Decanoic acid, mixed esters with heptanoic acid, octanoic acid and trimethylolpropane

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 April 2014 to 02 July 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD test guidelines in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Test material

Test material information

Constituent 1

Test material form:

other: liquid

Details on test material:

Identity: H2925 (CAS No, 68130-53-0), Lot# 2013090401Supplied By: Chemtura CorporationDate Received: 18 Mar 2014Storage: Room temperature and humidityDescription: Clear yellow liquid

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

The bovine eyes were received from Spear Products on 10 Apr 2014 and transported to MB Research in Hank's Balanced Salt Solution with Penstrep in a refrigerated container.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

Using the closed chamber method, a volume of 0.75 ml of the ethanol, MEM or liquid test article was applied to the epithelium of each of the three positive controls, three negative controls, or three test article-treated corneas in a manner, which ensured the entire cornea was covered.

Duration of treatment / exposure:

10 minutes

Observation period (in vivo):

2 hours

Number of animals or in vitro replicates:

9 cornea's used in total (3 positive control, 3 negative controls and 3 test article treated)

Details on study design:

Pretest Procedures
Fresh assay solutions were prepared prior to use. MEM solution was prepared by stirring together one jar of MEM powder (sufficient to make one liter of solution), 2.2 g Sodium Bicarbonate, 0.292 g L-Glutamine, 10 ml of Fetal Bovine Serum (FBS) and brought to a final volume of 1000 ml with distilled water. The MEM solution was kept in a 32°C (± 1°C) incubator for the duration of testing. Hanks Balanced Salt Solution (HBSS) was prepared by stirring together one jar of HBSS powder (sufficient to make one liter), 0.35 g Sodium Bicarbonate and brought to a final volume of 1000 ml with distilled water. HBSS was maintained at room temperature.
In addition, MEM solution with Phenol Red was prepared by stirring together 9.3 g MEM with Phenol Red (sufficient to make one liter), 2.2 g Sodium Bicarbonate, 0.292 g L-Glutamine, 10 mL Fetal bovine Serum and brought to a final volume of 1000 mL with distilled water. The MEM solution with Phenol Red was kept in a 32°C (± 1°C) incubator for the duration of testing.
The eyes were examined prior to use on the day of dosing. Any eye with a cornea exhibiting evidence of vascularization, pigmentation, opacity or scratches was discarded.
Corneas from eyes that were free of defects were dissected from the surrounding tissues. A 2-3 mm rim of sclera was left attached to each cornea. The Corneas were then placed in a container of fresh HBSS.
The dissected Corneas were mounted in specially designed holders that were separated into anterior and posterior chambers and filled separately. Each Cornea was mounted allowing the epithelium of the Cornea to project into the anterior chamber. The posterior chamber was filled with MEM solution ensuring contact with the endothelium. The anterior chamber was filled with MEM solution, ensuring contact with the epithelium. Each Cornea was visually inspected again to ensure there were no defects.
The entire holder was incubated at 32°C (± 1°C) and allowed to equilibrate for at least one hour but not longer than two hours.
Following the equilibration period, a pre-exposure (baseline) determination of opacity was made for each cornea by measuring each against the blank supplied with the opacitometer. Any cornea with a value greater than 7 opacity units was discarded.

Study Procedure
Following the pretest observations, the MEM solution was removed from the anterior chamber. Using the closed chamber method, a volume of 0.75 ml of the ethanol, MEM or liquid test article was applied to the epithelium of each of the three positive controls, three negative controls, or three test article-treated corneas in a manner, which ensured the entire cornea was covered.
All holders and corneas were placed in a horizontal position (anterior side up) in the 32°C (± 1°C) incubator. After 10 (± 1) minutes, the test article, ethanol or MEM solution in the controls were removed from the epithelium of the cornea and anterior chamber of the holder by washing with MEM solution containing phenol red. Following the 1O-minute (±1 minute) exposure, the corneal epithelium was thoroughly rinsed with MEM solution containing phenol red until the test article, ethanol, or MEM solution (in the negative controls) were washed off. A final rinse was made with MEM without phenol red. The anterior and posterior chambers of the holders were refilled with fresh MEM solution. Opacity measurements were made following the 10-minute exposure and MEM solution refill.
All corneas were incubated at 32°C (± 1°G) for an additional two hours at which time the MEM solution in the anterior and posterior chambers was removed and the holders refilled with fresh MEM solution. A measurement of opacity was taken with each treated cornea compared to the blank supplied with the OPKIT. This reading was used in the final IVIS calculations.
The corneas were visually inspected for abnormalities immediately following the 10-minute exposure period, and again at 2 hours post-exposure. There were no abnormalities noted.
Immediately following the two-hour opacity measurement, the MEM solution was removed from the anterior chamber and replaced with 1.0 ml of 0.4% sodium. fluorescein solution in Dulbecco's Phosphate Buffered Saline (PBS). Each holder was returned to the 32°C (±1°C) incubator in a horizontal position (anterior side up) ensuring contact of the fluorescein with the cornea.
After 90 (± 5) minutes, the fluid from the posterior chamber was removed and the amount of dye, which passed through the cornea (permeability), was measured as the optical density at 490 nm by a spectrophotometer. A 1: 1000 dilution of the fluorescein was prepared and measured in the spectrophotometer as a measure of consistency.

Analysis of Data
Individual corrected opacity scores were calculated by subtracting the pretest score from the ten minute and two-hour scores. Corrected mean opacity scores were calculated by averaging the individual two-hour corrected opacity scores for a given dose group and subtracting the mean opacity score for the negative control group. A corrected mean opacity score was not calculated for the negative control rather only the mean of the individual two-hour corrected opacity scores were calculated (with no subtraction of mean opacity score for negative control).
Individual corrected optical densities were calculated by subtracting the mean optical density for the negative control group from the individual optical density values. Individual corrected optical densities were not calculated for the negative control group. Corrected mean optical densities were calculated by averaging the individual corrected optical density values for a given dose group. A corrected mean optical density was not calculated for the negative control, rather only the mean of the individual optical densities.
The In Vitro Irritancy Score (IVIS) for the test article and positive control were calculated by adding the corrected mean opacity score to fifteen times the corrected mean optical density as shown by the equation below. The calculations to obtain an IVIS for the positive control was performed in the same manner as the test article.

In Vitro Irritancy Score = Corrected Mean + 15 (Corrected Mean Optical Density Score)
(IVIS) Opacity Score

OECD Guideline #437 defies a substance, which produces an IVIS of >55.1 as Category 1, a substance that causes “Serious eye damage”.

IVIS UN GHS
≤ 3 No Category
>3; ≤55 No prediction can be made
>55 Category 1

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

-2.42

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

RESULTS

TEST ARTICLE – H2925 (CAS No. 68130-53-0), Lot# 2013090401

 

INDIVIDUAL TEST VALUES

CORNEA#	Pretest Opacity Scores	10-Minute Scores	2-Hour Scores	O.D. ay 490 nm (Permeability)
7	2	2	2	0.028
8	2	2	3	0.020
9	1	1	0	0.007

 

INDIVIDUAL AND MEAN CALCULATED VALUES

CORNEA#	Corrected Opacity Scores		Individual Corrected O.D.^2
	Individual 10-Minute Corrected Opacity Score^1	Individual 2-Hour Corrected Opacity Score^1
7	0	0	0.004
8	0	1	-0.004
9	0	-1	-0.017
Corrected Mean Opacity Density^3=			-0.006
2-Hour Corrected Mean Opacity Score^4=			-2.33

¹Individual Corrected Opacity Score = 10-minute or 2-hour opacity score minus the pretest opacity score

²Individual Corrected Opacity Density = Individual test article OD minus the mean OD for negative control group. No correction was made for the negative control group.

³Corrected Mean Optical Density = Mean of the individual corrected optical density values for a given dose group.

⁴2-Hour Corrected Mean Opacity Score = Mean of the individual 2-hour corrected opacity scores for a given dose group minus the mean opacity score for the negative control group.

 

NEGATIVE CONTROL – MEM

INDIVIDUAL AND MEAN CALCULATED VALUES

Cornea#	Pretest Opacity Score	10-Minute Scores	10-Minute Corrected Opacity Score^1	2-Hour Scores	2-Hour Corrected Opacity Score^1	O.D. at 490 nm (Permeability)
C1 (neg)	2	3	1	4	2	0.027
C2 (neg)	2	3	1	3	1	0.027
C3 (neg)	5	5	0	9	4	0.018
Mean of Individual Optical Density =						0.024
2-Hour Corrected Mean Opacity Score =						2.33

 

POSITIVE CONTROL – ETHANOL

INDIVIDUAL AND MEAN CALCULATED VALUES

Cornea#	Individual Pretest Opacity Score	Individual 10-Minute Scores	Individual 10-Minute Corrected Opacity Score^1	Individual 2-Hour Scores	Individual 2-Hour Corrected Opacity Score^1	O.D. at 490 nm (Permeability)	Individual Corrected O.D.^2
C4 (pos)	3	9	6	3	0	0.192	0.168
C5 (pos)	3	34	31	34	31	0.266	0.242
C6 (pos)	1	32	31	30	29	0.175	0.151
Corrected Mean Optical Density^3=							0.187
2-Hour Corrected Mean Opacity Score^4=							17.67

¹Individual Corrected Opacity Score = 10-Minute or 2-Hour opacity score minus pretest opacity score.

²Individual Corrected Optical Density = Individual positive control OD minus the mean OD for negative control group.

³Corrected Mean Optical Density = Mean of the individual corrected optical density values for a given dose group.

⁴2-Hour Corrected Mean Opacity Score = Mean of the individual 2-hour corrected opacity scores for a given dose group minus the mean opacity score for the negative control group.

 

CALCULATED IN VITRO IRRITATION SCORES

Test Article (H2925 (CAS No. 68130-53-0), Lot# 2013090401) -2.33 + 15 (-0.006) -2.33 + (-0.09)      IVIS:                 -2.42

Negative Control (MEM) 2.33 + 15 (0.024) 2.33 + 0.36      IVIS:                 2.69

Positive Control (Ethanol) 17.67 + 15 (0.187) 17.67 + 2.805      IVIS:                  20.48

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Based on the In Vitro Irritation Score, H2925 (CAS No. 68130-53-0), Lot# 2013090401, is not classfied as an eye irritant in accordance with the CLP Regulation.

Executive summary:

Objective: To determine the potential for ocular irritation using an alternative to the Draize methodology.

This protocol is based on the methodology described in the current OECD Guideline for the Testing of Chemicals #437.

Method Synopsis: Three bovine corneas per group were dosed with 0.75 ml of H2925 (CAS No. 6813053-0), Lot# 2013090401, Minimal Essential Media (MEM) (negative control), or 100% Ethanol (positive control). Following a ten-minute exposure for each group of dosed corneas, opacity measurements and sodium fluorescein permeability were determined.

Summary/Conclusion:

Test Article: The corrected mean opacity score was -2.33. The corrected mean optical density (permeability) score was -0.006. The in vitro irritancy score (IVIS) was calculated as -2.42.

Negative Control: The corrected mean opacity score was 2.33. The mean optical density (permeability) score was 0.024. The IVIS was calculated as 2.69.

Positive Control: The corrected mean opacity score was 17.67. The corrected mean optical density (permeability) score was 0.187. The IVIS was calculated as 20.48.

All controls were within normal limits.

Based on the In Vitro Irritation Score, H2925 (CAS No. 68130-53-0), Lot# 2013090401, is not classfied as an eye irritant in accordance with the CLP Regulation.