Resin acids and Rosin acids, barium salts

Endpoint summary

Description of key information

The substance was found to be non irritating to skin in a GLP-compliant in-vitro study (OECD 439). The substance was found to cause no serious eye damage in a GLP-compliant in-vitro study (OECD 437). Since the result does not exclude an eye irritation potential of the test substance, a weight of evidence approach was conducted to determine hazard classification.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Feb - Apr 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

18 June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: BASF SE, Ludwigshafen Germany
- lot/batch number of test material: EXP NR.3757-01
- Expiration date: 31 Dec 2029
- Purity: 100 % UVCB

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerator
- Stability under storage conditions: The stability was guaranteed over the study period

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

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

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi 200
- Tissue batch number(s): 30854

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C, 1 hour
- Post-incubation temperature and time: 37°C, 18 ± 2 hours

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: yes
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg / mL MTT diluent
- Incubation time: 3 hours
- Spectrophotometer: SunriseTM Absorbance Reader
- Wavelength: 570 nm
- Filter: without reference filter

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD570, accetable
- Barrier function: Lower acceptance limit: ET50 = 4.0 hours, Upper acceptance limit: ET50 = 8.7 hours
- Contamination: no

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- freeze-killed tissues
- Procedure used to prepare the killed tissues: freezing
- N. of replicates : 3
- Method of calculation used: The individual tissue OD570 is calculated by subtracting the mean blank value of the respective microtiter plate from the respective individual tissue OD570 value. The mean OD570 for a test group of three tissues treated in the same way is calculated.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the mean tissue viability after exposure is less than 50%.
- The test substance is considered to be borderline irritant (inconclusive) to skin if the mean tissue viability after exposure is 45-55%.
- The test substance is considered to be non-irritant to skin if the viability after exposure is greater than or equal to 50%.
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439: The “borderline“ evaluation (50 ± 5%) was statistically determined by using historic BASF data and hence considers the variance of the test method. This evaluation is confirming the borderline range provided in OECD Guideline 439.

Control samples:

yes, concurrent negative control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg per tissue
- Concentration (if solution):

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution):

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5% (w/v)

Duration of treatment / exposure:

25 minutes at room temperature and 35 minutes in the incubator

Duration of post-treatment incubation (if applicable):

ca. 42

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 1

Value:

104.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 2

Value:

99.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 3

Value:

96.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Table 1: Individual and mean OD570 values, individual and mean viability values, standard deviations

and coefficient of variation

Test substance identification			tissue 1	tissue 2	tissue 3	mean	SD	CV [%]
NC	viable tissues	mean OD570	2.118	2.093	2.054	2.088
		viability [% of NC]	101.4	100.2	98.3	100.0	1.5	1.5
	KC tissues	mean OD570	0.059	0.072	0.054	0.061
		viability [% of NC]	2.8	3.4	2.6	2.9	0.4	15.2
Barium resinate	viable tissues	mean OD570	2.189	2.085	2.008	2.094
		viability [% of NC]	104.8	99.8	96.1	100.3	4.4	4.3
	KC tissues*	mean OD570KC NC corrected	0.000	0.000	0.000	0.000
		viability [% of NC]	0.0	0.0	0.0	0.0	0.0
	Final mean viability of tissues after KC correction [% of NC]:					100.3
PC	viable tissues	mean OD570	0.043	0.043	0.049	0.045
		viability [% of NC]	2.1	2.0	2.3	2.1	0.2	8.1

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results observed and by applying the evaluation criteria described, it was concluded that the test substance does not show a skin irritation potential in the EpiDermTM in vitro skin irritation test under the test conditions chosen.

Executive summary:

The potential of the test substance to cause dermal irritation was assessed by a topical application of ca. 2 x 25 µL bulk volume of the undiluted test substance to a reconstructed three-dimensional human epidermis model (EpiDerm™). Due to the chemical and physical properties of the test substance, a double bulk volume (corresponding to ca. 10 mg per tissue) was required for covering the whole tissue surface.

Three EpiDerm™ tissues were incubated with the test substance for 1 hour followed by a 42-hour post-incubation period. In addition to the test substance, 30 µL per tissue of a negative control (PBS) and of a positive control (5% SDS) were applied to three tissues each.

Tissue destruction was determined by measuring the metabolic activity of the tissue 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 quotient of both values indicates the relative tissue viability.  

The following results were obtained in the EpiDerm skin irritation test:

The test substance is able to reduce MTT directly. Therefore, an additional MTT reduction control KC (freeze-killed control tissues) was introduced. The final relative mean viability of the tissues treated with the test substance determined after an exposure period of 1 hour with an about 42-hour post-incubation was 100.3%. The acceptance criteria for the variability of the tissues are met. Application of the positive control 5% SDS showed a relative mean viability of the tissues of 2.1% and reflects the expected sensitivity of the tissues. The mean OD570 of the negative control (PBS) fulfills the acceptance criteria and demonstrates the validity of the assay.

Based on the results observed and by applying the evaluation criteria, it was concluded that the test substance does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation test under the test conditions chosen.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Mar - Apr 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

ADAPTATION ACCORDING TO REACH ANNEX XI, section 1 - See attached justification for WoE: Eye irritation

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:

09 Oct 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: BASF SE Ludwigshafen, Germany
- lot/batch number of test material: EXP NR.3757-01
- Expiration date of the lot/batch: 31 Dec 2029
- Purity: 100 % UVCB
- Physical state: Solid

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerator
- Stability under storage conditions: was guaranteed over the study period

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Freshly slaughtered cattles, Schlachthof Alzey, Emil Färber GmbH & Co. KG, Robert-Bosch-Strasse 23, 55232 Alzey, Germany
- Characteristics of donor animals (age): minimum 12 months, maximum 60 months
- indication of any existing defects or lesions in ocular tissue samples: Corneas free of defects (opacity, scratches, pigmentation etc.)

Vehicle:

water

Remarks:

Deionized

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL, NC AND PC
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 20% test material / Imidazole (w/v) suspension in deionized water

Duration of treatment / exposure:

4 hours

Number of animals or in vitro replicates:

Each treatment group (test substance, NC and PC) consisted of 3 corneas

Details on study design:

SELECTION AND PREPARATION OF CORNEAS :
Corneas free of defects (opacity, scratches, pigmentation etc.) were dissected with a
2 to 3 mm rim of sclera. Isolated corneas were mounted in cornea holders that consist of anterior and posterior chambers. Both chambers were filled to excess with pre-warmed 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 pre-warmed medium, and initial corneal opacity readings were taken for each cornea with an opacitometer.

QUALITY CHECK OF THE ISOLATED CORNEAS :
Any corneas that showed macroscopic tissue damage or an opacity value < 553 opacity units2 were discarded. The remaining corneas were then distributed into negative control, positive control and treatment groups.

NUMBER OF REPLICATES : 3

NEGATIVE CONTROL USED : yes

POSITIVE CONTROL USED : yes

APPLICATION DOSE AND EXPOSURE TIME : 750µL, 4 hours

TREATMENT METHOD: open chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 3

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: yes,
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry (OD490)
- Others (pertinent visual observations): Before measurement, each cornea was observed visually and observations were recorded. Final corneal opacity readings were taken for each cornea with an opacitometer.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: according to the OECD Guideline 437 (adopted 09 Oct 2017) for evaluation of results.
- IVIS ≤ 3: NO Category
- IVIS > 3; ≤ 55: No prediction can be made
- IVIS > 55 : Category 1
- IVIS 1,5 - 4,5: "borderline" result
- IVIS 45 - 65: "borderline" result
A “borderline“ evaluation was determined statistically using historic BASF data and takes the test facility-specific variance of the test method into account. This evaluation is an amendment to the evaluation provided in OECD Guideline 437.

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

8.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No indication of corrosion

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

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

Table 1: In Vitro Irritancy score (IVIS) of the test substance, the NC and the PC

Test substance identification	Cornea- No.	Opacity per cornea	Permeability per cornea	per cornea	IVIS per group
					mean	SD
	7	0.0	0.191	2.9
19/0421-1	8	0.0	0.462	6.9	8.1	5.8
	9	8.1	0.420	14.4
	1	9.3	0.003	9.3
NC	2	3.4	0.005	3.5	6.8	3.0
	3	7.5	0.008	7.6
	4	84.7	1.451	106.4
PC	5	105.3	2.193	138.2	118.0	17.5
	6	81.4	1.880	109.5

Interpretation of results:

study cannot be used for classification

Conclusions:

The test method according to the regulatory accepted protocol at the time of reporting does not allow for the evaluation of eye irritation. The result does not exclude an irritation potential of the test substance. For final assignment of a risk phrase at present, results from another study would be needed.

Executive summary:

The potential of the test substance to cause ocular irritation or serious damage to the eyes was assessed by a single topical application of 750 µL 20% test-substance preparation to the epithelial surface of isolated bovine corneas.

Three corneas were treated with the test-substance preparation for an exposure period of 4 hours. In addition to the test substance, a negative control (NC; deionized water) and a positive control (PC; 20% imidazole in deionized water) were applied to three corneas each.

Corneal opacity was measured quantitatively as the amount of light transmitted 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.

The mean IVIS (8.1) of the corneas treated with the 20% test-substance preparation does not indicate serious eye damage (BCOP cut-off threshold 55). The results of the test substance, the negative control (deionized water) and the positive control (20% imidazole) fulfill the acceptance criteria and demonstrate the validity of the assay.

Based on the results observed and by applying the evaluation criteria described, it was concluded that the test substance does not cause serious eye damage in the Bovine Corneal Opacity and Permeability Test (BCOP Test) under the test conditions chosen. The test method according to the regulatory accepted protocol at the time of reporting does not allow for the evaluation of eye irritation. The result does not exclude an irritation potential of the test substance. For final assignment of a risk phrase at present, results from another study would be needed.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

ADAPTATION ACCORDING TO REACH ANNEX XI, section 1 - See attached justification for WoE: Eye irritation

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%).

Reference 3

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-12-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

ADAPTATION ACCORDING TO REACH ANNEX XI, section 1 - See attached justification for WoE: Eye irritation

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 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.

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

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.

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

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

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

ADAPTATION ACCORDING TO REACH ANNEX XI, section 1 - see justification attached to IUCLID section 7.3.2 Eye irritation

- Justification for WoE: Eye irritation

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008:

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. A GLP-compliant EpiDerm skin irritation test (SIT, OECD Guideline 439) is available for skin irritation. According to the guideline, the scores for the test item treated tissues were below the thresholds for classification as an irritant. For eye irritation, a GLP-compliant IATA testing strategy including experimental data of the Bovine Corneal Opacity and Permeability (BCOP) Test (OECD TG 437) and read-across data of the Reconstructed human Cornea-like Epithelium (RhCE) test (OECD TG 492) are available. Experimental data on the test item itself clearly indicate the absence of irreversible damage to eyes. Test results from read-across regarding OECD TG 492 indicate that the test substance is an irritant and has to be classified for Cat. 2. As a result, the substance is not considered to be classified for skin irritation and classified for eye irritation Cat. 2 under Regulation (EC) No. 1272/2008, as amended for the fourteenth time in Regulation (EC) No. 2020/217.