

Test Group	IVIS	Mean IVIS	Relative Standard Deviation IVIS
Negative Control HBSS	1.52	1.30	14.60%
	1.17
	1.21
Test Item LUMULSE GMT-K	0.14	1.35	83.16%
	2.35
	1.56
Positive Control DMF undiluted	133.11	110.48	21.85%
	113.27
	85.07

Note: The high relative standard deviation of the IVIS of test item is due to mathematical reasons, as the respective means are very small.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

This in vitro study was performed to assess corneal damage potential of LUMULSE GMTK by quantitative measurements of changes in opacity and permeability in a bovine cornea.
The test item LUMULSE GMT-K was applied onto the cornea of a bovine eye which previously had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been determined. The test item was incubated on the cornea for 10 minutes at 32 ± 1 °C. After removal of the test item and 2 hours post-incubation, opacity and permeability values were measured.
The test item was tested neat.
Under the conditions of this test, the test item LUMULSE GMT-K showed no effects on the cornea of the bovine eye. The calculated IVIS is 1.35.
According to OECD Guideline no. 437 (Oct. 2017), a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.
The negative control (HBSS) and the positive control (undiluted dimethylformamide) have met the validity criteria.
No observations were made which might cause doubts concerning the validity of the study outcome. The test is considered valid.

Executive summary:

Title of Study: Evaluation of LUMULSE GMT-K in the Bovine Corneal Opacity and Permeability (BCOP) Test Method following OECD Guideline 437 resp. EU Method B.47

 

Findings and Results:

One valid experiment was performed.

Bovine corneas were used. They were collected from slaughtered cattle that were between 12 and 60 months old.

The test item LUMULSE GMT-K was applied onto the cornea of a bovine eye which had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been measured.

The test item was incubated on the cornea for 10 minutes at 32 ± 1 °C. After removal of the test item and 2 hours post-incubation, opacity and permeability values were measured.

Hank’s Balanced Salt Solution (HBSS) was used as negative control. The negative control showed no irritating effect on the cornea and the calculated IVIS is 1.30.

Dimethylformamide (DMF) undiluted was used as positive control. The positive control induced serious eye damage on the cornea and was within two standard deviations of the current historical mean. The calculated IVIS is 110.48.

Under the conditions of this study, the test item LUMULSE GMT-K showed no effects on the cornea of the bovine eye. The calculated IVIS is 1.35.

According to OECD Guideline No. 437 (Oct. 2017), a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.