Ammonium 2-ethylhexyl sulphate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2019-06-05 to 2019-06-05

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:

2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 440/2008, Annex Part B, B.40Bis: “In Vitro Skin Corrosion: Human Skin Model Test”, Official Journal of the European Union No. L142, dated May 31st, 2008.

Version / remarks:

2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: INVITTOX Protocol No. 118; “EPISKINTM Skin Corrosivity Test” updated December 2011 / February 2012 (ECVAM Database Service on Alternative Methods to Animal Experimentation).

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Ammonium 2-ethylhexyl sulphate

EC Number:

274-625-4

EC Name:

Ammonium 2-ethylhexyl sulphate

Cas Number:

70495-37-3

Molecular formula:

C8H18O4S.H3N

IUPAC Name:

ammonium 2-ethylhexyl sulfate

Test material form:

cream / paste

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The EPISKIN model has been validated for corrosivity testing in an international trial; it is considered to be suitable for this study (STATEMENT ON THE SCIENTIFIC VALIDITY OF THE EPISKINTM TEST (AN IN VITRO TEST FOR SKIN CORROSIVITY); ECVAM JRC Environment Institute, European Commission; Ispra; 03 April 1998).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM Small Model (EpiSkinTMSM), EPISKIN SNC Lyon, France
- Tissue batch numbers: 19-EKIN-023 (used for first experiment, 4h exposure); 19-EKIN-025 (used for additional experiment, 4h, 1h and 3 min exposure)
- Expiry date: 10 June 2019 (batch no. 19-EKIN-023); 24 June 2019 (bacth no. 19-EKIN-025)

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

REMOVAL OF TEST MATERIAL AND CONTROLS : After the incubation time the EpiSkinTMSM units were rinsed thoroughly with approximately 25 mL PBS 1x solution to remove the test item from the epidermal surface. The rest of the PBS was removed from the epidermal surface with suitable pipette tip linked to a vacuum source (care was taken to avoid the damage of epidermis).

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE :
After the exposure of test item was terminated by rinsing with PBS, the EpiSkinTMSM units were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well) and then incubated for 3 hours (± 5 min) at 37±1 °C in an incubator with 5±1 % CO2 protected from light, ≥95 % humidified atmosphere.
At the end of incubation with MTT a formazan extraction was undertaken:
A disk of epidermis was cut from the unit (this involves the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube of 500 µL acidified isopropanol (one tube corresponding to one well of the tissue culture plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material with the acidified isopropanol then incubated overnight at room temperature protected from light for formazan extraction. At the end of the incubation period, each tube was additionally mixed using a vortex mixer to help extraction.

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE : None. No colour change was observed after three hours of incubation during the check-test for possible direct MTT reduction with test item. The test item did not interact with the MTT, therefore additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin Optional sub category 1A if the viability after 3 minutes exposure is less than 35%
- The test substance is considered to be corrosive to skin Optional sub category 1B and 1C if the viability after 3 minutes exposure is less than or equal 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 35% and the viability after 4 hours exposure is less than 35%.
- The test substance is considered to be non-corrosive to skin if the viability after 4 hours exposure is greater than or equal to 35%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied:
The test item is a highly viscous material. Therefore exact weighting of treatment volume was not be performed. The necessary volume of the test item which can be covered the epidermal surface totally (52.6 mg/cm2) was applied evenly to the epidermal surface of the two test skin units respectively. Subsequently, 100 µL NaCl solution (9 g/L saline) was added topically (to each unit) to ensure good contact with the epidermis. The test item was spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary.

Duration of treatment / exposure:

First experiment: 4 hours
Additional experiment: 4 hours, 3 minutes and 1 hour

Number of replicates:

2

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean (2 replicates)

Value:

9

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

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

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes. The technical proficiency was demonstrated in a separate study (study no.: 392-431-4224) using the twelve Proficiency Chemicals according to OECD Test Guideline No. 431.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. In the first experiment the mean OD value of the two negative control tissues was 0.728 after 4 hours exposure. In the additional experiment the mean OD value of the two negative control tissues was 1.014 at 4 hours exposure, 1.065 at 1 hour exposure and 1.058 at 3 minutes exposure.
- Acceptance criteria met for positive control: Yes. In the first experiment the positive control result showed 1 % viability. In the additional experiment the positive control result showed 3 % viability.
- Acceptance criteria met for variability between replicate measurements: Yes. In the first rexperiment the difference of viability between the two tissue replicates was 0.7 % to 9.6 %. In the additional experiment the difference of viability between the two tissue replicates was 0.0% at 4 hours exposure, 2.6% to 7.8% at 1 hour exposure and 0.4 % to 5.3 % at 3 minutes exposure.

Any other information on results incl. tables

Results of first experiment

Table 1: OD values and viability percentages of the controls (first experiment)

Controls	Optical Density (OD)		Viability (%)	Δ%
Negative Control: NaCl (9 g/L saline)	1	0.693	95	96
	2	0.763	105
	mean	0.728	100
Positive Control: Glacial acetic acid	1	0.013	2	0.7
	2	0.007	1
	mean	0.010	1

 

 Table 2: OD values and viability percentages of the test item (incudinng corrected values; first experiment):

Controls	Optical Density (OD)		Viability (%)	Δ%
Test item: Ammonium 2-ethylhexyl sulfate	1	0.073	10	2.1
	2	0.057	8
	mean	0.065	9

 Remark Δ%: The difference of viability between the two relating tissues.

Results of additional experiment

Table 3: OD values and viability percentages of the controls (additional experiment)

Controls	Optical Density (OD)		Viability (%)	Δ%
Negative Control: NaCl (9 g/L saline) 4 h exposure time	1	1.014	100	0.0
	2	1.014	100
	mean	1.014	100
Negative Control: NaCl (9 g/L saline) 1 h exposure time	1	1.078	101	2.6
	2	1.051	99
	mean	1.065	100
Negative Control: NaCl (9 g/L saline) 3 min exposure time	1	1.086	103	5.3
	2	1.030	97
	mean	1.058	100
Positive Control: Glacial acetic acid 4 h exposure time	1	0.035	3	0.0
	2	0.035	3
	mean	0.035	3

Table 4: OD values and viability percentages of the test item (additional experiment)

Controls	Optical Density (OD)		Viability (%)	Δ%
Test item: Ammonium 2-ethylhexyl sulfate 4 h exposure time	1	0.118	12	0.0
	2	0.118	12
	mean	0.118	12
Test item: Ammonium 2-ethylhexyl sulfate 1 h exposure time	1	0.118	11	7.8
	2	0.200	19
	mean	0.159	15
Test item: Ammonium 2-ethylhexyl sulfate 3 min exposure time	1	0.182	17	0.4
	2	0.178	17
	mean	0.180	17

 RemarkΔ%: The difference of viability between the two relating tissues.

Applicant's summary and conclusion

Interpretation of results:

Category 1A (corrosive) based on GHS criteria

Conclusions:

Based on the results obtained under the conditions of this study according to OECD guideline 431, the test item is considered as corrosive to skin in accordance with UN GHS (optional sub-category 1A), as the mean percentage tissue viability was significantly reduced (below 35%) in comparison to the negative control after 4 hours, 1 hour and 3 minutes.

Executive summary:

The skin corrotion potential of ammonium 2-ethylhexyl sulfate was evaluated using the in vitro EpiSkinTM SM test by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 431.
According to the first experiment results, the test item was corrosive after 4 hours (±10 min) exposition. Therefore, an additional experiment was necessary with additional exposure times [testing at 3 minutes and 1 hour (±5 min)]. In this case, the 4 hours procedure was repeated during the additional experiment, but with additional 3 minutes and 1 hour exposure periods. Disks of EPISKIN (two units) were treated with test item and incubated for 4 hours (±10 min) at room temperature on June 05, 2019 (first experiment). Furthermore, disks of EPISKIN (two units / exposure time) were treated with the test item and incubated for 4 hours (±10 min), 1 hour (±5 min) and 3 min at room temperature on June 19, 2019 (additional experiment). Exposure of the test material was terminated by rinsing with PBS 1x solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37±1 °C in 5±1 % CO2 in a ≥ 95 % humidified atmosphere and protected from light. The formazan precipitated was then extracted using acidified isopropanol and quantified spectrophotometrically. NaCl (9 g/L saline) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively. For each treated tissue viability was expressed as a % relative to negative control. The test item is considered to be non-corrosive to skin, if the mean relative viability after 4 hours of exposure is above or equal 35 % of the negative control. The test item showed significantly reduced cell viability (below 35 %) in comparison to the negative control after 4 hours of exposure in first and additional experiment. In the first experiment (June 06, 2019) the average test item treated tissue relative viability was 9 % at 4 hours of exposure and in the additional experiment (June 20, 2019) it was 12 % at 4 hours of exposure. In the additional experiment (June 20, 2019), the test item treated tissue viabilities were below 35 % of the mean negative control value after 1 hour and 3 min of exposure. The average test item treated tissue relative viability was 15 % at 1 hour of exposure and 17 % at 3 minutes of exposure.
In conclusion, in this in vitro skin corrosion test in EPISKIN model (OECD 431) with Ammonium 2-ethylhexyl sulfate the results indicate that the test item is corrosive to skin after 4 hours, 1 hour and 3 minutes of exposure time. According to the UN GHS classification systems, Ammonium 2-ethylhexyl sulfate has been categorized as “Corrosive: Optional Sub- category 1A”.