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Administrative data

Description of key information

The substance was found to be not irritating to eyes in an in-vitro study (OECD 492,GLP). Skin irriation was assessed in a category approach and is based on in vivo data performed either according to or similar to OECD 404. All category members consistently show the absence of a skin irritation potential.

Key value for chemical safety assessment

Skin irritation / corrosion

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
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
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
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
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
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.
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:

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%).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Key eye irritation data is available for Resin acids and rosin acids, hydrogenated, calcium salts (CAS# 68554-12-1) and is summarised below. In addition, key and supportive data is available for other members of the category 'Rosins and their salts'.

 

In a key Guideline (OECD 492) in vitro eye irritation study (BASF 2018a RSS), the potential of Resin acids and rosin acids, hydrogenated, calcium salts (CAS# 68554-12-1) to cause ocular irritation was assessed by a single topical application of ca. 50 µL bulk volume (about 9 mg) of the undiluted test material to a reconstructed three-dimensional human cornea model (EpiOcular™). The test material was not able to reduce MTT directly. The mean viability of the tissues treated with the test material 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 material 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 it was concluded that Resin acids and Rosin acids, hydrogenated, calcium salts do not demonstrate the potential for eye irritation under the conditions chosen in the in vitro EpiOcular™ eye irritation test. The results of both test runs are close to the cut-off value (mean percent tissue viability equal to 60 ± 5%).

Skin irritation

The irritation potential of Rosin, hydrogenated (also known as Hydrogral, Foral AX, and Staybelite Resin-E) was evaluated based on the results of a Human Repeat Insult Patch Test (Industrial Bio-Test Laboratories Inc., 1977). Although the available study predates current regulatory guidelines, the procedure followed standard scientific methodology in effect at the time the study was conducted. There was no evidence of an irritation response in 53 male and female test subjects exposed three times per week for three weeks to 0.2 mL of a 50% solution of the test material in corn oil applied under occlusive patch for twenty-three (23) hours. The conditions of exposure were significantly more stringent than the 4-hour exposures used in current animal testing. There were no signs of test material-related irritation in any subject at any time during the study.

In a primary dermal irritation study on Hydrogenated rosin (Rosin, hydrogenated), 12 New Zealand white rabbits were dermally exposed to 0.5 gram of test substance for twenty-four (24) hours on intact (n=6) or abraded (n=6) skin (CIVO-Institutes TNO, 1982a). Animals were observed for seventy-two (72) hours. On intact skin, the test substance caused the appearance of very slight to well-defined erythema and only very slight edema following twenty-four (24) hour contact. By forty-eight (48) hours after patch removal, edema was absent and erythema was barely perceptible. Hydrogenated rosin was therefore not irritating to rabbit skin.

In a primary dermal irritation study, three male New Zealand white rabbits were dermally exposed on intact skin to 0.5 gram of Gum Rosin (Rosin)for 4 hours under semi-occlusive contact (Phycher Bio Developpement, 2010c). Animals were then observed for a period of 72 hours post-treatment. Irritation was scored by the method described in OECD Guideline 404. Except for slight erythema noted at the application site of a single rabbit one hour after termination of exposure, no signs of skin irritation (erythema and/or edema) were evident during 24-, 48-, or 72-hour examinations. Based on mean values of 0 for both erythema and edema at the 24-, 48-, and 72-hour observations, Gum Rosin was not irritating to rabbit skin.

In a primary dermal irritation study, six young adult New Zealand white rabbits (2 males, 4 females) were exposed to 0.5 g of UNITAC 70 (Rosin, reaction products with formaldehyde) applied for 24 hours under occluded contact to intact and abraded skin (Food & Drug Research Laboratories, Inc., 1985c). Animals were then observed for a period of 72 hours post-treatment. Irritation was scored by the method of Draize. The conditions of exposure in this study were significantly more stringent, i. e., 24 hours on intact and abraded skin, than the current OECD 404 guidelines that specify a 4-hour exposure period on intact skin with a semi-occlusive dressing. Examinations at 24.5 and 72 hours post-treatment with the undiluted test substance moistened with saline indicated no signs of erythema or edema on the abraded or intact skin of the six rabbits. Based on these findings, UNITAC 70 was not considered to be a skin irritant to rabbits.

The potential for Resin acids and rosin acids, hydrogenated, potassium salts to cause skin irritation has been evaluated in vitro and in vivo. In the in vitro investigation, the test substance was found to be inactive in a CORROSITEX™ assay (Eastman Kodak Company, 2004b); a satisfactory response was obtained with the positive and negative control substances included in the test. In the in vivo test (OECD Guideline 404), three New Zealand white rabbits were exposed to 0.5 g of Resin acids and rosin acids, hydrogenated, potassium salts for 4 hours under occluded contact with intact skin and observed for 72 hours. There were no signs of test material-related erythema or edema at any time during the study. In addition, no signs of erythema or edema were observed in a dermal toxicity study in which male and female rats were exposed to the test material under occluded contact for 24 hours (see Eastman Kodak Company, 2004b). Based on these data, Resin acids and rosin acids, hydrogenated, potassium salts is not considered to be a primary skin irritant.

Justification for classification or non-classification