Ammonium 2-ethylhexyl sulphate

Endpoint summary

Description of key information

Skin irritation/corrosion:

In an in vitro skin irritation study according to OECD guideline 439 the test substance was identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1).

In an in vitro skin corrosion study according to OECD guideline 431, the test item was 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.

A study with the trade product according to OECD guideline 431 was performed. The trade products was determined to be corrosive after 4 hours and 1 hour exposure, but not after 3 minutes. Therefore, the trade product is to be classified as “Corrosive: Optional Sub-categories 1B and 1C”.

Eye irriation:

Based on the results obtained in the Bovine Corneal Opacity and Permeability Test (OECD 437), the test item induced an IVIS of 47.4 after 10 minutes of treatment. As the test item induced an IVIS >3 and <55, the resulting classification is equivocal and no prediction can be made regarding the corrosivity or severe irritancy to Bovine corneas.

 

Based on the results obtained using the EpiOcular™ model (OECD 492), the test item Ammonium 2 -ethylhexyl sulfate is identified requiring classification and labeling UN GHS UN GHS Category 1 or Category 2 as the mean percentage tissue viability is less than 60 % of the negative control.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

no

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

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 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

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.

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.

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”.
 
 

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2019-06-13 to 2019-06-15

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)

Version / remarks:

July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM DB-ALM Protocol n° 131 (09 June 2012): EpiSkinTM Skin Irritation Test 15 min – 42 hours

Version / remarks:

June 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

other: reconstructed human epidermis

Cell type:

non-transformed keratinocytes

Justification for test system used:

The EPISKIN model has been validated for irritation testing in an international trial and is considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM Small Model (EpiSkinTMSM), manufactured by EPISKIN SNC Lyon, France
- Tissue batch number: 19-EKIN-024
- Expiry date: 17 June 2019

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS : After the incubation time the EpiSkinTMSM units were removed and rinsed thoroughly with approximately 25 mL PBS 1x solution to remove all of the test material 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
- MTT concentration: 2 mL of 0.3 mg/mL MTT per well
- Incubation time: 3 hours
- Spectrophotometer: Thermo Scientific; Multiscan FC
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE : None. No colour change was observed after three hours of incubation. The test material 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
- The test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean tissue viability after exposure and post-treatment incubation is ≤50%.
- The test substance is considered as non-irritant to skin in accordance with UN GHS (No Category), if the tissue viability after exposure and post-treatment incubation is >50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied:
The epidermal surface was first moistened with 5 µL deionised water in order to improve further contact between powder and epidermis. Subsequently, 10 mg of the test item was applied evenly to the epidermal surface. The test item was spread gently with a curved flat spatula in order to cover evenly all the skin surface if necessary.

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Remarks:

Mean (3 replicates)

Run / experiment:

8

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: standard deviation 1.99

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.: 392.554.2938) using the ten Proficiency Chemicals according to OECD Test Guideline No. 439.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. The mean OD of the three negative control tissues was 0.923.
- Acceptance criteria met for positive control: Yes. The mean oD value obtained for the positive control was 0.098 and this result corresponds to 11% viability when compared to the results obtained from the negative control.
- Acceptance criteria met for variability between replicate measurements: Yes. Each calculated standard deviation value (SD) for the % viability was below 18.

Table 1: OD values and viability percentages of the controls and test item

Substance	Optical density (OD)		Viability (%)
Negative Contol: 1 x PBS	1	0.967	105
	2	0.980	106
	3	0.822	89
	Mean	0.923	100
	Standard deviation (SD)		9.46
Positive control: SDS (5% aq.)	1	0.090	10
	2	0.120	13
	3	0.085	9
	Mean	0.098	11
	Standard deviation (SD)		2.08
Test Item: Ammonium 2-ethylhexyl sulfate	1	0.065	7
	2	0.059	6
	3	0.094	10
	Mean	0.073	8
	Standard deviation (SD)		1.99

Interpretation of results:

other: Category 1 or 2

Conclusions:

As a result of the available study, the test substance was identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1). Further information is required to distinguish between Category 1 and 2.

Executive summary:

The skin irritation 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. 439.
Disks of EPISKIN (three units) were treated with test item and incubated for 15 minutes (± 0.5 min) at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37±1°C for 42 hours (± 1h) in an incubator with 5±1% CO2, ≥95% humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours (± 5 min) with MTT solution at 37±1°C in 5±1% CO2 protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically.
SDS (5% aq.) and 1×PBS treated (three units / positive and negative control) epidermis were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control.
The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control.
The test item showed significantly reduced cell viability in comparison to the negative control (mean value: 8 %). All obtained test item viability results were below 50 % when compared to the viability values obtained from the negative control. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.
The results obtained from this in vitro skin irritation test, using the EPISKIN model, with the test item ammonium 2-ethylhexyl sulfate indicated that the test item is Irritant (UN GHS Category 2) and/or Corrosive (UN GHS Category 1). Since this test method (OECD 439) cannot resolve between UN GHS Categories 1 and 2, further testing is necessary.

Reference 3

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2020-01-07 to 2020-05-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 14 June 2019

Deviations:

no

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

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 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: 20-EKIN-003 (used for first experiment, 4 h exposure); 20-EKIN-008 (used for additional experiment, 4 h, 1 h and 3 min exposure)
- Expiry date: 20 January 2020 (batch no. 20-EKIN-003); 24 February 2020 (bacth no. 20-EKIN-008)

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 1xPBS 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.

Following the formazan extraction, 2×200 μL sample from each tube was placed into the wells of a 96-well plate (labelled appropriately) and read the OD (Absorbance / Optical Density) of the samples in a 96-well plate spectrophotometer at 570 nm (± 10 nm; Read out range: 0 - 3.5 Abs, Linearity range: 0.2720 – 3.4218) using acidified isopropanol solution as the blank (6×200 μL).

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: The test item has an intrinsic colour (light yellow). If the test item is not white, off white, almost white and/or colorless the additional controls are automatically used and based on the OD results of additional controls the Non Specific Colour % (NSCliving %) is determined. This step prevents the false viability which can possibly be caused by the stained surface of the tissues by the test item.

In addition to the normal procedure, two additional test item-treated tissues were used for the non-specific OD evaluation. These tissues followed the same treatment steps as the other tissues except for the MTT step: MTT incubation was replaced by incubation with fresh assay medium. OD reading was made following the same conditions as for the other tissues.

PREDICTION MODEL / DECISION CRITERIA
- 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: A volume of 50 μL test item was applied evenly to the epidermal surface of the two test skin units, respectively. The test item was spread gently with the pipette tip in order to cover evenly all the epidermal surface.

Duration of treatment / exposure:

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

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean (2 replicates)

Run / experiment:

3 minutes

Value:

104

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean (2 replicates)

Run / experiment:

1 h

Value:

28

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean (2 replicates)

Run / experiment:

4 h

Value:

6

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 1.063 after 4 hours exposure. In the additional experiment the mean OD value of the two negative control tissues was 0.708 at 4 hours exposure, 0.631 at 1 hour exposure and 0.795 at 3 minutes exposure.
- Acceptance criteria met for positive control: Yes. In the first experiment the positive control result showed 2 % viability. In the additional experiment the positive control result showed 8 % viability.
- Acceptance criteria met for variability between replicate measurements: Yes. In the first experiment the difference of viability between the two tissue replicates was 0.3 % to 5.5 %. In the additional experiment the difference of viability between the two tissue replicates was 0.1 % to 3.8 % at 4 hours exposure, 4.5 % to 11.1 % at 1 hour exposure and 13.0 % to 24.1 % at 3 minutes exposure.

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	1.034	97	5.5
	2	1.093	103
	mean	1.063	100
Positive Control: Glacial acetic acid	1	0.022	2	0.4
	2	0.026	2
	mean	0.024	2

 

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

Test item	Optical Density (OD)		Viability (%)	Δ%
Thiotan RMFM eco liq	1	0.099	9	0.3
	2	0.103	10
	mean	0.101	9

 

 Table 3: OD values and NSC_living % of additional control (first experiment)

Test item	Optical Density (OD)		Non Specific Colour % (NSCliving %)	Δ%
Thiotan RMFM eco liq	1	0.005		0.2
	2	0.002	0.34
	mean	0.004

Remark: Δ%: The difference of viability between the two relating tissues
Mean blank OD values were 0.039
Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

 

Results of additional experiment

 

Table 4: 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	0.716	101	2.3
	2	0.700	99
	mean	0.708	100
Negative Control: NaCl (9 g/L saline) 1 h exposure time	1	0.645	102	4.5
	2	0.617	98
	mean	0.631	100
Negative Control: NaCl (9 g/L saline) 3 min exposure time	1	0.846	106	13.0
	2	0.743	94
	mean	0.795	100
Positive Control: Glacial acetic acid 4 h exposure time	1	0.067	9	3.8
	2	0.040	6
	mean	0.053	8

 

Table 5: OD values and viability percentages of the test item (additional experiment)

Test item	Optical Density (OD)		Viability (%)	Δ%
Thiotan RMFM eco liq 4 h exposure time	1	0.046	7	0.1
	2	0.046	6
	mean	0.046	6
Thiotan RMFM eco liq 1 h exposure time	1	0.139	22	11.1
	2	0.209	33
	mean	0.174	28
Thiotan RMFM eco liq 3 min exposure time	1	0.729	92	24.1
	2	0.920	116
	mean	0.824	104

 

 Table 6: OD values and NSC_living % of additional control (additional experiment)

Test item	Optical Density (OD)		Non Specific Colour % (NSCliving %)	Δ%
Thiotan RMFM eco liq 4 h	1	0.012		0.8
	2	0.018	2.15
	mean	0.015
Thiotan RMFM eco liq 1 h	1	0.020		0.6
	2	0.016	2.91
	mean	0.018
Thiotan RMFM eco liq 3 min	1	0.014		0.9
	2	0.007	1.28
	mean	0.010

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

Mean blank OD values were 0.039 (plate 1) and 0.038 (plate 2)
Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

Interpretation of results:

other: other: Category 1B or 1C (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 1B and 1C), as the mean percentage tissue viability was significantly reduced (below 35 %) in comparison to the negative control after 4 hours and 1 hour but not after 3 minutes.

Executive summary:

EpiSkinTM SM test of the test item Thiotan RMFM eco liq has been performed to predict its corrosion potential by measurement of its cytotoxic effect, as reflected in the MTT assay according to the OECD TG 431.

According to the first experiment results, the test item was corrosive after 4 hours (± 10 min) exposure. 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 the test item and incubated for 4 hours (± 10 min) at room temperature (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 (additional experiment).

Exposure of the test material was terminated by rinsing with 1x PBS 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.

The test item has an intrinsic colour (light-yellow), therefore, two additional test item treated tissues were used for the non-specific OD evaluation (NSCliving).

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 to 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 the first and after 4 hours and 1 hour of exposure in the additional experiment. In the first experiment the average test item treated tissue relative viability was 9 % at 4 hours of exposure and in the additional experiment the average test item treated tissue relative viability was 6 % at 4 hours of exposure and it was 28 % at 1 hour of exposure.

In the additional experiment, the test item treated tissue viabilities after 3 min of exposure were above 35 % of the mean negative control value. The average test item treated tissue relative viability was 104 % at 3 minutes of exposure.

Positive and negative controls showed the expected cell viability values within acceptable limits in the first and in the additional experiment. All assay acceptance criteria were met, the experiment was considered to be valid.

In conclusion, the results indicate that the test item is corrosive to skin after 4 hours and 1 hour of exposure time and not corrosive after 3 minutes of exposure time. According to the UN GHS classification systems, Thiotan RMFM eco liq has been categorized as “Corrosive: Optional Sub-categories 1B and 1C”.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-05-07 to 2019-05-07

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 i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

9th October 2017

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)

Version / remarks:

9th December 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Tumkur slaughter house
- Characteristics of donor animals: 3.5 to 4.5 years old
- Storage, temperature and transport conditions of ocular tissue: To prevent exposure of the eyes to potentially irritant substances, the heads of the animals were not rinsed with detergent. Eyes were enucleated as soon as possible after death and immersed in the Hank's Balanced Salt Solution (HBSS) with 1% antibiotics (Penicillin and Streptomycin) in a suitable container and were transported to the test facility by placing in cool packs.

Vehicle:

water

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 750 µL
- Concentration: 10% w/v

VEHICLE
- Amount applied: 750 µL of distilled water

Duration of treatment / exposure:

The corneas were incubated for an exposure period of approximately 10 minutes. Similar procedure was followed for the vehicle and positive controls.

Duration of post- treatment incubation (in vitro):

1 hour and 55 minutes

Number of animals or in vitro replicates:

3

Details on study design:

QUALITY CHECK OF THE ISOLATED CORNEAS : Upon arrival to the test facility, eyes were examined for defects including opacity, scratches and neovascularization. Only corneas free of such defects were used in the experiment.

NUMBER OF REPLICATES : 3

NEGATIVE CONTROL USED : No

SOLVENT CONTROL USED: Yes (distilled water)

POSITIVE CONTROL USED : Yes, Imidazole

APPLICATION DOSE AND EXPOSURE TIME : 750 µL, 10 min exposure

TREATMENT METHOD: closed chamber

REMOVAL OF TEST SUBSTANCE : After the exposure periode the test item and the vehicle and positive control substance were removed from the anterior chamber and the epithelium was washed with EMEM containing phenol red until no visual evidence of the test item was observed.

- POST-EXPOSURE INCUBATION: yes, 1 hour and 55 minutes

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
A test item was classified based on the obtained IVIS and categorized as mentioned below:
- Test item of IVIS score <3 would be considered as not requiring classification for eye irritation or serious eye damage according to UN GHS.
- Test item of IVIS score >3 and <55 would be considered as equivocal, subsequently testing with any other adequate method remains at the discretion of the sponsor.
- Test item of IVIS score >55 would be considered as severe irritant causing serious eye damage and classified as UN GHS category 1 without further testing.
A single testing run was considered as sufficient for assessment as the results were met according to the following criteria:
- Two of the three replicates did not give discordant predictions from the mean.
- None of the three replicates gave discordant prediction from the mean of all three rep-licates and the IVIS of the discordant replicate was not higher than 65.

Irritation parameter:

in vitro irritation score

Remarks:

mean

Run / experiment:

1-3

Value:

47.4

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Study Acceptance Criteria

The study was accepted:
- As the vehicle/negative control response resulted in opacity and permeability values less than the established upper limits of background opacity and permeability values for bovine corneas treated with the respective negative/vehicle control.
- As the positive control gave an IVIS that falls within two standard deviations of the historical mean.

Interpretation of results:

other: Equivocal

Conclusions:

Based on the results obtained in the Bovine Corneal Opacity and Permeability Test, the test item Ammonium 2-ethylhexyl sulfate induced an IVIS of 47.4 after 10 minutes of treatment. As the test item induced an IVIS >3 and <55, the resulting classification is equivocal and no prediction can be made regarding the corrosivity or severe irritancy to Bovine corneas.

Executive summary:

The test item, Ammonium 2-ethylhexyl sulfate was evaluated for ocular corrosivity or severe irritancy according to OECD guideline 437. Eyes of cattle were collected from a slaughter house by immersing them in the Hank's Balanced Salt Solution (HBSS) with antibiotics (penicillin and streptomycin) in a suitable container and transported to the test facility by placing on cool packs. Eye balls free of defects were selected for the experiment. Empty cornea holder's opacity with pre-warmed Eagle's Minimum Essential Medium was measured and the mean opacity value obtained was determined as I0. Cornea holders with selected Corneas were equilibrated at 32±1°C for 1 hour 3 minutes with Eagle's Minimum Essential Medium with 1% Fetal Bovine Serum supplemented with 1% antibiotics and baseline opacity was recorded for each cornea. Corneas with opacity units less than 7 were selected and used for the study and distributed for the treatment groups.

A volume of 750 µL of 10% w/v test item, vehicle (distilled water) and positive control (20% w/v imidazole) was introduced into anterior chamber in triplicates to the designated cornea holders and incubated at 32±1 °C for 10 minutes. Treated corneas were washed till no visual evidence of test item observed with EMEM containing phenol red and finally with EMEM without phenol red. The anterior chamber was then refilled with fresh EMEM without phenol red. Opacity was measured with the aid of opacitometer and permeability was determined spectrophotometrically at 490 nm (OD490) using 4 mg/mL sodium fluorescein, post incubation of 85 minutes at 32±1 °C.

The test item resulted in the mean corrected opacity and mean corrected permeability values of 36.93 and 0.700, respectively. The in vitro Irritancy Score (IVIS) of test item resulted in 47.4 and thus the result is considered as equivocal (no prediction can be made). Whereas the positive control resulted in mean corrected opacity and mean corrected permeability values of positive control are 83.56 and 1.601 respectively where the in vitro Irritancy Score (IVIS) of 107.6, indicating corrosivity or severe irritancy to Bovine cornea.

Based on the results obtained in the Bovine Corneal Opacity and Permeability Test, the test item Ammonium 2-ethylhexyl sulfate induced an IVIS of 47.4 after 10 minutes of treatment. As the test item induced an IVIS >3 and <55, the resulting classification is equivocal and no prediction can be made regarding the corrosivity or severe irritancy to Bovine corneas.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2019-06-05 to 2019-06-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Strain:

not specified

Details on test animals or tissues and environmental conditions:

- Justification of the test method :
The EpiOcular™ Eye Irritation Test (EIT) was validated by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 2013. It was concluded that the EpiOcular™ EIT is able to correctly identify substances and mixtures not requiring classification and labelling for eye irritation or serious eye damage according to UN GHS, and the test method was recommended as scientifically valid for that purpose.

- Description of the cell system used:
The EpiOcular™ human cell construct (OCL-200-EIT) procured from MatTek Corporation was used as test system. EpiOcular™ human cell construct (EPI-200-SIT) is composed of stratified human keratinocytes in a three-dimensional structure. Its use for ocular irritation potential involves the topical application of test materials to the surface of the EpiOcular™ human cell construct, and the subsequent assessment of their effects on cell viability. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT (Fentem et al., 1998).

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 mg

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

25 minutes (post-exposure soak) + 18 hours and 10 minutes (post-treatment incubation)

Number of animals or in vitro replicates:

2 replicates per test item, negative control and positive control, respectively.

Details on study design:

- RhCE tissue construct used : Reconstructed Human EpiOcular™ tissues (OCL-200), Lot No: 30609
- Doses of test chemical and control substances used : 50 mg of test item, 50 µL of negative control and positive control
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods:
exposure: 6 hours, 37 °C; post-exposure immersion (post-soak): 25 minutes, room temperature; post-exposure incubation: 18 hours and 10 minutes, 37 °C
- Number of tissue replicates used per test chemical and controls: Test item; 2 tissue replicates; positive control: 2 tissue replicates; negative control: 2 tissue replicates
- Wavelength used for quantifying MTT formazan: 570 nm
- Description of the method used to quantify MTT formazan : The optical density (OD) of the MTT extracts in a 96-well plate was read in plate reader at wavelength of 570 nm.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean percent tissue viability after exposure and post-exposure incubation is less than or equal (≤) to 60 % of the negative control. However, the OECD 492 cannot resolve between UN GHS Categories 1 and 2, further testing with other test methods will be required to decide on its final classification.
Depending on the regulatory framework in member countries, the test chemical is identified as not requiring classification and labelling according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60 %. In this case no further testing in other test methods is required.
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals : Yes
- Acceptable variability between tissue replicates for positive and negative controls : yes
- Acceptable variability between tissue replicates for the test chemical: yes

Irritation parameter:

other: mean percent viability [%]

Run / experiment:

1-2

Value:

10

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: None

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

Table 1: Summary of optical density (OD) and viability (%)

Treatment		OD	Viability (%)	Viability difference between tissues	Classification
Negative Control (Sterile deionized water)	Mean	2.086	100.00	0.44	No Category
	±SD	0.019	0.89
	n	2	2	2
Positive Control (Methyl acetate)	Mean	0.043	2.0	0.04	Cat 2 or Cat 1
	±SD	0.002	0.07
	n	2	2	2
Test item	Mean	0.210	10.0	0.02	Cat 2 or Cat 1
	±SD	0.001	0.05
	n	2	2	2

Interpretation of results:

other: UN GHS Category 1 or Category 2

Conclusions:

Based on the results obtained and under the laboratory testing conditions using the EpiOcular™ model, the test item Ammonium 2-ethylhexyl sulfate is identified requiring classification and labeling UN GHS Category 1 or Category 2 as the mean percentage tissue viability is less than 60% of the negative control.

Executive summary:

The eye irritation potential of Ammonium 2-ethylhexyl sulfate was evaluated using the in vitro EpiOcular™ model (OCL-200-EIT) according to OECD guideline 492.

The test item did not develop any colour when dissolved in distilled water/isopropanol and was considered as non-reducer of MTT as no purple colour was developed when mixed and incubated with MTT solution. Tissues were equilibrated for 15 minutes at room temperature and then transferred to 6-well plates prefilled with 1.0 mL of assay medium and incubated in CO2 incubator at 37±1°C and 5±1% CO2 for 17 hours and 20 minutes. Tissues were pre-wetted with 20 µL DPBS and tapped to ensure that DPBS spreads all over the tissues surface. The tissues were incubated at standard culture conditions for 30 minutes. Post 30 minutes of incubation, 50 mg each of test item, 50 µL of negative control and positive control were dispensed directly atop EpiOcular™ tissues at 1 minute intervals to facilitate rinsing after exposure. The tissues were incubated at standard culture conditions for 6 hours. All the treatments were maintained in duplicates. At the end of treatment time with test item, negative control and positive control, tissue inserts were dipped into the first tubes of DPBS, swirled in a circular motion in the liquid for 2 seconds, lifted out so that the inserts were mostly filled with DPBS, and the liquid was decanted back into the container. This process was performed two additional times in the first tube. The cultures were then rinsed in the second and third tubes of DPBS. After rinsing, the tissues were immediately transferred to and immersed in 5 mL of previously warmed assay medium in a prelabeled 12-well plate for 25 minutes immersion incubation (Post-Soak) at room temperature which was intended to remove any test item absorbed. At the end of the post-soak immersion, each insert was removed from the assay medium, the medium was decanted off the tissue, and the insert was blotted onto the absorbent material, and transferred to the appropriate well of the prelabeled 6-well plate containing 1 mL of warm assay medium. The tissues were incubated for 18 hours and 10 minutes in CO2 incubator at 37±1°C and 5±1% CO2 (Post-treatment Incubation). Post 18 hours and 10 minutes of incubation with the assay medium, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into 24-well plate containing 0.3 mL of MTT solution (1 mg/mL) and incubated for 3 hours at 37±1°C and 5±1% CO2. Post incubation, the tissue inserts were removed and blotted onto the tissue paper and transferred to a prelabeled 24-well plate containing 2.0 mL of isopropanol in each designated. The plates were placed on an orbital plate shaker and shaken for 2 hours at room temperature. At the end of the extraction period, the tissue was pierced with an injection needle and the liquid within each insert was decanted into the well from which it was taken.The optical density of the extracted formazan was measured in 96-well plate spectrophotometer at 570 nm. Viability of tissues was calculated.

Mean percentage viability of negative control, positive control and test item were 100±0.89, 2.0±0.07 and 10.0±0.05 respectively. As the mean percentage viability of the test item was less than 60% of the negative control it is identified as requiring classification and labeling UN GHS (Cat 2 or Cat 1). Similarly the percentage viability of positive control is less than 50% of negative control. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.
Based on the results obtained and under the laboratory testing conditions using the EpiOcular™ model , the test item Ammonium 2-ethylhexyl sulfate is identified requiring classification and labeling UN GHS UN GHS Category 1 or Category 2 as the mean percentage tissue viability is less than 60% of  the  negative control.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

OECD 439

The skin irritation 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. 439.
Disks of EPISKIN (three units) were treated with test item and incubated for 15 minutes (± 0.5 min) at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37 ± 1 °C for 42 hours (± 1h) in an incubator with 5 ± 1 % CO2, ≥95 % humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours (± 5 min) with MTT solution at 37 ± 1 °C in 5 ± 1 % CO2 protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically.
SDS (5 % aq.) and 1×PBS treated (three units / positive and negative control) epidermis were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control.
The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % of the negative control.
The test item showed significantly reduced cell viability in comparison to the negative control (mean value: 8 %). All obtained test item viability results were below 50 % when compared to the viability values obtained from the negative control. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.
The results obtained from this in vitro skin irritation test, using the EPISKIN model, with the test item ammonium 2-ethylhexyl sulfate indicated that the test item is Irritant (UN GHS Category 2) and/or Corrosive (UN GHS Category 1). Since this test method (OECD 439) cannot resolve between UN GHS Categories 1 and 2, further testing was necessary.

 

Skin corrosion

OECD 431

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”.
 

The test item was evaluated for its skin irritation (OECD 439) and skin corrosion potential (OECD 431) in validated in vitro test methods using a bottom-up approach. The results obtained from this in vitro skin irritation test (OECD 439), using the EPISKIN model, with the test item Ammonium 2-ethylhexyl sulfate indicated that the test item is irritant (UN GHS Category 2) or corrosive (UN GHS Category 1). This test method (OECD 439) cannot resolve between UN GHS Categories 1 and 2. However, the results obtained from this in vitro skin corrosion test (OECD 431), using the EPISKIN model, indicated that the test item is corrosive to skin after 4 hours, 1 hour and 3 minutes of exposure time under the utilised testing conditions. In conclusion, the test item Ammonium 2 -ethylhexyl sulfate can be classified as corrosive and is categorized as “Corrosive: Optional Sub- category 1A”.

 

OECD 431 - Trade product

EpiSkinTM SM test of the test item Thiotan RMFM eco liq (trade product) has been performed to predict its corrosion potential by measurement of its cytotoxic effect, as reflected in the MTT assay according to the OECD TG 431.

According to the first experiment results, the test item was corrosive after 4 hours (± 10 min) exposure. 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 the test item and incubated for 4 hours (± 10 min) at room temperature (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 (additional experiment).

Exposure of the test material was terminated by rinsing with 1x PBS 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.

The test item has an intrinsic colour (light-yellow), therefore, two additional test item treated tissues were used for the non-specific OD evaluation (NSCliving).

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 to 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 the first and after 4 hours and 1 hour of exposure in the additional experiment. In the first experiment the average test item treated tissue relative viability was 9 % at 4 hours of exposure and in the additional experiment the average test item treated tissue relative viability was 6 % at 4 hours of exposure and it was 28 % at 1 hour of exposure.

In the additional experiment, the test item treated tissue viabilities after 3 min of exposure were above 35 % of the mean negative control value. The average test item treated tissue relative viability was 104 % at 3 minutes of exposure.

Positive and negative controls showed the expected cell viability values within acceptable limits in the first and in the additional experiment. All assay acceptance criteria were met, the experiment was considered to be valid.

In conclusion, the results indicate that the test item is corrosive to skin after 4 hours and 1 hour of exposure time and not corrosive after 3 minutes of exposure time. According to the UN GHS classification systems, Thiotan RMFM eco liq has been categorized as “Corrosive: Optional Sub-categories 1B and 1C”.

 

Eye irritation

OECD 437

The test item, Ammonium 2-ethylhexyl sulfate was evaluated for ocular corrosivity or severe irritancy according to OECD guideline 437. Eyes of cattle were collected from a slaughter house by immersing them in the Hank's Balanced Salt Solution (HBSS) with antibiotics (penicillin and streptomycin) in a suitable container and transported to the test facility by placing on cool packs. Eye balls free of defects were selected for the experiment. Empty cornea holder's opacity with pre-warmed Eagle's Minimum Essential Medium was measured and the mean opacity value obtained was determined as I0. Cornea holders with selected Corneas were equilibrated at 32 ± 1 °C for 1 hour 3 minutes with Eagle's Minimum Essential Medium with 1% Fetal Bovine Serum supplemented with 1 % antibiotics and baseline opacity was recorded for each cornea. Corneas with opacity units less than 7 were selected and used for the study and distributed for the treatment groups.

A volume of 750 µL of 10 % w/v test item, vehicle (distilled water) and positive control (20 % w/v imidazole) was introduced into anterior chamber in triplicates to the designated cornea holders and incubated at 32 ± 1 °C for 10 minutes. Treated corneas were washed till no visual evidence of test item observed with EMEM containing phenol red and finally with EMEM without phenol red. The anterior chamber was then refilled with fresh EMEM without phenol red. Opacity was measured with the aid of opacitometer and permeability was determined spectrophotometrically at 490 nm (OD490) using 4 mg/mL sodium fluorescein, post incubation of 85 minutes at 32 ± 1 °C.

The test item resulted in the mean corrected opacity and mean corrected permeability values of 36.93 and 0.700, respectively. The in vitro Irritancy Score (IVIS) of test item resulted in 47.4 and thus the result is considered as equivocal (no prediction can be made). Whereas the positive control resulted in mean corrected opacity and mean corrected permeability values of positive control are 83.56 and 1.601 respectively where the in vitro Irritancy Score (IVIS) of 107.6, indicating corrosivity or severe irritancy to Bovine cornea.

Based on the results obtained in the Bovine Corneal Opacity and Permeability Test, the test item Ammonium 2-ethylhexyl sulfate induced an IVIS of 47.4 after 10 minutes of treatment. As the test item induced an IVIS >3 and <55, the resulting classification is equivocal and no prediction can be made regarding the corrosivity or severe irritancy to Bovine corneas.

 

OECD 492

The eye irritation potential of Ammonium 2-ethylhexyl sulfate was evaluated using the in vitro EpiOcular™ model (OCL-200-EIT) according to OECD guideline 492. The test item did not develop any colour when dissolved in distilled water/isopropanol and was considered as non-reducer of MTT as no purple colour was developed when mixed and incubated with MTT solution. Tissues were equilibrated for 15 minutes at room temperature and then transferred to 6-well plates prefilled with 1.0 mL of assay medium and incubated in CO2 incubator at 37 ± 1 °C and 5 ± 1 % CO2 for 17 hours and 20 minutes. Tissues were pre-wetted with 20 µL DPBS and tapped to ensure that DPBS spreads all over the tissues surface. The tissues were incubated at standard culture conditions for 30 minutes. Post 30 minutes of incubation, 50 mg each of test item, 50 µL of negative control and positive control were dispensed directly atop EpiOcular™ tissues at 1 minute intervals to facilitate rinsing after exposure. The tissues were incubated at standard culture conditions for 6 hours. All the treatments were maintained in duplicates. At the end of treatment time with test item, negative control and positive control, tissue inserts were dipped into the first tubes of DPBS, swirled in a circular motion in the liquid for 2 seconds, lifted out so that the inserts were mostly filled with DPBS, and the liquid was decanted back into the container. This process was performed two additional times in the first tube. The cultures were then rinsed in the second and third tubes of DPBS. After rinsing, the tissues were immediately transferred to and immersed in 5 mL of previously warmed assay medium in a prelabeled 12-well plate for 25 minutes immersion incubation (Post-Soak) at room temperature which was intended to remove any test item absorbed. At the end of the post-soak immersion, each insert was removed from the assay medium, the medium was decanted off the tissue, and the insert was blotted onto the absorbent material, and transferred to the appropriate well of the prelabeled 6-well plate containing 1 mL of warm assay medium. The tissues were incubated for 18 hours and 10 minutes in CO2 incubator at 37 ± 1 °C and 5 ± 1 % CO2 (Post-treatment Incubation). Post 18 hours and 10 minutes of incubation with the assay medium, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into 24-well plate containing 0.3 mL of MTT solution (1 mg/mL) and incubated for 3 hours at 37 ± 1 °C and 5 ± 1 % CO2. Post incubation, the tissue inserts were removed and blotted onto the tissue paper and transferred to a prelabeled 24-well plate containing 2.0 mL of isopropanol in each designated. The plates were placed on an orbital plate shaker and shaken for 2 hours at room temperature. At the end of the extraction period, the tissue was pierced with an injection needle and the liquid within each insert was decanted into the well from which it was taken.The optical density of the extracted formazan was measured in 96-well plate spectrophotometer at 570 nm. Viability of tissues was calculated.

Mean percentage viability of negative control, positive control and test item were 100 ± 0.89, 2.0 ± 0.07 and 10.0 ± 0.05 respectively. As the mean percentage viability of the test item was less than 60 % of the negative control it is identified as requiring classification and labeling UN GHS (Cat 2 or Cat 1). Similarly the percentage viability of positive control is less than 50 % of negative control. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.
Based on the results obtained and under the laboratory testing conditions using the EpiOcular™ model , the test item Ammonium 2-ethylhexyl sulfate is identified requiring classification and labeling UN GHS UN GHS Category 1 or Category 2 as the mean percentage tissue viability is less than 60 % of  the  negative control.

 

The test item was evaluated for serious eye effects (OECD 437) and for eye irritation (OECD 492) in validated in vitro test methods using a top-down approach. No clear prediction can be made based on both test systems. However, considering the low cell viablity of 10 % in the OECD 492 test, the IVIS of 47.4 which is just below the threshold of 55 triggering the UN GHS category 1 and the fact that the test item is corrosive to skin, it is considered to cause serious eye damage (category 1).

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. Based the test results of the in vitro tests, the substance is classified to be corrosive to skin (sub-category 1A, H314) and to cause serious damage to eyes (category 1, H318) according to Regulation (EC) No 1272/2008 (CLP), as amended for the fifteenth time in Regulation (EU) No 2020/1182.