9-Octadecenoic acid (Z)-, sulfonated, potassium salts

Endpoint summary

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:

From February 18, 2014 to February 24, 2014

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Species:

other: human three dimensional epidermal model (i.e., EPISKIN Small Model (EPISKIN-SMTM))

Type of coverage:

open

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 10.0 to 11.7 mg of solid test substance was applied directly on top of the skin tissue

Duration of treatment / exposure:

15 minutes

Observation period:

After a 42 h post-incubation period, determination of the cytotoxic (i.e., irritancy) effect was performed

Details on study design:

- Test for reduction of MTT by the test substance:
Test substance was checked for possible direct MTT reduction before the study was started. 11.9 mg of the test substance was added to 2 mL of MTT solution (i.e., 0.3 mg/mL in PBS). The mixture was incubated for 3 h at 37°C. A negative control, sterile Milli-Q water was tested concurrently.

- Application/Treatment of the test substance:
The test was performed on a total of 3 tissues per test substance, negative and positive controls. 10.0 to 11.7 mg of solid test substance (i.e., with a small glass weight boat) was added into 12-well plates on top of the skin tissues. The skin was moistened with 5 μL Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test substance to the tissue. Three tissues were treated with 25 μL PBS (i.e., negative control) and 3 tissues with 25 μL 5% sodium dodecyl sulphate (SDS) (i.e., positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test substance. After rinsing the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 h at 37°C.

- Cell viability measurement:
After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 mL MTT-medium (i.e., 0.3 mg/mL). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 μL isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for approximately 69 h. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation potential of the test substance was classified according to remaining cell viability following exposure of the test substance.

Irritation / corrosion parameter:

other: Cell viability (%)

Run / experiment:

15 minutes treatment time followed by 42 h post-incubation period.

Value:

95

Negative controls validity:

valid

Remarks on result:

other: Basis: mean

Other effects / acceptance of results:

The relative mean tissue viability obtained after 15 minutes treatment with test substance compared to the negative control tissues was 95%. Since the mean relative tissue viability for test substance was above 50%, the test substance was considered to be non-irritant.

The absorption at 570 nm measured after treatment with test substance and controls are presented below:

Table 1: Individual OD measurements at 570 nm.

Test system	A (OD570)	B (OD570)	C (OD570)
Negative control
OD570 measurement 1	1.402	1.004	1.152
OD570 measurement 2	1.311	1.037	1.160
Test substance
OD570 measurement 1	1.137	1.062	1.132
OD570 measurement 2	1.154	1.058	1.175
Positive control
OD570 measurement 1	0.196	0.441	0.226
OD570 measurement 2	0.234	0.452	0.229

Table 2: Mean absorption values of test substance, negative and positive controls.

 

 

Test System	A (OD570)	B (OD570)	C (OD570)	Mean±SD (OD570)
Negative control	1.315	0.979	1.114	1.136±0.169
Test substance	1.104	1.018	1.112	1.078±0.052
Positive control	0.173	0.404	0.186	0.254±0.130

OD = optical density

SD = Standard deviation

Triplicate exposures are indicated by A, B and C.

In this table the values are corrected for background absorption (0.042). Isopropanol was used to measure the background absorption.

 

The absolute mean OD₅₇₀ of the negative control tissues was within the laboratory historical control data range.

Mean tissue viability obtained after 15 minutes treatment with test substance compared to the negative control tissues is given below:

Table 3: Mean tissue viability values of test substance, negative and positive controls.

 

Test System	Mean tissue viability (percentage of control)
Negative control	100
Test substance	95
Positive control	22

 

The standard deviation value of the percentage viability of three tissues treated identically was less than 15%, indicating that the test system functioned properly.

Result of test for reduction of MTT by the test substance:

As no colour change was observed in the assay, it was concluded that test substance did not interact with MTT.

Acceptability of the assay:

 

The test is considered acceptable because it meets the following criteria:

a) The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability should be ≤18.

b) The mean relative tissue viability of the positive control should be ≤ 40% relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤18.

c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤18.

Data evaluation and statistical procedures

A test substance is considered irritant in the skin irritation test if:

The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 h of post incubation is ≤ 50% of the mean viability of the negative controls.

A test substance is considered non-irritant in the in vitro skin irritation test if:

The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 h of post incubation is >50% of the mean viability of the negative controls.

The preceding criteria are not absolute and other modifying factors may enter into the final evaluation decision.

Interpretation of results:

not irritating

Conclusions:

Under the study conditions, the test substance was considered to be non-irritating to skin.

Executive summary:

A study was conducted to determine the skin irritation potential of the test substance in a human three dimensional epidermal model (EPISKIN Small Model (EPISKIN-SMTM)) according to OECD Guideline 439, in compliance with GLP. Skin tissue was moistened with 5 μL of Milli-Q water and 10.0 to 11.7 mg of test substance was applied on top of the tissue for 15 minutes. After a 42 h post-incubation period, determination of the cytotoxic (irritancy) effect was performed using the MTT assay. The relative mean tissue viability, compared to the negative control, was 95%, therefore the substance was considered to be non-irritating. The positive control had a mean cell viability of 22% after 15 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. Under the study conditions, the test substance was considered to be non-irritating to skin (Verbaan, 2014).

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:

From February 10, 2014 to August 12, 2014

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 Ocular Corrosives and Severe Irritants)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The OTWG of ICCVAM and the NICEATM, Background Review Document (BRD): current status of in vitro test methods for identifying ocular corrosives and severe irritants: The Bovine Corneal Opacity and Permeability (BCOP) Test Method, March 2006.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: In Vitro Techniques in Toxicology Database (INVITTOX) protocol 127. Bovine Opacity and Permeability (BCOP) Assay, 2006.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Gautheron P., Dukic M., Alix D. and Sina J.F., Bovine corneal opacity and permeability test: An in vitro assay of ocular irritancy. Fundam Appl Toxicol 18:442-449, 1992.

Deviations:

no

GLP compliance:

yes

Species:

other: Bovine eyes

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

750 µL 10% (w/w) solution of test substance

Duration of treatment / exposure:

10±1 minutes

Observation period (in vivo):

After the completion of the incubation period (i.e., 120±10 minutes) opacity determination was performed

Details on study design:

Preparation of corneas

- The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by submersing them in physiological saline and holding them in the light. Those exhibiting defects were discarded.

- The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Invitrogen Corporation, Breda, The Netherlands) containing 1% (v/v) L-glutamine (Invitrogen Corporation) and 1% (v/v) Foetal Bovine Serum (Invitrogen Corporation)). The isolated corneas were mounted in a corneal holder (i.e., one cornea per holder) of MC2 (Clermont, France) 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.

Irritation parameter:

in vitro irritation score

Run / experiment:

10 minutes

Value:

22

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The corneas treated with the test substance showed opacity values ranging from 4 to 7 and permeability values ranging from 0.603 to 1.752. The corneas were translucent after the 10 minutes of treatment with the test substance. A pH effect of the test substance was observed on the rinsing medium, the corneas were rinsed until no colour change of the medium was observed. Hence, the in vitro irritancy scores ranged from 13 to 33 after 10 minutes of treatment with test substance.

Negative and positive controls:

The individual in vitro irritancy scores for the negative controls ranged from -1.0 to 0.1. The individual positive control in vitro irritancy scores ranged from 164 to 186. The corneas treated with the positive control were turbid after the 10 minutes of treatment.

In Vitro irritancy score

Eye	Negative control correctedFinal Opacity	Negative control correctedFinal OD490	In vitro Irritancy Score^1
Negative control
1	-1	-0.002	-1.0
2	0	-0.005	-0.1
3	0	0.008	0.1
Positive control
4	115	4.596	183.9
5	114	4.806	186.1
6	82	5.430	163.5
Test substance
7	7	1.752	33.3
8	5	1.073	21.1
9	4	0.603	13.0

 

¹ In vitro irritancy score (IVIS) = opacity value + (15 x OD₄₉₀value).

Summary of opacity, permeability and in vitroscores (10% solution, 10 minutes treatment)

Treatment	Mean Opacity	Mean Permeability	Mean In vitro Irritation Score^1, 2
Negative control	0	0.000	0
Positive control	104	4.944	178
Test substnace	5	1.143	22

1 Calculated using the negative control mean opacity and mean permeability values.

2 In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD₄₉₀value).

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Under the study conditions, mean in vitro irritancy score for the test substance was established at 22, and hence no prediction can be made regarding its eye irritation potential.

Executive summary:

A study was conducted to determine the eye irritation potential of the test substance according to OECD Guideline 437 (Bovine Corneal Opacity and Permeability test), in compliance with GLP. The test substance (750 µL of a 10% w/w solution) was applied topically onto the epithelium of the bovine cornea for 10±1 minutes at 32±1°C. After exposure, the corneas were washed with MEM (Minimum Essential Medium), phenol red and cMEM (Eagle's Minimum Essential Medium), then incubated for 120±10 minutes at 32±1°C. Opacity and permeability were determined. The negative control responses for opacity and permeability were within laboratory historical control ranges. The mean in vitro irritancy score of the positive control (10% w/v benzalkonium chloride) was 178, which was also within the historical control range. Under the study conditions, mean in vitro irritancy score for the test substance was established at 22, and hence no prediction can be made regarding its eye irritation potential (Verspeek, 2014).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

A study was conducted to determine the skin irritation potential of the test substance in a human three dimensional epidermal model (EPISKIN Small Model (EPISKIN-SMTM)) according to OECD Guideline 439, in compliance with GLP. Skin tissue was moistened with 5 μL of Milli-Q water and 10.0 to 11.7 mg of test substance was applied on top of the tissue for 15 minutes. After a 42 h post-incubation period, determination of the cytotoxic (irritancy) effect was performed using the MTT assay. The relative mean tissue viability, compared to the negative control, was 95%, therefore the substance was considered to be non-irritating. The positive control had a mean cell viability of 22% after 15 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. Under the study conditions, the test substance was considered to be non-irritating to skin (Verbaan, 2014).

Eye irritation

A study was conducted to determine the eye irritation potential of the test substance according to OECD Guideline 437 (Bovine Corneal Opacity and Permeability test), in compliance with GLP. The test substance (750 µL of a 10% w/w solution) was applied topically onto the epithelium of the bovine cornea for 10±1 minutes at 32±1°C. After exposure, the corneas were washed with MEM (Minimum Essential Medium), phenol red and cMEM (Eagle's Minimum Essential Medium), then incubated for 120±10 minutes at 32±1°C. Opacity and permeability were determined. The negative control responses for opacity and permeability were within laboratory historical control ranges. The mean in vitro irritancy score of the positive control (10% w/v benzalkonium chloride) was 178, which was also within the historical control range. Under the study conditions, mean in vitro irritancy score for the test substance was established at 22, and hence no prediction can be made regarding its eye irritation potential (Verspeek, 2014).

Respiratory irritation

Due to the low vapour pressure of 9-Octadecenoic acid (Z) -, sulfonated, potassium salts, exposure via inhalation is not expected to occur under the conditions of normal and foreseeable handling and use and therefore irritation to the respiratory tract is considered unlikely.

Justification for classification or non-classification

The available in vitro study on 9-Octadecenoic acid (Z)-, sulfonated, potassium salts indicates that the substance is not a skin irritant and does not meet the requirement for classification according to CLP (EC 1272/2008) criteria.

In the available in vitro BCOP eye irritation study, 9-Octadecenoic acid (Z)-, sulfonated, potassium salts did not meet the criteria as corrosive, severe irritant or ‘not classified’. Due to the limitations of the test, the results cannot be used to establish a definitive classification as mildly irritating or irritating to eyes (Category 2, GHS). This is because the test cannot correctly identify all chemicals requiring a Category 1 or non-classification. The following classification is therefore proposed: Eye Irritation Category 2 - H319 (causes serious eye irritation) according to CLP (EC 1272/2008) criteria.