Resin acids and Rosin acids, aluminum salts

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-12-15

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 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE Experimental Toxicology and Ecology 67056 Ludwigshafen, Germany

Test material

Test material information

Constituent 1

Reference substance name:

Resin acids and Rosin acids, aluminum salts

EC Number:

263-075-0

EC Name:

Resin acids and Rosin acids, aluminum salts

Cas Number:

61789-65-9

Molecular formula:

Unspecified

IUPAC Name:

Resin acids and Rosin acids, aluminum salts

impurity 1

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Water

Test material form:

solid

Details on test material:

Name of test substance: Resin acids and Rosin acids, aluminium salts
Batch identification: V11794/4M
Identity: confirmed
Purity: The calculated purity based on the analyzed carbon content was found to be 94%.
The calculated purity based on the analyzed aluminium content was found to be 100%.
Physical state, appearance: solid, off-white
Storage conditions: refrigerator

Specific details on test material used for the study:

Name of test material: Resin acids and Rosin acids, aluminium salts
Purity: 94%
Water content: 1.2% (Karl-Fischer Titration)
Physical state/colour: solid, off-white
storage in the refrigerator
Expiry date: 2018

pH value: Ca. 5 (undiluted test substance moistened with deionized water, determined in the lab prior to start of the GLP study)
The test substance was homogeneous by visual inspection. The identity was confirmed.

Test animals / tissue source

Species:

human

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

The EpiOcularTM model (OCL-200) is a three-dimensional non-keratinized tissue construct composed of normal human derived epidermal keratinozytes used to model the human corneal epithelium. The EpiOcularTM tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs, 10 mm diameter) and are commercially available as kits (EpiOcular™ 200), containing 24 tissues on shipping agarose.

To assess the ability of the test material to directly reduce MTT a pretest was performed.

Tissue lot number: 23789

Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.05 mL (about 13 mg)

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

18h

Number of animals or in vitro replicates:

Two tissue samples were used per group.

Details on study design:

Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the testsubstance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability.

The substance showed no potency for direct reduction of MTT by the test substance as determined in a pre-test.

On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 1 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour, the pre-incubation medium was replaced by fresh medium and preconditioning continued in the incubator at standard culture conditions for 16 – 24 hours.
After pre-incubation, the tissues were pretreated with 20 µL PBS in order to wet the tissue surface. The tissues were incubated at standard culture conditions for 30 minutes.
By using a sharp spoon, a bulk volume of ca. 50 µL test material was applied covering the whole tissue surface.

Control tissues were concurrently applied with 50 µL sterile deionized water (NC) or with 50 µL of methyl acetate (PC).
After application, the tissues were placed into the incubator until the total exposure time of 6 hours was completed.

^In order to remove the test substance, the tissues were washed with sterile PBS. For this purpose, the tissues were immersed and swiveled three times in each of three beakers filled with PBS. Washed tissues were immediately immersed into 12-well plates pre-filled with
5 mL/well pre-warmed medium (post-soak immersion) in order to remove residual test substance.
After 25 minutes of post-soak immersion, each tissue was dried on absorbent paper and transferred to fresh 6-well plates filled with 1 mL/well pre-warmed medium.
Subsequently, the tissues were incubated at standard culture conditions for 8 hours (post-incubation period).


After the post-incubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours.
After incubation, the tissues were washed with PBS to stop the MTT incubation.
The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol at room temperature overnight or for at least 2 hours on a plate shaker. The optical density at a wavelength of 570 nm (OD570) of the extracts was spectrophotometrically determined. Blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.

Results and discussion

In vitro

Results

Irritation parameter:

other: viability (%)

Run / experiment:

mean of both tissues

Value:

92.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: not applicable.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The laboratory has successfully demonstrated proficiency. Historical control data is included in the report.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Any other information on results incl. tables

Table 1: Results

		Tissue 1	Tissue 2	mean	Intertissue variability (%)
negative control (NC)	mean OD570	1,847	1,785	1,816
	viability [% of NC]	101.7	98,3	100	3,4
test substance	mean OD570	1,641	1,714	1,678
	viability [% of NC]	90,4	94,4	92,4	4,0
positive control	mean OD570	0,283	0,282	0,283
	viability [% of NC]	15,6	15,5	15,6	0

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The mean viability of the substance of 92.4% is above the threshold of 60% given for irritants in OECD TG 492. Therefore, the substance is evaluated as non-irritating according to GHS criteria.

Executive summary:

The potential of Resin acids and Rosin acids, aluminium salts to cause ocular irritation was assessed by a single topical application of ca. 50 µL bulk volume (about 13 mg) of the undiluted test substance to a reconstructed three-dimensional human cornea model (EpiOcular™).

Two EpiOcular™ tissues were incubated with the test substance for 6 hours followed by an 18-hour post-incubation period.

 

Tissue destruction was determined by measuring the metabolic activity of the tissues after exposure/post-incubation by using a colorimetric test. The reduction of mitochondrial dehydrogenase activity measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the epidermal tissues treated with the test substance is compared to that of negative control tissues. The ratio of the values indicates the relative tissue viability.

 

The test substance is not able to directly reduce MTT directly. The mean viability of the tissues treated with the test substance was 92.4%. Based on the results observed and by applying the evaluation criteria it was concluded that Resin acids and Rosin acids, aluminium salts does not show an eye irritation potential in the in vitro EpiOcular™ eye irritation test under the test conditions chosen.