Alkaline earth metal and zinc hydroxy(substituted)alkanoate and oxoalkanoate

Skin irritation / corrosion

Some information in this page has been claimed confidential.

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-08-21 to 2018-11-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted on 29 July 2016

Deviations:

yes

Remarks:

please refer to "Principles of method if other than guideline"

Principles of method if other than guideline:

OD values obtained in the main experiment fell out the spectrophotometer linear range, defined by the MTT formazan curve performed on the day of the experiment. As soon as this issue was noted, within the same week of the experiment, a second calibration curve was prepared using a higher maximum concentration. Thus, a spectrophotometer linear range including OD values obtained in the main experiment was obtained. No other deviations occurred during the study.

GLP compliance:

yes (incl. QA statement)

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

solid: particulate/powder

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: adult donors

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:
Commercial Name: EPISKIN™ - 0.38 cm2
Supplier: SkinEthic Laboratories (4, rue A. Fleming – 69366 Lyon – France)
- Tissue batch number(s): 17-EKIN-041 (alive tissues) and 17-EKIN-019 (killed tissues)
- Delivery date: 10 October 2017 and 10 May 2017
- Date of initiation of testing: 12 October 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 mL (1x)
- Modifications to validated SOP: OD values obtained in the main experiment fell out the spectrophotometer linear range, defined by the MTT formazan curve performed on the day of the experiment. As soon as this issue was noted, within the same week of the experiment, a second calibration curve was prepared using a higher maximum concentration. Thus, a spectrophotometer linear range including OD values obtained in the main experiment was obtained. No other deviations occurred during the study.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h +- 5 min
- Spectrophotometer:
- Wavelength: 595 nm
- Linear OD range of spectrophotometer: yes

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: IC50 (SDS, MTT test) => 1.5 <=3
- Morphology: Well-differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum
- Contamination: no

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- N. of replicates : 2 replicates of killed tissue
- Method of calculation used: NSMT T = 100 × (ODtreated killed tissues − ODnon-treated killed tissues)/(ODnegative control tissues)
The NSMTT values were higher than 5%, but lower than 50% at all treatment times, thus these values were subtracted from the concurrent mean OD value of the test item treated tissues, to evaluate the actual viability.

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 corrosive to skin (sub-category 1A) if the viability after 3 minutes exposure is less than 35%
- The test substance is considered to be corrosive to skin (sub-category 1B/C) if the viability after 3 minutes exposure is greater than or equal to 35 % and the viability after 1 hour exposure is less than 35% or if the viability after 1 hour exposure is greater than or equal to 35 % and the viability after 3 hours exposure is less than 35%
- The test substance is considered to be non-corrosive to skin if the viability after 3 hours exposure is greater than or equal to 35%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

test item: treatment level of 20 ± 2 mg/epidermis unit, each measuring 0.38 cm2 (treatment level: 52.6 mg/cm2)
positive and negative control: treatment level of 50 μL/epidermis unit

Duration of treatment / exposure:

exposure period of 3, 60 and 240 minutes

Number of replicates:

2

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min

Value:

40

Negative controls validity:

valid

Positive controls validity:

not examined

Remarks on result:

other: Positive control was included only at the longest treatment time of 240 minutes, while a negative control was included for each treatment time.

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 min

Value:

79

Negative controls validity:

valid

Positive controls validity:

not examined

Remarks on result:

other: Positive control was included only at the longest treatment time of 240 minutes, while a negative control was included for each treatment time.

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

240 min

Value:

75

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Positive control was included only at the longest treatment time of 240 minutes, while a negative control was included for each treatment time.

Other effects / acceptance of results:

In the Main Assay, the negative controls gave the expected baseline value (Optical Density values ≥ 0.6 and ≤ 1.5) and variability (difference of viability between the two replicates lower than 30%), at each treatment time, in agreement with the guideline indications. For each treatment time, the concurrent negative control mean value is considered the baseline value of the treatment series and thus represents 100% of cell viability. The positive control caused the expected cell death (1% of cell viability, when compared to the negative control). Based on the stated criteria, the assay was regarded as valid.

Applicant's summary and conclusion

Interpretation of results:

other: EU GHS criteria not met

Conclusions:

The mean cell viability of the test item treated tissues, after the blank subtraction, was higher than 35% at all treatment times. Based on the results obtained, the test item TP 1646 is identified as non-corrosive to the skin.

Executive summary:

The potential of the test item to be corrosive to the skin was investigated through an in vitro skin corrosion study, using a commercial reconstructed human epidermis (RhE) model named EPISKIN™. The experimental procedures are based on the OECD Guideline for testing of chemicals no. 431 and was performed under GLP conditions. The test item, as well as controls, were tested for their ability to impair cell viability after an exposure period of 3, 60 and 240 minutes. The final endpoint of the assay is the colorimetric measurement of MTT reduction (blue formazan salt) in the test system, being this reaction an index of cell viability. The test item was tested as supplied by the Sponsor.

A preliminary test was carried out to evaluate the compatibility of the test item with the test system. In a first step, the test item was assayed for the ability of reducing MTT per se. After addition of the test item to the MTT solution, a blue colour was noted indicating that the test item could direct interact with MTT. In a second step, the test item was assayed for the ability of colouring water per se. A colourless solution was observed, indicating that the test item has no potential interfering ability. Based on these results, an additional control for non specific MTT reduction (NSMTT) was added in the Main Assay.

In the Main Assay, for each treatment time, the test item (physical state: solid) was applied as supplied in two replicates, at the treatment level of 20 ± 2 mg/epidermis unit, each measuring 0.38 cm² (treatment level: 52.6 mg/cm²). Positive and negative controls (glacial acetic acid and physiological saline, respectively) were concurrently tested, in the same number of replicates and test conditions at the treatment level of 50 μL/epidermis unit. Positive control was included only at the longest treatment time of 240 minutes, while a negative control was included for each treatment time.

In the Main Assay, the negative controls gave the expected baseline value (Optical Density values ≥ 0.6 and ≤ 1.5) and variability (difference of viability between the two replicates lower than 30%), at each treatment time, in agreement with the guideline indications. For each treatment time, the concurrent negative control mean value is considered the baseline value of the treatment series and thus represents 100% of cell viability.

The positive control caused the expected cell death (1% of cell viability, when compared to the negative control).

Based on the stated criteria, the assay was regarded as valid.

The NSMTT values were higher than 5%, but lower than 50% at all treatment times, thus these values were subtracted from the concurrent mean OD value of the test item treated tissues, to evaluate the actual viability.

The mean cell viability of the test item treated tissues, after the blank subtraction, was higher than 35% at all treatment times. Based on the results obtained, the test item TP 1646 is identified as non-corrosive to the skin.