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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in Section 13.
Cross-reference
Reason / purpose:
read-across: supporting information
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in Section 13.
Reason / purpose:
read-across source
Irritation parameter:
other: viability (%)
Run / experiment:
mean of two independent experiments (each with two tissues)
Value:
62
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Not irritating according to criteria outlayed in OECD TG 492.
Remarks:
Within the range of 60 +/- 5% for which the predictivity of the test is low.
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: not applicable.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The laboratory has demonstrated technical 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

Individual viabilities:

Experiment 1, tissue 1: 60.1%

Experiment 1, tissue 2: 66.4%

Experiment 2, tissue 1: 59.7 %

Experiment 2, tissue 2: 61.7%

Result of experiment 1

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 1,206 1,091 1,149
viability [% of NC] 66,6 60,1 63,3 6,3
positive control mean OD570 0,283 0,282 0,283
viability [% of NC] 15,6 15,5 15,6 0

Result of experiment 2

Tissue 1 Tissue 2 mean Intertissue variability (%)
negative control (NC) mean OD570 1,474 1,289 1,382
viability [% of NC] 106,7 93,3 100 13,4
test substance mean OD570 0,825 0,852 0,838
viability [% of NC] 59,7 31,7 60,7 2,0
positive control mean OD570 0,243 0,306 0,275
viability [% of NC] 17,6 22,2 19,9 4,6
Interpretation of results:
GHS criteria not met
Conclusions:
According to the criteria outlied in OECD TG 492, a viability score of >60% is evaluated as "non-classified" for eye irritation. The mean viability value of 62% is within the later assessed borderline-range of 55 - 65%. In this range, the predictive performance (ie both over- and underclassification) is low. Since a second experiment confirmed that the viability is slightly higher than 60%, the overall result is treated as "non irritating" according to GHS criteria.
Executive summary:

This data is being read across from the source study that tested Resin and rosin acids, hydrogenated, calcium salt based on category read across that is explained in the category justification document attached in Section 13 of the dossier.

The potential of Resin acids and Rosin acids, hydrogenated calcium salts to cause ocular irritation was assessed by a single topical application of ca. 50 µL bulk volume (about 9 mg) of the undiluted test substance to a reconstructed three-dimensional human cornea model (EpiOcular™).

Two test runs were performed. Two EpiOcular™ tissues per test run 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 was not able to directly reduce MTT directly.

The mean viability of the tissues treated with the test substance for the 1st test run was 63.3% (viability values for single tissues: 66.4% and 60.1%).

Due to the borderline result a 2nd test run was performed to verify the result.

The mean viability of the tissues treated with the test substance for the 2nd test run was 60.7% (viability values for single tissues: 59.7% and 61.7%).

Based on the results observed and by applying the evaluation criteria described in chapter 3.8, it was concluded that Resin acids and Rosin acids, hydrogenated calcium salts does not show an eye irritation potential in the EpiOcular™ in vitro eye irritation test under the test conditions chosen. The results of both test runs are close to the cut-off value (mean percent tissue viability equal to 60 ± 5%).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to
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. certificate)
Remarks:
BASF SE Experimental Toxicology and Ecology 67056 Ludwigshafen, Germany

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Type:
impurity
Test material form:
solid: particulate/powder
Details on test material:
Name in report: Resin acids and Rosin acids, hydrogenated, calcium salts
Lot No. ResinateCa1.2014
Storage conditions: refridgerator
The identity of Hydrogenated rosin, Calcium salt was confirmed with IR, UVVis and mass spectroscopy analysis
Purity: 100 % UVCB
Specific details on test material used for the study:
Name in report: Resin acids and Rosin acids, hydrogenated, calcium salts
Purity: 98.7%
Storage conditions: refrigerator
The identity of Hydrogenated rosin, Calcium salt was confirmed with IR, UVVis and mass spectroscopy analysis
Expiry date: 20 Feb 2024
Physical state / color: Solid / off-white

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 (test run 1) and 23797 (test run 2)
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

Test system

Vehicle:
unchanged (no vehicle)
Controls:
other: yes (tissue incubations for positive and negative controls included)
Amount / concentration applied:
0.05 mL (about 9 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.
Two seperate experiments were performed
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, me
asured 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. Was
hed 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 two independent experiments (each with two tissues)
Value:
62
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Not irritating according to criteria outlayed in OECD TG 492.
Remarks:
Within the range of 60 +/- 5% for which the predictivity of the test is low.
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: not applicable.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The laboratory has demonstrated technical 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

Individual viabilities:

Experiment 1, tissue 1: 60.1%

Experiment 1, tissue 2: 66.4%

Experiment 2, tissue 1: 59.7 %

Experiment 2, tissue 2: 61.7%

Result of experiment 1

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 1,206 1,091 1,149
viability [% of NC] 66,6 60,1 63,3 6,3
positive control mean OD570 0,283 0,282 0,283
viability [% of NC] 15,6 15,5 15,6 0

Result of experiment 2

Tissue 1 Tissue 2 mean Intertissue variability (%)
negative control (NC) mean OD570 1,474 1,289 1,382
viability [% of NC] 106,7 93,3 100 13,4
test substance mean OD570 0,825 0,852 0,838
viability [% of NC] 59,7 31,7 60,7 2,0
positive control mean OD570 0,243 0,306 0,275
viability [% of NC] 17,6 22,2 19,9 4,6

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
According to the criteria outlied in OECD TG 492, a viability score of >60% is evaluated as "non-classified" for eye irritation. The mean viability value of 62% is within the later assessed borderline-range of 55 - 65%. In this range, the predictive performance (ie both over- and underclassification) is low. Since a second experiment confirmed that the viability is slightly higher than 60%, the overall result is treated as "non irritating" according to GHS criteria.
Executive summary:

The potential of Resin acids and Rosin acids, hydrogenated calcium salts to cause ocular irritation was assessed by a single topical application of ca. 50 µL bulk volume (about 9 mg) of the undiluted test substance to a reconstructed three-dimensional human cornea model (EpiOcular™).

Two test runs were performed. Two EpiOcular™ tissues per test run 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 was not able to directly reduce MTT directly.

The mean viability of the tissues treated with the test substance for the 1st test run was 63.3% (viability values for single tissues: 66.4% and 60.1%).

Due to the borderline result a 2nd test run was performed to verify the result.

The mean viability of the tissues treated with the test substance for the 2nd test run was 60.7% (viability values for single tissues: 59.7% and 61.7%).

Based on the results observed and by applying the evaluation criteria described in chapter 3.8, it was concluded that Resin acids and Rosin acids, hydrogenated calcium salts does not show an eye irritation potential in the EpiOcular™ in vitro eye irritation test under the test conditions chosen. The results of both test runs are close to the cut-off value (mean percent tissue viability equal to 60 ± 5%).