Esterification products of fatty acids, C18 (unsaturated) alkyl and adipic acid with pentaerythritol

Endpoint summary

Description of key information

- In vitro skin corrosion (OECD 431, GLP): non-corrosive

- In vitro skin irritation (OECD 439, GLP): non-irritant

- In vitro eye irritation (OECD 437, GLP): non-irritant

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 - 22 Nov 2021

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:

adopted in 2021

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EPISKIN Small Model™ (EPISKIN-SM™)

Source strain:

other: not applicable (human skin model)

Details on animal used as source of test system:

not applicable (human skin model)

Justification for test system used:

The used test system is in line with OECD 439.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small Model™ (EPISKIN-SM™), EPISKIN, Lyon, France
- Tissue batch number(s): 21-EKIN-046
- Suggested expiration date: 22 Nov 2021
- Date of initiation of testing: 16 Nov 2021

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37° ± 1.0 °C (5% CO2 and 80 - 100% relative humidity)

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: At the end of the exposure period, the test item was washed from the skin surface with Phosphate buffered saline (PBS). No information on volume given.
- Observable damage in the tissue due to washing: not reported

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

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Histology: A histological evaluation of the human epidermis was provided in the Certificate of Analysis of the EPISKIN Small Model™. An HES stained paraffin section demonstrated that the model was satisfactory and described as "Multi-layered, highly differentiated epidermis consisting of organized basal, spinous and granular layers, and multilayered stratum corneum". In addition, it consisted of 8 cell layers, which is in line with the determined specification (≥ 4 cell layers).
- Viability: A MTT cell viability test was performed by EpiSkin. The results were provided in the Certificate of Analysis of the EPISKIN Small Model™. The determined IC50 was 1.8 mg/mL, which is within the specified range of 1.5 mg/mL ≤ IC50 ≤ 3.0 mg/mL.
- Contamination: EpiSkin verified the absence of HIV1 and 2 antibodies, hepatitis C antibodies, and hepatitis B antigen HBs in the blood of the donors. In addition, EpiSkin verified the absence of bacteria, fungus, and mycoplasma on the cells form the donors.

NUMBER OF REPLICATE TISSUES
3 tissues were used per treatment, negative, and positive control (12 tissues in total).

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test item has been tested previously for possible direct MTT reduction and color interference in the skin corrosion test using Epiderm as a Skin Model™ (Report no. 20326170). The solutions were not turned blue / purple nor a blue / purple precipitate was observed in the presence of MTT, therefore it was concluded that the test item did not interfere with the MTT endpoint. The Optical Density (OD) for the test item solution was ≤ 0.08, therefore it was concluded that the test item did not interact with the MTT measurement.

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

PREDICTION MODEL / DECISION CRITERIA
- According to the EU and GHS classification, an irritant is predicted if the mean relative tissue viability of three individual tissues exposed to the test substance is reduced below or equal to 50% of the mean viability of the negative controls.
mean tissue viability ≤ 50% Irritant (I), (CLP / GHS Category 2)
mean tissue viability > 50% non-irritant (NI).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 25 µL

NEGATIVE CONTROL
- Amount(s) applied: 25 µL

POSITIVE CONTROL SUBSTANCE:
- Amount(s) applied: 25 µL
- Concentration: 5% aqueous solution

Duration of treatment / exposure:

15 ± 0.5 min at room temperature

Duration of post-treatment incubation (if applicable):

42 ± 1 h at 37 °C

Number of replicates:

3 for treatment and control groups

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of 3 tissues (percentage of control)

Run / experiment:

15 min exposure

Value:

103

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:
- Direct-MTT reduction: No direct interaction with the MTT assay reagent was observed.
- Colour interference with MTT: The test item has been tested previously for possible direct MTT reduction and color interference in the skin corrosion test using Epiderm as a Skin Model™ (Report no. 20326170). The solutions were not turned blue / purple nor a blue / purple precipitate was observed in the presence of MTT, therefore it was concluded that the test item did not interfere with the MTT endpoint. The Optical Density (OD) for the test item solution was ≤ 0.08, therefore it was concluded that the test item did not interact with the MTT measurement.
- Visible damage on test system: not reported

DEMONSTRATION OF TECHNICAL PROFICIENCY:
No demonstration of proficiency shown in the study report. However, the acceptance criteria were all met (see below) and the optical density (OD) values of the negative and positive controls are all within the historical control value ranges, indicating that the test system functioned properly.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD of the tissue replicates treated with the negative control was ≥ 0.6 and ≤ 1.5 (value: 1.157, see 'Any other information on results incl. tables', table 1).
- Acceptance criteria met for positive control: The mean viability of the tissue replicates treated with the positive control was < 40% compared to the negative control (value: 2.5%, see 'Any other information on results incl. tables', table 2).
- Acceptance criteria met for standard deviation: The standard deviation calculated from individual % tissue viabilities of the 3 identically treated replicates was below the limit of acceptance of 18% (< 9%, refer to table 2).

HISTORICAL CONTROL DATA
See 'Any other information on results incl. tables', table 4.

Table 1: Mean Absorption in the In Vitro Skin Irritation Test

	A (OD570)	B (OD570)	C (OD570)	Mean (OD570)	± SD
Negative control	1.046	1.181	1.243	1.157	0.101
Test item	1.164	1.254	1.150	1.189	0.056
Positive control	0.026	0.031	0.029	0.028	0.003
OD = Optical density SD = Standard deviation Triplicate exposures are indicated by A, B, and C. Values are corrected for background absorption (0.044). Isopropanol was used to measure the background absorption.

 

Table 2: Mean Tissue Viability in the In Vitro Skin Irritation Test

	Mean tissue viability (percentage of control)	± SD (percentage)
Negative control	100	8.7
Test item	103	4.9
Positive control	2.5	0.2
SD = Standard deviation

 

Table 3: Individual OD Measurements at 570 nm

	A (OD570)	B (OD570)	C (OD570)
Negative control
OD570 measurement 1	1.0475	1.1989	1.2956
OD570 measurement 2	1.1326	1.2517	1.2792
Test item
OD570 measurement 1	1.1743	1.3139	1.2163
OD570 measurement 2	1.2426	1.2817	1.1722
Positive control
OD570 measurement 1	0.0682	0.0739	0.0692
OD570 measurement 2	0.0713	0.0755	0.0771
OD = Optical density Triplicate exposures are indicated by A, B, and C.

 

Table 4: Historical Control Data for In Vitro Skin Irritation Studies (Data Collection Period From May 2018 To May 2021)

	Negative control (absorption; OD570)	Positive control (absorption; OD570)
Range	0.507 - 1.478	0.026 - 0.549
Mean	1.060	0.106
SD	0.153	0.085
n	159	159
SD = Standard deviation n = Number of observations

Interpretation of results:

other: Non-irritant

Conclusions:

Under the present test conditions, Esterification products of fatty acids, C18 (unsaturated) alkyl and adipic acid with pentaerythritol tested at an exposure time of 15 min and a 42 h post-treatment incubation period, was a non-irritant to reconstructed human epidermis tissue in the EPISKIN Small Model™.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 Sep - 24 Sep 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

adopted in 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: EpiDerm™ Skin Model (EPI-200)

Source strain:

other: not applicable (human skin model)

Details on animal used as source of test system:

not applicable (human skin model)

Justification for test system used:

The used test system is in line with OECD 431.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ (EPI-200) (MatTek Corporation, MA, USA)
- Tissue batch number(s): 35665
- Date of initiation of testing: 21 Sep 2021

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (3-min exposure), 37 ± 1.0 °C (5% CO2 and 80 - 100% humidity; 1-h exposure)
- Temperature of post-treatment incubation: incubation with MTT assay solution at 37 ± 1.0 °C (5% CO2 and 80 - 100% humidity)

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: At the end of the exposure period, the test item was carefully washed from skin surface with phosphate buffered saline (PBS). No information on volume given.
- Observable damage in the tissue due to washing: not reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3-h
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: A MTT cell viability test (4-h; n = 3) was performed by MatTek. The results were provided in the Certificate of Analysis of the EpiDerm™ Reconstructed Human Epidermis. The determined OD (540 - 570 nm) was 1.801 ± 0.03 (pass). The negative control tissue has been shown to be stable in culture (provided similar viability measurements) for the duration of the test exposure period (refer to 'Results and discussion' /'Test Acceptance Criteria'). The tissue employed has been shown to demonstrate reproductivity over time and between laboratories.

- Barrier function: A barrier function test was performed by MatTek. The results were provided in the Certificate of Analysis of the EpiDerm™ Reconstructed Human Epidermis. The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 1% Triton X-100. The ET-50 value was determined to be 5.17 h (pass).

- Morphology: The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.

- Contamination: A sterility test was performed by MatTek. The cells used to produce the EpiDerm™ tissue were screened long term in antibiotic and antimycotic free culture. No contamination was detected (specific contaminants not reported).

NUMBER OF REPLICATE TISSUES
2 tissues were used per treatment, negative and positive control included, and exposition time (12 tissues in total).

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
No possible interference with the MTT measurement (OD 570 nm), due to colour changes or direct interacting with the MTT assay reagent, was noted. The solutions were not turned blue / purple nor a blue / purple precipitate was observed in the presence of MTT, therefore it was concluded that the test item did not interfere with the MTT endpoint. The OD for the test item solution was ≤ 0.08, therefore it was concluded that the test item did not interact with the MTT measurement. Therefore, no additional control tissues were needed.

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

PREDICTION MODEL / DECISION CRITERIA
- The test item is considered to be corrosive to skin if the viability after 3-min exposure is less than 50%. Additionally, CLP / GHS sub-category 1A if the viability after 3-min exposure is less than 25%.
- The test item is considered to be corrosive to skin if the viability after 3-min exposure is greater than or equal to 50% and the viability after 1-h exposure is less than 15%. Additionally, CLP / GHS sub-category 1B and 1C if the viability after 3-min exposure is greater than or equal to 25%.
- The test item is considered to be non-corrosive to skin, if the viability after 3-min exposure is greater than or equal to 50% and the viability after 1-h exposure is greater than or equal to 15%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 µL

NEGATIVE CONTROL
- Amount(s) applied: 50 µL

POSITIVE CONTROL SUBSTANCE:
- Amount(s) applied: 50 µL
- Concentration: 8 N KOH

Duration of treatment / exposure:

3 and 60 min

Duration of post-treatment incubation (if applicable):

3h (for cell viability measurement)

Number of replicates:

2 tissues were used per treatment, negative and positive control included, and exposition time (12 tissues in total).

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of 2 tissues (percentage of control)

Run / experiment:

3 min exposure

Value:

88

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of 2 tissues (percentage of control)

Run / experiment:

60 min exposure

Value:

100

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:
- Direct-MTT reduction: No direct interaction with the MTT assay reagent was observed.
- Colour interference with 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 0.0045 and 0.0128, respectively. Therefore it was concluded that the test item did not induce color interference.
- Visible damage on test system: not reported

DEMONSTRATION OF TECHNICAL PROFICIENCY:
No demonstration of proficiency shown in the study report. However, the acceptance criteria were all met (see below) and the optical densitiy (OD) values of the negative and positive controls are all within the historical control value ranges, indicating that the test system functioned properly.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean optical density (OD) of the negative control of 2 tissues was 1.824 (3-min exposure) or 1.708 (1-h exposure) and was well within the acceptable range of ≥ 0.8 to ≤ 2.8 (see 'any other information on results incl. tables', table 1, respectively).
- Acceptance criteria met for positive control: The mean viability of cells treated with the positive reference item 8 N KOH was 6.4% or 9.0% (3-min or 1-h exposure, respectively) of the negative control and, hence well below the 15% cut-off value at the 1-h exposure (see 'any other information on results incl. tables', table 2, respectively).
- Acceptance criteria met for variability between replicate measurements: The difference of viability between the 2 tissue replicates (at 20 – 100% viability) was below the limit of acceptance of 30% (refer to table 3).

HISTORICAL CONTROL DATA
See 'Any other information on results incl. tables', table 5

Table 1: Mean Absorption in the in vitro Skin Corrosion Test

	3-min application				1-h application
	A (OD570)	B (OD570)	Mean (OD570)	± SD	A (OD570)	B (OD570)	Mean (OD570)	± SD
Negative control	1.960	1.688	1.824	0.193	1.707	1.709	1.708	0.001
Test item	1.540	1.686	1.613	0.103	1.732	1.684	1.708	0.034
Positive control	0.104	0.131	0.117	0.019	0.148	0.161	0.155	0.009
SD = Standard deviation Duplicate exposures are indicated by A and B. Values are corrected for background absorption (0.0424). Isopropanol was used to measure the background absorption.

 

Table 2: Mean Tissue Viability in the in vitro Skin Corrosion Test

	3-min application viability (percentage of control)	1-h application viability (percentage of control)
Negative control	100	100
Test item	88	100
Positive control	6.4	9.0

 

Table 3: Coefficient of Variation between Tissue Replicates

	3-min	1-h
Negative control	14	0.1
Test item	8.6	2.7
Positive control	21	8.0
CV (%) = 100 – [(lowest OD570 / highest OD570) x 100%]

 

Table 4: Individual OD Measurements at 570 nm

	3-min application (OD570)		1-h application (OD570)
	A	B	A	B
Negative control
OD570 measurement 1	2.0002	1.6996	1.7501	1.7549
OD570 measurement 2	1.9956	1.7627	1.7562	1.7390
OD570 measurement 3	2.0125	1.7283	1.7427	1.7591
Test item
OD570 measurement 1	1.5643	1.6853	1.7719	1.7350
OD570 measurement 2	1.5897	1.7439	1.7721	1.7292
OD570 measurement 3	1.5936	1.7550	1.7786	1.7161
Positive control
OD570 measurement 1	0.1452	0.1716	0.1893	0.2029
OD570 measurement 2	0.1469	0.1735	0.1919	0.2054
OD570 measurement 3	0.1460	0.1740	0.1902	0.2019
OD = Optical density Duplicate exposures are indicated by A and B

 

Table 5: Historical Control Data for in vitro Skin Corrosion Studies (Data Collection Period From May 2018 to May 2021)

	Negative control		Positive control
	3-min application (OD570)	1-h application (OD570)	3-min application (OD570)	1-h application (OD570)
Range	1.375 - 2.226	1.317 - 2.182	0.080 - 0.512	0.032 - 0.319
Mean	1.718	1.746	0.161	0.132
SD	0.172	0.151	0.065	0.046
n	126	126	126	126
SD = Standard deviation n = Number of observations

 

Interpretation of results:

other: non-corrosive

Conclusions:

Under the present test conditions, Esterification products of fatty acids, C18 (unsaturated) alkyl and adipic acid with pentaerythritol tested at two exposure periods of 3 min or 1 h was non-corrosive to reconstructed human epidermis tissue in the EpiDerm™ model. There is regulatory acceptance in the EU that a substance can be considered corrosive (Skin Corrosive Cat.1A) based on a positive result in the human epidermis model test. Negative in vitro corrosivity responses are not conclusive with respect to non-classification or classification as irritant and shall therefore be subject to further evaluation. A follow-up in vitro skin irritation test using the EPISKIN Small Model™ (Report no. 20326171) demonstrated no skin irritation.

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:

31 Aug 2021

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:

adopted in 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: obtained from slaughterhouse (Vitelco, 's-Hertogenbosch, The Netherlands)
- Characteristics of donor animals: young cattle; no further characteristics given in the report
- 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. No further transport conditions given in the report.
- Indication of any existing defects or lesions in ocular tissue samples: no; only corneas from eyes free of defects were used
- 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) Foetal 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-h at 32 ± 1°C.
- Quality check of the isolated corneas: 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(s) applied: 750 µL (also for neg. and pos. controls)

Duration of treatment / exposure:

10 ± 1 min

Observation period (in vivo):

Not applicable

Duration of post- treatment incubation (in vitro):

120 ± 10 min

Number of animals or in vitro replicates:

Triplicates for each treatment and control group

Details on study design:

TREATMENT METHOD
- The medium from the anterior compartment was removed and 750 µL of either the negative control, positive control (ethanol) or test item was introduced onto the epithelium of the cornea. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or the test item over the entire cornea. Corneas were incubated in a horizontal position for 10 ± 1 min at 32 ± 1°C.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Eagle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM. Possible pH effects of the test item on the corneas were recorded.

POST-EXPOSURE INCUBATION:
After the washing steps, the medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120 ± 10 min at 32 ± 1 °C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was determined by the diminution of light passing through the cornea measured quantitatively via an opacitometer (BASF-OP3.0, Duratec GmbH). 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 cornea treated 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: Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Sigma-Aldrich, Germany) was evaluated. The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 mL of 4 mg Na-fluorescein (Sigma-Aldrich Chemie GmbH, Germany) / mL cMEM solution. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 ± 5 minutes at 32 ± 1 °C. 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).

SCORING SYSTEM:
In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
Test substance with an IVIS > 55 was regarded as serious eye damage and labelled Category 1 according to CLP / GHS, respectively.
Test substance with an IVIS ≤ 3 was regarded as non-irritant and labelled in no category.
Test substance with an IVIS > 3; ≤ 55 no prediction can be made.

Irritation parameter:

in vitro irritation score

Remarks:

mean value of 3 corneas

Run / experiment:

10 min exposure

Value:

0

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 reported
- There were no pH effects.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the negative control resulted in mean opacity (3.0) and permeability (0.003) values that were less than the established upper limits for background opacity and permeability values for bovine corneas treated with the respective negative control (see 'Any other information on results incl. tables', table 1 and 6).
- Acceptance criteria met for positive control: Yes, the positive control resulted in an IVIS (55) which was within two standard deviations of the current historical mean (see 'Any other information on results incl. tables', table 1 and 6).

HISTORICAL CONTROL DATA
See 'Any other information on results incl. tables', table 6

Table 1: Summary of Opacity, Permeability and In Vitro Scores

Treatment	Mean opacity^1	Mean permeability^1	Mean in vitro irritation score^1,2
Negative control	3.0	0.003	3.0
Positive control	23	2.079	55
Test item	0.0	-0.001	0.0
^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).

 

Table 2: Opacity Scores

Treatment	Opacity before treatment	Opacity after treatment	Final opacity^1	Negative control corrected final opacity^2	Mean final opacity
Negative control	0.1	3.1	3.0		3.0
	1.1	3.9	2.8
	0.6	3.8	3.2
Positive control	1.4	29	27	24	23
	1.7	21	19	16
	1.5	35	33	30
Test item	-0.1	3.9	4.0	1.0	0.0
	-0.2	3.0	3.2	0.2
	-0.6	1.2	1.8	-1.2
Calculations are made without rounding off. ^1 Final Opacity = Opacity after treatment – Opacity before treatment. ^2Negative control corrected Final Opacity = Final opacity – Mean final opacity negative control.

 

Table 3: Permeability Score Individual Values (Uncorrected)

Treatment	Dilution factor	OD490 1	OD490 2	OD490 3	Average OD	Final OD	Mean final negative control
Negative control	1	-0.003	-0.001	-0.002	-0.002	-0.002	0.003
	1	0.013	0.015	0.017	0.015	0.015
	1	-0.005	-0.003	-0.007	-0.005	-0.005
Positive control	6	0.632	0.428	0.447	0.502	3.014
	1	1.168	1.161	1.146	1.158	1.158
	8	0.361	0.364	0.324	0.350	2.098
Test item	1	0.006	0.009	0.003	0.006	0.006
	1	0.002	0.001	0.001	0.002	0.002
	1	0.001	-0.005	-0.005	-0.003	-0.003
Calculations are made without rounding off.

 

Table 4: Permeability Score Individual Values (Corrected)

Treatment	Dilution factor	Negative control corrected OD490 1^1	Negative control corrected OD490 2^1	Negative control corrected OD490 3^1	Negative control corrected Average OD490	Negative control corrected Final OD490	Average OD
Positive control	6	0.629	0.425	0.444	0.500	2.998	2.079
	1	1.165	1.158	1.143	1.156	1.156
	6	0.358	0.361	0.321	0.347	2.082
Test item	1	0.003	0.006	0.000	0.003	0.003	-0.001
	1	-0.001	-0.002	-0.002	-0.001	-0.001
	1	-0.002	-0.008	-0.008	-0.006	-0.006
Calculations are made without rounding off. ^1 OD490 values corrected for the mean final negative control permeability (0.003).

 

Table 5: In Vitro Irritancy Scores (IVIS)

Treatment	Final Opacity^2	Final OD490^2	IVIS score^1
Negative control	3.0	-0.002	3.0
	2.8	0.015	3.0
	3.2	-0.005	3.1
Positive control	24	2.998	69
	16	1.156	34
	30	2.082	61
Test item	1.0	0.003	1.0
	0.2	-0.001	0.2
	-1.2	-0.006	-1.2
^1 In vitro irritancy score (IVIS) = opacity value + (15 x OD490 value). ^2 Positive control and test item are corrected for the negative control.

 

Table 6: Historical Control Data for the BCOP Studies (Data Collection Period From May 2018 To May 2021)

	Negative control			Positive control
	Opacity	Permeability	IVIS	IVIS
Min	-2.50	-0.017	-2.50	26
Max	3.70	0.065	4.00	86
Mean	0.86	0.002	0.89	50
SD	1.26	0.009	1.28	14
n	143	143	143	143
IVIS = In vitro irritancy score SD = Standard deviation n = Number of observations

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.

Conclusions:

Under the present test conditions, Esterification products of fatty acids, C18 (unsaturated) alkyl and adipic acid with pentaerythritol tested at an exposure time of 10 min and a 120 min post-treatment incubation period, was a non-irritant to bovine eyes in the Bovine Corneal Opacity and Permeability (BCOP) test. Application of the test substance to bovine corneas resulted in a calculated mean IVIS of 0.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin corrosion

The potential of Esterification products of fatty acids, C18 (unsaturated) alkyl and adipic acid with pentaerythritol to induce skin irritation was examined in vitro according to OECD 431 (EpiDerm™ skin model) and GLP principles (Charles River, 2022b). The test item was applied undiluted (50 µL), directly on top of reconstructed human epidermis (RhE) tissue for 3 min and 1 h. Determination of the cytotoxic (corrosion) effect was performed by the MTT cell viability assay. Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3 min and 1 h treatment with the test item compared to the negative control tissues was 88% and 100%, respectively. Since the mean relative tissue viability for the test item was above 50% after 3 min treatment and above 15% after 1 h treatment, the test item is considered to be non-corrosive. The positive control had a mean cell viability of 9% after 1 h exposure and was considered valid. The mean OD570 (optical density at 570 nm) values of the negative control tissues were ≥ 0.8 and ≤ 2.8 for every exposure time. The difference of viability between the 2 tissue replicates (at 20 – 100% viability) was below the limit of acceptance of 30%, indicating that the test system functioned properly. In conclusion, the test item is a non-corrosive in the in vitro skin corrosion test under the experimental conditions described.

 

Skin irritation

The potential of Esterification products of fatty acids, C18 (unsaturated) alkyl and adipic acid with pentaerythritol to induce skin irritation was examined in vitro according to OECD 439 (EPISKIN™ Small model (EPISKIN-SM™)) and GLP principles (Charles River, 2022c). The test item was applied undiluted (25 µL), directly on top of reconstructed human epidermis (RhE) tissue for 15 ± 0.5 min. After a 42 ± 1 h post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity was measured by the MTT cell viability assay. 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 min treatment with the test item (and 42 ± 1 h post-incubation period) compared to the negative control tissues was 103%. Since the mean relative tissue viability for the test item was above 50% after 15 ± 0.5 min treatment the test item is considered to be non-irritant. The positive control had a mean cell viability of 2.5% after 15 ± 0.5 min exposure and was considered valid. 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. In conclusion, the test item is a non-irritant in the in vitro skin irritation test under the experimental conditions described.

 

Conclusion:
Based on the available in vitro skin corrosion and skin irritation data, it can be concluded that Esterification products of fatty acids, C18 (unsaturated) alkyl and adipic acid with pentaerythritol is a non-irritant and no classification according to the CLP Regulation (EC) No. 1272/2008 is required.

 

Eye irritation

The potential of Esterification products of fatty acids, C18 (unsaturated) alkyl and adipic acid with pentaerythritol to induce eye irritation was examined in vitro according to OECD 437 (Bovine Corneal Opacity and Permeability Test) and GLP principles (Charles River, 2021a). For this purpose 750 µL of the undiluted test substance was topically applied to bovine corneas for 10 ± 1 min. After exposure the corneas were thoroughly rinsed to remove the test item and transferred to fresh medium for incubation (120 ± 10 min) prior to the determination of the irritancy effect. Corneal opacity was determined by the diminution of light passing through the cornea measured quantitatively via an opacitometer at 490 nm. Corneal permeability was determined by the passage of sodium fluorescein dye measured quantitatively with the aid of a microplate reader (OD490). An in-vitro irritancy score (IVIS) was then calculated based on the corneal opacity and permeability values (IVIS = mean opacity value + (15 x mean permeability OD490 value)). Three tissues for each treatment and control group were examined. Since the mean IVIS for the test item (0) was ≤ 3, the test item is considered to be a non-irritant. The positive control, ethanol, had a mean IVIS of 55 and was considered valid (within two standard deviations of the current historical mean). The negative control, saline, resulted in mean opacity (3.0) and permeability (0.003) values that were less than the established upper limits for background opacity and permeability values for bovine corneas treated with the respective negative control, indicating that the test system functioned properly. In conclusion, the test item is a non-irritant in the in vitro eye irritation test under the experimental conditions described and no classification is required according to CLP Regulation (EC) No. 1272/2008.

Justification for classification or non-classification

The available in vitro data on skin irritation/corrosion and eye irritation conducted with the registration substance do not meet the classification criteria according to the CLP Regulation (EC) No. 1272/2008 and are therefore conclusive but not sufficient for classification.