Benzenesulfonic acid, C10-24-branched and linear alkyl derivs., magnesium salts, overbased

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 February 2019 - 08 February 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 July 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test". Official Journal of the European Union No. L142,

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Phosphonic acid, P-octadecyl-, dibutyl ester

EC Number:

846-068-2

Cas Number:

52235-56-0

Molecular formula:

C26 H55 O3 P

IUPAC Name:

Phosphonic acid, P-octadecyl-, dibutyl ester

Test material form:

liquid

Details on test material:

Physical Description: Clear solution
Storage Conditions: At room temperature

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Justification for test system used:

Recommended test system in international guidelines (OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model
- Tissue batch number(s): 29944, Kit I and J
- Surface: 0.6 cm^2
- Pretreatment: The tissues were kept on agarose and stored in the refrigerator on the day they were received. The next day, at least one hour before the assay was started the tissues were transferred to 6-well plates containing 0.9 ml DMEM per well. The level of the DMEM was just beneath the tissue. The plates were incubated for approximately one hour at 37.0 ± 1.0ºC. The medium was replaced with fresh DMEM just before X-20777 was applied.

INTERFERENCE WITH THE MTT ENDPOINT:
- Test for color interference by the test item: 50 μL test item was added to 0.3 mL Milli-Q water and the mixture was incubated for approx. one hour at 37.0 ± 1.0°C in the dark. At the end of the exposure time the mixture was shaken and it was checked if a blue/purple color change was observed. A negative control (Milli-Q water) was tested concurrently.
- Test for reduction of MTT by the test item: 50 μL of the test item was added to 1 mL MTT solution (1 mg/mL) in phosphate buffered saline. The mixture was incubated for approx. one hour at 37.0 ± 1.0°C. At the end of the exposure time it was checked if a blue/purple color change was observed or a blue/purple precipitate was observed. A negative control (Milli-Q water) was tested concurrently.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment of 3 minutes: room temperature
- Temperature of 1 hour and post-treatment incubation: 37.2 - 37.5°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: after exposure and after incubation tissues were washed with phosphate buffered saline
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- Amount of MTT-medium: 300 μL
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: two tissues treated with test item + two replicates for the negative control and the positive control per exposure duration

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: one experiment per exposure period (two in total) with 3 independent OD570 measurements per replicate.

ACCEPTABILITY CRITERIA
The in vitro skin corrosion test is considered acceptable if it meets the following criteria:
- The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range.
- The mean relative tissue viability following 1-hour exposure to the positive control should be <15 %.
- In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30%.

DECISION CRITERIA (see table 1 in 'any other information on materials and methods')
A test item is considered corrosive in the in vitro skin corrosion test if:
- The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
- In addition, a test item considered non-corrosive (viability ≥50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%.

A test item is considered non corrosive in the in vitro skin corrosion test if:
- The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
- In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 50 μL
- The test item was applied directly on top of the skin tissues

NEGATIVE CONTROL
- Amount applied: 50 μL (Milli-Q water)

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

Duration of treatment / exposure:

3 minutes and 1 hour

Duration of post-treatment incubation (if applicable):

3 hours in MTT medium

Number of replicates:

2 tissues per test item per exposure time (4 tissues in total)

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

Mean of 2 replicates

Run / experiment:

3-minute exposure

Value:

87

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

Mean of 2 replicates

Run / experiment:

1-hour exposure period

Value:

94

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- RESULTS
Because the mean relative tissue viability for X-20777 was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment, X-20777 is considered to be not corrosive.

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS: (see table 2 and 3 in 'any other information on results' for historical data and mean OD570 absorption data.):

- Acceptance criteria met for negative control: yes, the absolute mean OD570 of the two tissues of the negative control was within the laboratory historical control data range (i.e 1.444 and 1.428 for the 3-minute and 1-hour exposure respectively).
- Acceptance criteria met for positive control: yes, the mean relative tissue viability following 1-hour exposure to the positive control was <15 % (i.e. 13%).
- Acceptance criteria met for variability between replicate measurements: yes, the Coefficient of Variation between tissue replicates was ≤30% for the negative control (3-minute: 9.0%; 1-hour: 14%) and test item (3-minute 1.3%).
The acceptance criteria for variability between replicate measurements were not met for the positive control, the Coefficient of Variation between tissue replicates at the 3-minute exposure was 36%. However both individual tissues were clearly positive therefore it was concluded that the test system functioned properly.

Any other information on results incl. tables

Table 1 Decision criteria

Viability measured after 3-minutes and 1 hour	Prediction to be considered
Step 1
< 50% after 3 minute exposure	Corrosive
≥ 50% after 3 minute exposure AND < 15% after 1 hour exposure	Corrosive
≥ 50% after 3 minute exposure AND ≥ 15% after 1 hour exposure	Non-corrosive
Step 2 (for substances/mixtures identified as Corrosive in step 1)
< 25% after 3 minute exposure	Optional Sub-category 1A
≥ 25% after 3 minute exposure	A combination of optional Sub-categories 1B and 1C

Table 2 Historical control data

	Negative control		Positive control
	3-minute treatment (OD570)	1-hour treatment (OD570)	3-minute treatment (OD570)	1-hour treatment (OD570)
Range	1.258 – 2.615	1.371 – 2.371	0.092 – 0.56	0.046 – 0.339
Mean	1.73	1.78	0.19	0.14
SD	0.24	0.21	0.09	0.05
n	116	119	114	112

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 November 2015 to November

Table 3 Mean Absorption in the in vitro Skin Corrosion Test with X-20777

	3-minute application			1-hour application
	Mean A (OD570) B (OD570) (OD570)		SD	Mean A (OD570) B (OD570) (OD570)		SD
Negative control	1.376         1.512	1.444±	0.097	1.317        1 .539	1.428±	0.157
X-20777	1.255         1.272	1.263±	0.012	1.335          1.345	1.340±	0.007
Positive control	0.512         0.326	0.419±	0.132	0.251         0.118	0.185±	0.094

SD = Standard deviation

Duplicate exposures are indicated by A and B.

In this table the values are corrected for background absorption (0.0463). Isopropanol was used to measure the background absorption.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Remarks:

Study is part of a tiered approach, no conclusion can be drawn for classification on skin corrosion/irritation.

Conclusions:

In an in vitro skin corrosion test performed according to OECD 431, X-20777 is found not corrosive to skin. The study is part of a tiered approach, no conclusion can be drawn on classification for skin corrosion/irritation from this study.