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

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Jan. 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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, edition adopted 26. Jul. 2013: “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:

Regulation (EU) No. 1152/2010 amending Regulation (EC) No. 440/2008, EU Method B. 47: “Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants” adopted 08. Dec. 2010

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:

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

EC Number:

270-471-7

EC Name:

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

Cas Number:

68441-67-8

Molecular formula:

Not applicable - UVCB

IUPAC Name:

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

Test material form:

liquid

Details on test material:

Designation in Test Facility: 16102602G
Date of Receipt: 26. Oct. 2016
Condition at Receipt: Room temperature, in proper conditions

Specification
Name Hatcol 3178
Batch no. 2011280182
Appearance: Clear liquid
CAS No.: 68441-67-8
EINECS-No.: 270-471-7
Molecular formula: C33H60O8 to C41H76O8 (in CH2 unit increments)
Molecular weight: 692 daltons
Purity: 99.6 %
Homogeneity: homogeneous liquid
Stability: H2O: unknown; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown
Solubility: H2O: unknown; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown
Production date: 18. Jul. 2011
Expiry date: 01. Nov. 2019
Storage: Room Temperature (20 ± 5°C)

Storage in Test Facility
The test item was stored in a closed flask at 15.8 – 22.0 °C, protected from humidity.

Specific details on test material used for the study:

No further details specified in the study report.

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Specification
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 Hank’s balanced salt solution (supplemented with 0.01% streptomycin and 0.01% penicillin). Then, the corneas were dissected and incubated in medium at 32 ± 1 °C in an incubation chamber for 1 h.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The respective substance (negative control solution, test item or positive control) was applied by pipetting 750 μL of the appropriate liquid.

Duration of treatment / exposure:

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

Duration of post- treatment incubation (in vitro):

The corneas were stored for additional 2 hours at 32 ± 1 °C (post-incubation).

Number of animals or in vitro replicates:

3 replicates per group (test item, negative control & positive control).

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 11. Jan. 2017
Incubation time 10 min.
Negative control HBSS solution
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 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 the post-incubation, 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.
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, the permeability of the liquid was measured with the spectrophotometer at 492 nm.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Test Item

Value:

-0.35

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Under the conditions of this test, the test item Hatcol 3178 showed no effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is -0.35.
The negative control (HBSS-solution) 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.

Any other information on results incl. tables

Illuminance Values

Parameter	Negative Control			Test item			Positive Control
(I) Measured values before exposure	1018	989	1016	1023	1033	1053	997	985	956
(I) Measured values after exposure	971	971	1001	1013	1013	1031	311	300	271

 

Opacity Values Negative Control

Parameter	Negative Control
Opacity before exposure	2.17	3.39	2.25
Opacity after exposure	4.18	4.18	2.87
Opacity Difference	2.01	0.79	0.62
Mean Opacity Difference	1.14

 

Opacity Values Test Item and Positive Control

Parameter	Test Item			Positive Control
Opacity before exposure	1.96	1.56	0.79	3.04	3.56	4.87
Opacity after exposure	2.37	2.37	1.64	96.71	101.70	116.80
Opacity Difference	0.41	0.81	0.86	93.66	98.14	111.93
Opacity Difference Corrected	-0.74	-0.33	-0.29	92.52	96.99	110.79

 

Optical density at 492 nm of Blank

Parameter	cMEM without phenol red
1. Measurement	0.033
2. Measurement	0.035
3. Measurement	0.043
Mean	0.037

 

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

Parameter	Negative Control			Test Item			Positive Control
1. Measurement	0.046	0.037	0.033	0.037	0.039	0.060	0.534	0.484	0.784
2. Measurement	0.043	0.035	0.033	0.035	0.040	0.060	0.546	0.482	0.784
3. Measurement	0.041	0.040	0.034	0.037	0.037	0.060	0.544	0.480	0.797
1. Measurement – blank	0.009 0	0.000 0	- 0.004 0	0.000 0	0.002 0	0.023 0	0.4970	0.4470	0.7470
2. Measurement – blank	0.006 0	- 0.002 0	- 0.004 0	- 0.002 0	0.003 0	0.023 0	0.5090	0.4450	0.7470
3. Measurement – blank	0.004 0	0.003 0	- 0.003 0	0.000 0	0.000 0	0.023 0	0.5070	0.4430	0.7600
Mean of each replicates	0.006 3	0.000 3	- 0.003 7	- 0.000 7	0.001 7	0.023 0	0.5043	0.4450	0.7513
Mean of the three replicates	0.0010			-			-
Corrected	-	-	-	- 0.0017	0.0007	0.0220	2.5207*	2.2240*	3.7557*

* Note: All values for the positive control were obtained by measurement of a 5-fold diluted solution and multiplication of the absorbances with factor 5.

 

IVIS

Test Group	IVIS	Mean IVIS	Relative Standard Deviation IVIS
Negative Control HBSS-solution	2.11	1.16	71.64%
	0.80
	0.57
Test item Hatcol 3178	-0.76	-0.35	116.10%
	-0.32
	0.04
Positive Control DMF undiluted	130.33	142.60	14.89%
	130.35
	167.13

 

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 Hatcol 3178 by quantitative measurements of changes in opacity and permeability in a bovine cornea.
The test item Hatcol 3178 was brought 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 pure.
Under the conditions of this test, the test item Hatcol 3178 showed no effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is -0.35.
According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.
The negative control (HBSS-solution) 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 Hatcol 3178 in the BCOP Test 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 which were between 12 and 60 months old.

The test item Hatcol 3178 was brought 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.

 

HBSS-solution was used as negative control. The negative control showed no irritating effect on the cornea and the calculated IVIS (in vitro irritancy score) is 1.16.

Dimethylformamide (DMF) undiluted was used as positive control. The positive control induced serious eye damage on the cornea and falls within two standard deviations of the current historical mean. The calculated IVIS (in vitro irritancy score) is 142.60.

 

Under the conditions of this study, the test item Hatcol 3178 showed no effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is -0.35.

According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.