[No public or meaningful name is available]

Endpoint summary

Description of key information

In vitro testing was conducted in accordance with the OECD Testing Guidelines 437 and 439. It was concluded the registered substance was not irritant or corrosive to the eye or the skin.

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:

1 September 2020 - 7 September 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

July 2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Adult donors

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small Model(TM)
- Tissue batch number: 20-EKIN-036
- Surface: 0.38 cm^2
- Expiration date: 7 September 2020

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during exposure: room temperature
- Temperature of incubation: 36.3 - 37.3°C (actual range)
- Humidity: 81 - 95% (actual range)

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: tissues were washed with phosphate buffered saline to remove residual test item (1 washing step)
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3 tissues per test item together with negative and positive
controls

NUMBER OF INDEPENDENT TEST SEQUENCES: 1 single run

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
A test item is considered irritant in the skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is = 50% of the mean viability of the negative controls.

A test item is considered non-irritant in the in vitro skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

ACCEPTABILITY CRITERIA
The in vitro skin irritation test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the acceptance limits of OECD439 (lower acceptance limit =0.6 and upper acceptance limit =1.5) and the Standard Deviation value (SD) of the % viability should be =18.
b) The mean relative tissue viability of the positive control should be =40% relative to the negative control and the Standard Deviation value (SD) of the % viability should be =18.
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be =18.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 10.6 to 24.3 mg

NEGATIVE CONTROL
- Amount applied: 25 µL PBS

POSITIVE CONTROL
- Amount applied: 25 µL 5% SDS

Duration of treatment / exposure:

15 ± 0.5 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of 3 replicates

Value:

110

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

COLOR INTERFERENCE AND MTT
The test item was checked for color interference in aqueous conditions. Addition of the test item to Milli-Q and isopropanol resulted after subtraction of the blank in an OD of -0.0019 and -0.0004, respectively. Therefore, it was concluded that the test item did not induce color interference.
In addition, because no color change was observed in the presence of MTT it was concluded that the test item did not interact with the MTT endpoint.

RESULTS
Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 110%. Since the mean relative tissue viability for the test item was above 50% the test item is considered to be non-irritant.

CONTROLS
The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 4%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was <9%, indicating that the test system functioned properly.

Interpretation of results:

GHS criteria not met

Conclusions:

In an in vitro skin irritation test, performed according to OECD guideline 439 and under GLP principles, Reaction products of 1,3-cyclohexanedimethanamine and 12-hydroxystearic
acid (BA-1801) showed to be non-irritant (mean tissue viability of 110%). Therefore, the substance does not need to be classified according to GHS and CLP criteria.

Executive summary:

In an in vitro skin irritation test using a human skin model (EPISKIN Standard Model), the influence of Reaction products of 1,3-cyclohexanedimethanamine and 12-hydroxystearic acid (BA-1801) on the viability of human skin was tested. Skin tissue was moistened with 5 μL of Milli-Q water and at least 10 mg of the test item was applied directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.
Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 110%. Since the mean relative tissue viability for the test item was above 50% after 15 ± 0.5 minutes treatment the test item is considered to be non-irritant.
The positive control had a mean cell viability of 4% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was <9%, indicating that the test system functioned properly.

Since the mean relative tissue viability after exposure to the test substance was above 50%, no classification is required for skin irritation according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

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:

12 October 2020 - 13 October 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

26 June 2020

Deviations:

no

GLP compliance:

yes

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, -'s Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as
soon as possible after slaughter.
- Storage, temperature and transport conditions of ocular tissue: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions and tested the day of arrival in the laboratory.
- Indication of any existing defects or lesions in ocular tissue samples: The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
- Indication of any antibiotics used: No
- Selection and preparation of corneas: The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Fetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of Duratec Analysentechnik GmbH (Hockenheim, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1 °C. The corneas were incubated for the minimum of 1 hour at 32 ± 1 °C.
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, Duratec GmbH). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 301.99 to 319.40 mg

NEGATIVE CONTROL: physiological saline (Merck KGaA, Darmstadt, Germany)
- Amount applied: 750 µL

POSITIVE CONTROL: 20% (w/v) Imidazole (Merck KGaA, Darmstadt, Germany)
- Amount applied: 750 µL
- Concentration: 20% (w/v) Imidazole solution

Duration of treatment / exposure:

240 ± 10 minutes at 32 ± 1 °C

Duration of post- treatment incubation (in vitro):

90 ± 5 minutes at 32 ± 1 °C

Number of animals or in vitro replicates:

3 replicates

Details on study design:

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

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea.
The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter.
The opacity value (measured with the device OP-KIT) was calculated according to: The opacity value (measured with the device OP-KIT) was calculated according to: Opacity = ((I0/I) - 0.9894) / 0.0251
With I0 the empirically determined illuminance through a cornea holder but with windows and medium, and I the measured illuminance through a holder with cornea.
The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each treated cornea with the test item or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test item or positive control treated cornea.
The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.
- Corneal permeability: After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 µL of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.
The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution has been performed, the OD490 of each reading of the positive control and the test item was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS): mean opacity value + (15 x mean OD490 value)

DECISION CRITERIA: Additionally the opacity and permeability values were evaluated independently to determine whether the test item induced irritation through only one of the two endpoints.
The IVIS cut-off values for identifying the test items as inducing serious eye damage (UN GHS Category 1) and test items not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given hereafter:
In vitro score range - UN GHS
= 3 - No Category
> 3; = 55 - No prediction can be made
>55 - Category 1

Irritation parameter:

in vitro irritation score

Value:

-0.9

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 142 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Table 1 Summary of opacity, permeability and in vitro score

1 Calculated using the negative control corrected mean opacity and mean permeability values for the positive control and test item.
2 In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

Treatment	Mean Opacity	Mean Permeability	Mean In vitro Irritation Score 1,2
Negative control	2.5	0.011	2.7
Positive control	118	1.593	142
Test item	-1.2	0.016	-0.9

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results of bovine corneal opacity and permeability test (BCOP test), performed according to OECD guideline 437 and GLP principles, the mean in vitro irritancy score (IVIS) was determined to be -0.9. As a consequence, Reaction products of 1,3-cyclohexanedimethanamine and 12-hydroxystearic acid (BA-1801) is not classified according to CLP criteria.

Executive summary:

The objective of this study was to evaluate the eye hazard potential of Reaction products of 1,3-cyclohexanedimethanamine and 12-hydroxystearic acid (BA-1801) as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test). The eye damage of the test item was tested through topical application for approximately 240 minutes. The study is is based on on the most recent OECD guideline and performed according to GLP principles.
The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 142 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.
The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -0.9 after 4 hours of treatment. As a consequence, Reaction products of 1,3-cyclohexanedimethanamine and 12-hydroxystearic acid (BA-1801) is not classified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin Irritation
In an in vitro skin irritation test using a human skin model (EPISKIN Standard Model), the influence of Reaction products of 1,3-cyclohexanedimethanamine and 12-hydroxystearic acid (BA-1801) on the viability of human skin was tested. Skin tissue was moistened with 5 μL of Milli-Q water and at least 10 mg of the test item was applied directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.
Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 110%. Since the mean relative tissue viability for the test item was above 50% after 15 ± 0.5 minutes treatment the test item is considered to be non-irritant.
The positive control had a mean cell viability of 4% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was <9%, indicating that the test system functioned properly.
Since the mean relative tissue viability after exposure to the test substance was above 50%, no classification is required for skin irritation.

 

Eye Irritation
The objective of this study was to evaluate the eye hazard potential of Reaction products of 1,3-cyclohexanedimethanamine and 12-hydroxystearic acid (BA-1801) as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test). The eye damage of the test item was tested through topical application for approximately 240 minutes. The study is is based on on the most recent OECD guideline and performed according to GLP principles.
The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 142 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.
The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -0.9 after 4 hours of treatment. As a consequence, no classification is required for eye irritation.

Justification for classification or non-classification

In vitro testing was conducted in order to assess the capacity of the registered substance to be irritant to the skin and/or the eye. It was concluded that it does not meet the criteria for classification according to Regulation (EC) No 1272/2008.