Fatty acids, tallow, compds. with triethanolamine

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 January 2016 to 29 January 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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:

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

Fatty acids, tallow, compds. with triethanolamine

EC Number:

263-158-1

EC Name:

Fatty acids, tallow, compds. with triethanolamine

Cas Number:

61790-67-8

IUPAC Name:

Fatty acids, tallow, esters of triethanolamine

Test material form:

liquid

Details on test material:

Appearance: Brown liquid
Storage: At ambient temperature, protected from light

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Eyes from adult cattle obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter.
- Characteristics of donor animals: Typically 12 to 60 months old.
- Storage, temperature and transport conditions of ocular tissue: They were transported to the test facility over ice packs on the same day of slaughter.
- Time interval prior to initiating testing: The corneas were refrigerated on arrival and used within 24 hours of receipt.
- indication of any existing defects or lesions in ocular tissue samples: All eyes were macroscopically examined before and after dissection. Only corneas free from damage were used.
- Indication of any antibiotics used: The eyes were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with penicillin at 100 IU/mL and streptomycin at 100 µg/mL.

Test system

Vehicle:

unchanged (no vehicle)

Remarks:

The test material was used as supplied.

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.75 mL

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

Three corneas with opacity values close to the median value of all corneas were allocated to the negative control. Three corneas were also allocated to the test material and three corneas to the positive control.

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 65 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

QUALITY CHECK OF THE ISOLATED CORNEAS
The medium from both chambers of each holder was replaced with fresh complete EMEM. A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated.
The condition of the cornea was visually assessed post treatment and post incubation.

NUMBER OF REPLICATES
Three corneas with opacity values close to the median value of all corneas were allocated to the negative control. Three corneas were also allocated to the test material and three corneas to the positive control material.

NEGATIVE CONTROL USED
0.75 mL 0.9 % w/v sodium chloride solution

POSITIVE CONTROL USED
0.75 mL ethanol

APPLICATION DOSE AND EXPOSURE TIME
The undiluted test material was applied for 10 minutes followed by an incubation period of 120 minutes.

TREATMENT METHOD:
The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test material or control materials were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the material over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 10 minutes.
At the end of the exposure period the test material and control materials were removed from the anterior chamber and the cornea was rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM. The anterior chamber was refilled with fresh complete EMEM. A post-treatment opacity reading was taken and each cornea was visually observed.
The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes.
After incubation the holders were removed from the incubator, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.
- Corneal permeability: Following the final opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes. After incubation the medium in the posterior chamber of each holder was decanted and retained.
360 µL of medium representing each cornea was applied to a designated well on a 96 well plate and the optical density at 492 nm (OD492) was measured. Passage of sodium fluorescein dye measured with the aid of the Anthos 2001 microtiter plate reader (OD492).
The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.
- Histopathology: The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre labelled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10 % neutral buffered formalin.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The following formula was used to determine the In Vitro Irritancy Score:

In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)

Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.

DECISION CRITERIA:
For an acceptable test the following positive control criterion should be achieved:
Neat ethanol was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean collated during 2014 for this testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 29.6 to 52.0.

For an acceptable test the following negative control criteria should be achieved:
0.9% w/v sodium chloride solution was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values during 2014 for bovine corneas treated with the respective negative control. When testing liquids the negative control limit for opacity should be =2.9 and for permeability =0.103.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

3.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: The corneas treated with the test material or negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control gave opacity of =2.9 and permeability =0.103. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The positive control In Vitro Irritancy Score was within the range of 29.6 to 52.0. The positive control acceptance criterion was therefore satisfied.

Any other information on results incl. tables

Table 1. In Vitro Irritancy Scores

Treatment	In Vitro Irritancy Score
Test Material	3.5
Negative Control	2.1
Positive Control	39.0

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the conditions of this study, the test material could not be categorised as it had an IVIS score greater than 3 and less than 55, therefore no prediction of eye irritation can be made.

Executive summary:

The eye irritancy potential of the test material was investigated in vitro in a study conducted through The Bovine Corneal Opacity and Permeability (BCOP) Assay in accordance with the standardised guidelines OECD 437 and EU Method B.47 under GLP conditions.

The undiluted test material was applied for 10 minutes followed by an incubation period of 120 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS). 

After test material exposure, the corneas treated with the test material or negative control item were clear post treatment and post incubation and the corneas treated with the positive control item were cloudy post treatment and post incubation. The negative and positive control acceptance criteria were satisfied as the negative control gave an opacity reading of =2.9 and permeability =0.103 and the positive control In Vitro Irritancy Score was within the range of 29.6 to 52.0. The test material gave an opacity reading of 3.3 and permeability of 0.008 (mean corrected values) and had an In Vitro Irritancy Score of 3.5.

Under the conditions of this study, the test material could not be categorised as it had an IVIS score greater than 3 but less than 55, therefore no prediction of eye irritation can be made.