Calcium di(octanoate)

Endpoint summary

Description of key information

The test item is not classified as irritant to the eye or skin as it had a mean IVIS score of 0.4 after 4 hours in a Bovine Corneal Opacity and Permeability test, and the relative mean tissue viability obtained after 15 ± 0.5 minutes treatment compared to the negative control tissues was 112% in an in vitro EPISKIN Reconstructed Human Epidermis Model study.

Based on the definitive results for classification purposes in the in vitro skin irritation study, according to the bottom-up approach of tiered testing, it was scientifically unjustified to conducted an in vitro skin corrosion study.

Furthermore as the results were considered to be sufficient for classification it was considered scientifically unjustified to conduct either an in vivo skin irritation study or in vivo eye irritation study.

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:

28 May 2018 to 04 June 2018

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 28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Amended by EC No. 640/2012 OJ No. L193, 20 July 2012.

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 ModelTM (EPISKIN-SMTM, 0.38 cm2)
- Tissue batch number(s): 18-EKIN-022
- Production date: 29 May 2018 (Cerification and release date)
- Shipping date: Not reported
- Delivery date: Not reported
- Date of initiation of testing: 28 May 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature
- Temperature of post-treatment incubation (if applicable): 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Not reported
- Observable damage in the tissue due to washing: None reported
- Modifications to validated SOP: None

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 mL MTT-solution (0.3 mg/mL in PBS)
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm
- Filter: None reported
- Filter bandwidth: Not applicable
- Linear OD range of spectrophotometer: Not reported

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE : The test item was checked for possible color interference and direct MTT reduction before the study was started. Some non-colored test items may change into colored items in aqueous conditions and thus stain the skin tissues during the exposure. To assess the color interference, at least 10 mg of the test item was added to 90 µL Milli-Q water. The mixture was mixed for approximately 15 minutes. A negative control, 10 µL Milli-Q water was tested concurrently. At the end of the shaking period a color check was performed. To assess the ability of the test item to reduce MTT, at least 10 mg of the test item was added to 2 mL MTT solution (0.3 mg/mL in PBS). The mixture was incubated for 3 hours at 37°C. A negative control, 25 µL sterile Milli-Q water was tested concurrently. At the end of the incubation period a color check was performed. Because no color changes were observed it was concluded that A027 did not interact with the MTT endpoint.

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 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.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): The solid test item was applied directly on top of the skin tissue and was spread to match the size of the tissue.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 µL
- Concentration (if solution): PBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 µL
- Concentration (if solution): 5% (aq) Sodium dodecyl sulfate in PBS

Duration of treatment / exposure:

15 ± 0.5 minutes; the positive control was re-spread after 7 minutes contact time.

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean

Value:

112

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: None reported
- Visible damage on test system: None reported
- Direct-MTT reduction: No color changes were observed, therefore it was concluded that the test item did not interact with the MTT endpoint
- Colour interference with MTT: No color changes were observed, therefore it was concluded that the test item did not interact with the MTT endpoint

DEMONSTRATION OF TECHNICAL PROFICIENCY: All validity criteria met

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
- Range of historical values if different from the ones specified in the test guideline: See table 4

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

	A (OD570)	B (OD570)	C (OD570)	Mean (OD570)	SD
Negative Control	0.998	0.972	1.039	1.003	0.034
Test item	1.282	1.107	0.986	1.125	0.148
Positive Control	0.042	0.055	0.041	0.046	0.008

OD = Optical density

SD = Standard Deviation

Values are corrected for background absoprtion (0.042); Isopropanol was used to measure the background absorption

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

	Mean tissue viability (% of control)	Standard Deviation (%)
Negative Control	100	3.4
Test item	112	15
Positive Control	4.6	0.8

Table 3: Individual OD Measurements at 570 nm

	OD570 Measurement	A (OD570)	B (OD570)	C (OD570)
Negative Control	1	1.0376	1.0264	1.0975
	2	1.3237	1.1762	1.0396
Test item	1	1.3237	1.1762	1.0396
	2	1.3233	1.1207	1.0171
Positive Control	1	0.0809	0.0971	0.0833
	2	0.0868	0.0972	0.0824

OD = Optical density

Table 4: Historical Control Data for In Vitro Skin Irritation Studies

	Negative Control (absorption; OD570)	Positive Control (absorption; OD570)
Range	0.422 - 1.547	0.023 - 0.437
Mean	0.98	0.13
SD	0.18	0.08
n	174	173

SD = Standard deviation

n = Number of observation

Historical control data range of the controls were obtained by collecting all data over the period November 2014 to November 2017

Interpretation of results:

GHS criteria not met

Conclusions:

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 112%. Since the mean relative tissue viability for the test item was above 50%, it is considered to be non-irritant.

Executive summary:

The skin irritancy potential of the test item was conducted in an in vitro study EPISKIN Reconstructed Human Epidermis Model test according to OECD 439 and EU B.46 test guidelines alongside Phosphate Buffer solution (PBS) and 5 % w/v Sodium dodecyl sulphate (SDS) as a negative and positive controls, respectively. The relative mean viability of the test item treated tissues was 112 % after a 15 minute +/- 0.5 minute exposure period followed by 42 hour post-exposure period. The test item was classified as non-irritant under the UN GHS classification criteria.

The study is a GLP compliant guideline experimental study with no restrictions and therefore fully adequate for assessment for this endpoint.

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:

30 April 2018 - 01 May 2018

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 26 July 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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.
- Number of animals: Not reported
- Characteristics of donor animals (e.g. age, sex, weight): Young cattle
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- Time interval prior to initiating testing: Not reported
- 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: None reported

Vehicle:

unchanged (no vehicle)

Remarks:

No workable suspension in physiological saline could be obtained, the test item was used as delivered by the sponsor and added pure on top of the corneas.

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 364.3 to 392.4 mg
- Concentration (if solution): Not applicable

Duration of treatment / exposure:

240 +/- 10 minutes

Number of animals or in vitro replicates:

3 per treatment

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The isolated corneas were stored in a petri dish with cMEM (Earle’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 BASF (Ludwigshafen, 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.

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, BASF, Ludwigshafen, Germany). 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.

NUMBER OF REPLICATES
Three corneas were selected at random for each treatment group.

NEGATIVE CONTROL USED
Physiological saline (Eurovet Animal Health, Bladel, The Netherlands)

SOLVENT CONTROL USED (if applicable)
Not applicable

POSITIVE CONTROL USED
20% (w/v) Imidazole solution prepared in physiological saline

APPLICATION DOSE AND EXPOSURE TIME
The medium from the anterior compartment was removed and 750 µl of the negative control and 20% (w/v) Imidazole solution (positive control) were introduced onto the epithelium of the cornea. The test item was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (364.3 to 392.4 mg).

TREATMENT METHOD: [closed chamber / open chamber] Open chamber

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: At least three times with MEM with phenol red (Earle’s Minimum Essential Medium Life Technologies)

- POST-EXPOSURE INCUBATION:
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 5 mg Na-fluorescein/mL cMEM solution (Sigma-Aldrich Chemie GmbH, Germany). 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.

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) measured with the device OP-KIT)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a micro plate reader] (OD490)
- Others (e.g, pertinent visual observations, histopathology): None reported

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used. Yes

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

0.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

cornea opacity score

Run / experiment:

Mean

Value:

0.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: mean permability

Run / experiment:

Mean

Value:

0.007

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: The corneas were clear after the 240 minutes treatment

DEMONSTRATION OF TECHNICAL PROFICIENCY: Positive and negative control criteria met

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes; the individual in vitro irritancy scores for the negative controls ranged from 2.1 to 4.1
- Acceptance criteria met for positive control: Yes, the individual positive control in vitro irritancy scores ranged from 158 to 177
- Range of historical values if different from the ones specified in the test guideline: 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 165 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 Scores

Treatment	Mean Opacity	Mean Permeability	Mean in vitro irritancy Score^1,2
Negative Control	2.7	0.021	3.0
Positive Control	121	2.936	165
Test item	0.3	0.007	0.4

¹    Calculated using the negative control 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 Score

Treatment	Opacity before treatment	Opacity after treatment	Final Opacity^1	Negative control corrected for Final Opacity^2	Mean Final Opacity
Negative Control	4.5	8.3	3.8		2.7
	4.1	5.9	1.8
	3.8	6.2	2.5
Positive Control	4.4	123.8	119.4	117	121
	5.2	138.1	132.9	130
	3.4	122.6	119.1	116
Test item	3.6	6.4	2.8	0.2	0.3
	3.4	5.6	2.2	-0.4
	3.2	7.1	3.9	1.3

Calculations are made without rounding off.

¹  Final Opacity = Opacity after treatment – Opacity before treatment.

²  Negative control corrected Final Opacity = Final opacity – Mean final opacity negative control³

 

Table 3: Permeability Score Individual Values (Uncorrected)

Treatment	Dilution Factor	OD490 1	OD490 2	OD490 3	Average OD	Final OD	Mran final negative control
Negative Control	1	0.020	0.021	0.020	0.020	0.020	0.021
	1	0.020	0.020	0.020	0.020	0.020
	1	0.021	0.029	0.021	0.024	0.024
Positive Control	6	0.510	0.508	0.530	0.516	3.096
	6	0.530	0.540	0.541	0.537	3.222
	6	0.468	0.476	0.493	0.479	2.874
Test item	1	0.035	0.036	0.042	0.038	0.038
	1	0.022	0.022	0.021	0.022	0.022
	1	0.027	0.026	0.026	0.026	0.026

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 OD490 Average	Negative control corrected final OD490	Average OD
Positive Control	6	0.489	0.487	0.509	0.495	2.968	2.936
	6	0.509	0.519	0.520	0.516	3.094
	6	0.447	0.455	0.472	0.458	2.746
Test item	1	0.014	0.015	0.021	0.016	0.016	0.007
	1	0.001	0.001	0.000	0.000	0.000
	1	0.006	0.005	0.005	0.005	0.005

Calculations are made without rounding off.

¹  OD490 values corrected for the mean final negative control permeability (0.021)

Table 5: In Vitro Irritancy Score

Treatment	Final Opacity^2	Find OD490^2	In vitro Irritancy Score^1
Negative Control	3.8	0.020	4.1
	1.8	0.020	2.1
	2.5	0.024	2.8
Positive Control	117	2.968	161
	130	3.094	177
	121	2.746	158
Test item	0.2	0.016	0.4
	-0.4	0.000	-0.4
	1.3	0.005	1.3

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

	Negative Control			Positive Control
	Opacity	Permeability	In vitro Irritancy Score	In vitro Irritancy Score
Range	-5.4 - 5.2	-0.011 - 0.205	-5.3 - 5.4	86.5 - 211.4
Mean	0.55	0.02	0.78	138.42
SD	1.84	0.02	1.90	26.57
n	166	166	166	166

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of May 2015 to May 2018.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item induced an IVIS ≤ 3 (actual: 0.4), therefore, no classification is required for eye irritation or serious eye damage.

Executive summary:

The eye irritation potential of the test item was investigated in an OECD 437 guideline study. The test item was applied without modification and was tested in conjunction with concurrent negative and postive controls. The test item induced a mean IVIS score of 0.4, and the negative and positive controls were within the acceptability limits and historic values. Therefore, no classification is required for eye irritation or serious eye damage.

The study is a GLP compliant guideline experimental study and is fully acceptable for assessment of this endpoint without restriction.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

A Bovine Corneal Opacity and Permeability Assay (BCOP) conducted according to OECD guideline 437 is available for the test item (Gijsbrechts 2018). The test item had an IVIS score of 0.4 after 4 hours of treatment.

An EPISKIN Reconstructed Human Epidermis Model test according to OECD guideline 439 and EU B.46 guideline is available for the test item (de Jong 2018). The relative mean viability of test item treated tissues was 112 % after 15 minute exposure period.

Justification for classification or non-classification

The test item is not classified as irritant to the eye or skin as in an in vitro eye irritation and an in vitro skin irritation study it did not exhibit classifiable responses.

As the results were considered to be sufficient for classification it was considered scientifically unjustified to conduct either an in vivo skin irritation study or in vivo eye irritation study.