Calcium di(octanoate)

Skin irritation / corrosion

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Test guideline 1

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

Test guideline 2

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 material

Test material information

Constituent 1

Reference substance name:

Calcium di(octanoate)

EC Number:

228-067-3

EC Name:

Calcium di(octanoate)

Cas Number:

6107-56-8

Molecular formula:

C8H16O2.1/2Ca

IUPAC Name:

calcium di(octanoate)

Test material form:

solid

Details on test material:

Purity: 100 %

In vitro test system

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

Results and discussion

In vitro

Results

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.