D-Glucose, reaction products with alcohols C16-18 (even numbered)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-03-01

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

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:

adopted July 26, 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

D-Glucose, reaction products with alcohols C16-18 (even numbered)

IUPAC Name:

D-Glucose, reaction products with alcohols C16-18 (even numbered)

Test material form:

solid: particulate/powder

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Abattoir A. Moksel AG, Buchloe, Germany
- Storage, temperature and transport conditions of ocular tissue: Eyes were collected on test day from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated.
- indication of any existing defects or lesions in ocular tissue samples: The eyes were carefully examined for defects and any defective eyes were discarded.
- Indication of any antibiotics used: Pen/Strep

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 20% in physiological saline (0.9% NaCl)

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 µL
- Lot/batch no. (if required): 609709

Duration of treatment / exposure:

4 h ± 5 min incubation

Duration of post- treatment incubation (in vitro):

90 min

Number of animals or in vitro replicates:

three corneae per dose group

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The isolated corneae were obtained from an abattoir. The eyes were collected and subsequently transported in HBSS containing Pen/Strep on ice. Immediately after arrival of the eyes in the laboratory cornea preparation was initiated.

QUALITY CHECK OF THE ISOLATED CORNEAS
The quality of the corneae was checked visually before the tissues were mounted in the corneal holders, they were examined for defects and any defect cornea had been discarded.

NUMBER OF REPLICATES
Three corneae per dose group were used including negative (vehicle) and positive control.

SOLVENT CONTROL USED (if applicable)
yes, vehicle (0.9 % NaCl)

POSITIVE CONTROL USED
yes, 20 % imidazole in 0.9% NaCl

APPLICATION DOSE AND EXPOSURE TIME
750 µl of a solution containing either no substance, 20 % test item or 20 % imidazole were placed in the anterior chamber of the cornea holder for 4 h ± 5 min. After the exposure time the corneas were rinsed at least three times to remove all of the test substance.

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: yes. If YES please specify duration: 90 min

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: At least three washing steps wit MEM medium containing phenol red and a final washing step with RPMI medium to substitute the phenol red containing medium.
- POST-EXPOSURE INCUBATION: 90 min

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: yes, using an Opacitometer (BASF-OP3.0, Duratec GmbH)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry (OD490)
- Others: pertinent visual observations

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used. - No

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean IVIS of three experiments

Value:

9.11

Vehicle controls validity:

valid

Remarks:

IVIS = 0.97 mean value of three corneae

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

IVIS = 153.69 mean value of three corneae

Other effects / acceptance of results:

None of the corneae treated with D-Glucose reaction products with alcohols C16-C18 showed opacity of the tissue.

Any other information on results incl. tables

Results of the Bovine Corneal Opacity and Permeability Assay with 20 % D-Glucose reaction products with alcohols C16-C18 after 4 h exposure

Test group	Cornea No.	Change of Opacity (final - initial opacity)	Corrected Opacity	Permeability at 490 nm	Corrected Permeability at 490 nm	In vitro Irritation Score (mean)	In vitro Irritation Scale
Negative control	1	0.45		0.007			Not severely irritating
	2	0.38		0.009
	3	1 .58		0.018
	MV	0.80		0.011		0.97
Positive control	4	103.36	102.56	3.060	3.049		Severely irritating
	5	99.33	98.52	4.660
	6	81.98	81.18	4.235	4.224
	MV	94.89	94.09	3.985	3.974	153.69
Test Item	7	2.38	1.57	0.332	0.321		Not conclusive
	8	1.69	0.88	0.493	0.482
	9	2.83	2.02	0.733	0.722
	MV	2.30	1.49	0.519	0.508	9.11

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

In the present study the treatment of bovine corneae with D-Glucose reaction products with alcohols C16-C18 for 4h at 32°C resulted in a mean IVIS of 9.11. There were no signs of opacity in none of the treated corneae after 4 h exposure. However, in accordance with OECD Testguideline 437 (adopted July 26, 2013) the following cut-off values for classification according to GHS criteria the IVIS must be < 3 for no classification and > 55 to identify a substance as inducing serious eye damage (UN GHS Category 1). All values between the afore mentioned values do not provide conclusive results, thus, no prediction can be made.

Executive summary:

This in vitro study was performed to assess the corneal irritation and damage potential of D-Glucose reaction products with alcohols C16-C18(20% in 0.9 % NaCl) by means of the BCOP assay using fresh bovine corneae according to OECD guideline 437, adopted 26 July 2013.

The corneae were incubated with the test substance and controls for 4 h ± 5 min. After rinsing with MEM and RPMI medium, the corneae were incubated for another 90 min at 32±1°C in order to measure the permeability. The test was performed in triplicates. Opacity and permeability were determined. The in vitro irritancy score (IVIS) were calculated as mean opacity value + (15 x mean OD490 value); a substance that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant.

A 20% dilution of the test substance in physiological saline caused no visually detectable increase of the corneal opacity. However, the calculated mean in vitro irritation score was 9.11.

The positive control (20% imidazole) increased the opacity and permeability of the corneae in both experiments (mean in vitro irritation score 153.69). 

With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed in both experiments (mean in vitro irritation score 0.97). Since the mean in vitro irritancy score of the test substance was <55.1 but > 3, no valid conclusion can be drawn after a 4h treatment of bovine corneae with a 20% dilution of D-Glucose reaction products with alcohols C16-C18 in pyhsiological saline under the experimental conditions described in this report.