Resin acids and Rosin acids, reaction products with formaldehyde, calcium salts

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 08 Oct 2012 to 24 Oct 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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 Ocular Corrosives and Severe Irritants)

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

Constituent 1

Reference substance name:

Resin acids and Rosin acids, reaction products with formaldehyde, calcium salts

EC Number:

696-130-6

Cas Number:

1364681-45-7

Molecular formula:

Unspecified

IUPAC Name:

Resin acids and Rosin acids, reaction products with formaldehyde, calcium salts

Test material form:

other: solid

Details on test material:

- Name of test material (as cited in study report): Resin acids and rosin acids, reaction products with formaldehyde, calcium salts
- Physical state: Solid
- Analytical purity: 96.0 % w/w
- Lot/batch No.: DMG 7245
- Expiration date of the lot/batch: July 05, 2016
- Stability under test conditions: The stability of the test item under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- Storage condition of test material: Room temperature
- Homogeneity: The test substance was homogeneous by visual inspection.
- Others:
pH-value: Ca. 5 (undiluted test substance, moistened with water)

Test animals / tissue source

Species:

other: in vitro test on isolated bovine cornea

Strain:

other: not applicable

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: not applicable

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): The solid test substance could not be prepared as a homogeneous 20% preparation in de-ionized water. Therefore 100 mg of the undiluted test substance was applied directly to the epithelial surface of the cornea covering the whole surface of the cornea.

Duration of treatment / exposure:

4 hours

Observation period (in vivo):

not applicable (in vitro test)

Number of animals or in vitro replicates:

not applicable (in vitro test); each treatment group consisted of 3 corneas

Details on study design:

TEST SYSTEM:
- Isolated bovine cornea: Target system is the isolated bovine cornea. Bovine eyes are obtained as a by-product of freshly slaughtered cattle (age of the animals: minimum 12 months, maximum 60 months).

OBJECTIVE:
- Corneal opacity was measured quantitatively as the amount of light transmission through the cornea.
- Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea.
- Both measurements were used to calculate an In Vitro Irritancy Score of the test substance relative to the control corneas.

EXPERIMENTAL PROCEDURE:
- Preparation of the bovine corneas and measurement of initial corneal opacity:
• Corneas free of defects (opacity, scratches, pigmentation etc.) were dissected with a 2 to 3 mm rim of sclera and isolated corneas were mounted in corneal holders consisting of anterior and posterior chambers. Both chambers were filled to excess with Eagles's MEM (without phenol red) and then equilibrated in a vertical position at about 32 °C for at least 1 hour.
• After the equilibration period the medium in both chambers was replaced with fresh medium and initial corneal opacity readings were taken for each cornea with an opacitometer. Any corneas that show macroscopic tissue damage or an opacity value < 512 opacity units were discarded.

- Application of the test substance and washing
• Before application the medium in the anterior chamber was removed using a syringe.
Thereafter, 100 mg of the test substance was applied directly to the epithelial surface of the cornea covering the whole surface of the cornea (open chamber method). Subsequently, the corneas were incubated at about 32 °C for approximately 4 hours (non-surfactant solids).

Control tissues:
• Negative control, NC: 750 μL of de-ionized water
• Positive control, PC: 750 μL of 20% (w/v) solution of Imidazole in de-ionized water

• After the incubation period, NC and PC were removed from the anterior chamber using a syringe and the epithelium was washed at least 3 times with Eagle’s MEM (containing phenol red) and once with Eagle’s MEM (without phenol red). Both chambers were then refilled with fresh Eagle’s MEM (without phenol red).
The epithelium of the test substance treated corneas was rinsed with the open chamber method.

- Measurement of final corneal opacity
• Before measurement, each cornea was observed visually and observations were recorded. Final corneal opacity readings were taken for each cornea with an opacitometer.

- Determination of permeability
• For determination of permeability the medium in the anterior chamber was replaced by sodium fluorescein solution (5 mg/mL for solid test substances) and incubated for 90 ± 5 min at about 32 °C. The amount of sodium fluorescein that permeated through the corneas into the posterior chamber was measured spectrophotometrically. Three aliquots per cornea were transferred to a 96-well microtiter plate and the optical density (OD490) was determined.

DATA EVALUATION
The mean corneal opacity and permeability values of each treatment group were used to calculate an In Vitro Irritancy Score (IVIS).

- Calculation of the corneal opacity value:
First, the opacity was calculated using the opacitometer specific algorithm:
• opacity value = a * Io/I + b
a and b: device specific;
Io: illuminance (lux) through the empty corneal holder with windows and liquid
I: illuminance (lux) through the holder with the cornea

Then the opacity change per cornea was calculated by subtracting the initial from the final
• opacity (opacity change per cornea = final opacity - initial opacity).

Subsequently, the corrected opacity change was calculated by subtracting the mean opacity change of the negative control
• corrected opacity change = opacity change - mean opacity change of NC).

Finally, the mean opacity value for each test substance could be determined as the mean of all corrected opacity changes per treatment group
• mean opacity value = mean of all corrected opacity changes per group).

- Calculation of permeability value:
First, the OD490 value was calculated by subtracting the mean blank OD490 (blank = Eagle´s MEM w/o phenol red) from the OD490 of each cornea.
• OD490 value = OD490 - mean blank OD490

If the OD490 value of the treated cornea was above 1.5, the OD490 of a 1:5 dilution was used to calculate the OD490 value:
• OD490 value = 5 * (OD490 of a 1:5 dilution - mean blank OD490)

Subsequently, the corrected OD490 value was calculated by subtracting the mean OD490 value of the negative control.
• corrected OD490 value = OD490 value - mean OD490 value of NC

Finally, the mean OD490 value for each test substance could be determined as the mean of all corrected OD490 values per treatment group.
• mean OD490 value = mean of all corrected OD490 values per group

- Calculation of the In Vitro Irritancy Score (IVIS)
The IVIS could be calculated per treated cornea and finally the mean IVIS per treatment group ± standard deviation was determined:
• IVIS per cornea = corrected opacity change + 15 * corrected permeability OD change
• IVIS per treatment group = mean opacity value + 15 * mean permeability OD value

ACCEPTANCE CRITERIA
- In case one of the below given acceptance criteria is not covered, repetition of the test was considered.
• A study is considered acceptable if the positive control gives an IVIS that falls within two standard deviations of the current historical mean.
• The negative control responses should result in opacity and permeability values that are less than the established upper limits.
• Since the IVIS per treatment group is determined from the mean of three single corneas, the variability between the corneas treated per test substance should be acceptably low. If no clear prediction is possible, e.g. different predictions are obtained for single corneas, the test will be repeated.

EVALUATION OF RESULTS
- Rules for assessment:
IVIS > 55: risk of serious damage to the eyes
IVIS ≤ 55: no risk of serious damage to the eyes

Results and discussion

In vivo

Results

Irritation parameter:

other: In Vitro Irritancy Score (IVIS)

Time point:

other: 4 h

Score:

-7.2

Reversibility:

other: not applicable

Remarks on result:

other: in vitro test on isolated bovine cornea

Irritant / corrosive response data:

IVIS NC: 6.1
IVIS PS: 117.5

Other effects:

No other effects.

Any other information on results incl. tables

Table 1: Opacity score

Test substance	Cornea No.	Initial Opacity	Final Opacity	Opacity Change	Corrected Opacity Change	Mean	SD
Test substance	25	3.7	1.6	-2.1	-8.2	-7.2	0.9
	26	3.7	2.7	-1.1	-7.2
	27	5.0	4.8	-0.2	-6.3
NC	19	4.6	11.3	6.7	NA	6.1	2.3
	20	3.3	6.9	3.6	NA
	21	3.8	11.8	8.0	NA
PC	22	3.9	93.5	89.6	83.5	67.2	14.2
	23	4.2	71.2	67.0	60.9
	24	3.1	66.3	63.2	57.2

 

Table 2: Permeability score

Test substance	Cornea No.	Mean OD490	Dilution Factor	Mean Corrected OD490	Mean	SD
Test substance	25	0.004	1	0.002	0.001	0.002
	26	0.004	1	0.002
	27	0.001	1	-0.001
NC	19	-0.001	1	NA	0.002	0.004
	20	0.007	1	NA
	21	0.000	1	NA
PC	22	0.584	5	2.916	3.354	0.383
	23	0.705	5	3.521
	24	0.725	5	3.625

 

Table 3:In Vitro Irritancy Score (IVIS)

Test substance	Cornea No.	Opacity per cornea	Permeability per cornea	IVIS
				Per cornea	Per group
					mean	SD
Test substance	25	-8.2	0.002	-8.1	-7.2	0.9
	26	-7.2	0.002	-7.1
	27	-6.3	-0.001	-6.4
NC	19	6.7	-0.001	6.7	6.1	2.2
	20	3.6	0.007	3.7
	21	8.0	0.000	8.0
PC	22	83.5	2.916	127.2	117.5	8.5
	23	60.9	3.521	113.7
	24	57.2	3.625	111.5

NC = negative control

PC = positive control

 

 

Applicant's summary and conclusion

Interpretation of results:

other: no serious eye damage