Sodium N-methyl-N-(1-oxotetradecyl)aminoacetate

Endpoint summary

Description of key information

Skin corrosion in vitro (OECD 431): non-corrosive
Skin irritation in vitro (OECD 439): irritating
Skin irritation in vivo (OECD 404): not irritating at a specific concentration of 30% (v/v)

Eye irritation in vitro (OECD 437): corrosive
Eye irritation in vivo (OECD 405): corrosive at a specific concentration of 30% (v/v)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

05 Apr - 08 Apr 1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

yes

Remarks:

no experimental 48 h reading performed

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: a USDA and FDRL approved supplier (no further information)
- Age at study initiation: young adults
- Weight at study initiation: 2-3 kg
- Housing: animals were housed individually in wire mesh bottom cages.
- Diet: NIH 09 Rabbit Ration (Zeigler Brothers, Inc., Gardners, PA, USA)
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Environment-controlled rooms per "Guide for the Care and Use of Laboratory Animals" (no further information).
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

occlusive

Preparation of test site:

other: shaved and abraded

Vehicle:

unchanged (no vehicle)

Controls:

other: not required, untreated sites of the same animal served as control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 mL

Duration of treatment / exposure:

4 h

Observation period:

72 h
Reading time points: 0,5, 24 and 72 h

Number of animals:

6

Details on study design:

TEST SITE
- Area of exposure: right and left dorsal side of the animal. The right dorsal side was abraded to penetrate the stratum corneum.
- Type of wrap if used: the test side was covered with a one square inch gauze patch, two single layers thick and held in place with a Micropore tape and was subsequently secured by a non-absorbant binder.

REMOVAL OF TEST SUBSTANCE
- The test areas were gently wiped using a soft clean gauze.
- Time after start of exposure: 4 h

SCORING SYSTEM: Draize scoring system

Irritation parameter:

erythema score

Basis:

mean

Remarks:

out of all 6 animals

Time point:

24/48/72 h

Score:

0.78

Max. score:

4

Reversibility:

not fully reversible within: 72 h

Remarks on result:

other: no 48 h data are available; for calculation of mean scores, the 48 h values were assumed to be the same as those at 24 h (worst case assumption: persistence of effects seen at 24 h over 48 h)

Irritation parameter:

edema score

Basis:

mean

Remarks:

out of all 6 animals

Time point:

24/48/72 h

Score:

0.44

Max. score:

4

Reversibility:

fully reversible within: 72 h

Remarks on result:

other: no 48 h data are available; for calculation of mean scores, the 48 h values were assumed to be the same as those at 24 h (worst case assumption: persistence of effects seen at 24 h over 48 h)

Table 1. Results of skin irritation study (intact skin).

Observation time	Rabbit no.
	1		2		3		4		5		6
	Erythema	Edema	Erythema	Edema	Erythema	Edema	Erythema	Edema	Erythema	Edema	Erythema	Edema
0.5 h	1	1	1	2	2	2	1	1	1	1	0	0
24 h	1	0	1	2	1	0	2	2	0	0	0	0
48 h	No experimental data available; for calculation of mean scores, the 48 h values were assumed to be the same as those at 24 h (worst case assumption)
72 h	0	0	1	0	1	0	1	0	0	0	1	0

Table 2. Calculation of mean scores (intact skin).

	Rabbit no.
	1		2		3		4		5		6
	Erythema	Edema	Erythema	Edema	Erythema	Edema	Erythema	Edema	Erythema	Edema	Erythema	Edema
Mean value 24 + 48 + 72 h*	0.67	0.00	1.00	1.33	1.00	0.00	1.67	1.33	0.00	0.00	0.33	0.00

*No 48 h data are available: for calculation of mean scores, the 48 h values were assumed to be the same as those at 24 h (worst case assumption: persistence of effects seen at 24 h over 48 h).

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

16 Oct 2012 - 22 Oct 2012

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:

(adopted 22 July 2010)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(adopted 2008)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Species:

human

Strain:

other: reconstructed human epidermis

Type of coverage:

other: Not applicable.

Preparation of test site:

other: intact reconstructed human epidermis

Vehicle:

unchanged (no vehicle)

Controls:

other: concurrent negative control tissue treated with Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca2+ and Mg2+, and concurrent positive control treated with Sodium Dodecyl Sulphate (SDS) 5% w/v

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: approximately 10 mg of the test item was applied to the epidermis surface. The epidermis surface had previously been moistened with 5 μL of sterile distilled water to improve contact between the solid test item and the epidermis.

Duration of treatment / exposure:

15 min

Observation period:

Not applicable: post-treatment incubation period: 42 h

Number of animals:

Not applicable. Tests were performed in triplicates for each treatment and control group.

Details on study design:

PRE-TEST
- MTT Dye Metabolism, Cell Viability Assay
The MTT assay, a colourimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide) to a blue formazan dye by mitochondrial succinate dehydrogenase in viable cells.

- Assessment of Direct Test Material Reduction of MTT
One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified.

- Test for Direct MTT Reduction
As specified, a test item may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, each test item is checked for the ability to directly reduce MTT according to the following procedure: 10 mg of the test item was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37ºC, 5% CO2 in air for 3 h. Untreated MTT solution was used as a control. If the MTT solution containing the test item turns blue, the test item is presumed to have reduced the MTT.

PRE-INCUBATION (Day 0: tissue arrival)
2 mL of maintenance medium, warmed to approximately 37°C, was pipetted into the first column of 3 wells of a pre-labelled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37°C, 5% CO2 in air overnight.

MAIN TEST
- Application of Test Material and Rinsing (Day 2)
2 mL of maintenance medium, warmed to approximately 37°C, was pipetted into the second column of 3 wells of the 12-well plate. Triplicate tissues were treated with the test item for an exposure period of 15 min. The test item was applied topically to the corresponding tissues ensuring uniform covering. Approximately 10 mg of the test item was applied to the epidermis surface. The epidermis surface had previously been moistened with 5 μL of sterile distilled water to improve contact between the solid test item and the epidermis. Triplicate tissues treated with 10 μL of DPBS served as the negative controls and triplicate tissues treated with 10 μL of SDS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the centre). After 7 min contact time the SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period. The plate(s) were kept in the biological safety cabinet at room temperature for 15 min. At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca2+ and Mg2+. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37ºC, 5% CO2 in air for 42 h.

- MTT Loading/Formazan Extraction (Day 3)
Following the 42-h post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 min to homogenise the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer at -14 to -30ºC for possible inflammatory mediator determination. 2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12 well plate(s). The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37°C, 5% CO 2 in air. At the end of the 3-h incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labelled 1.5 mL micro tubes containing 500 μL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10°C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.

- Absorbance/Optical Density Measurements (Day 6)
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous coloured solution. For each tissue, duplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 μL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 540 nm (without a reference filter) using the Anthos 2001 microplate reader.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 min

Value:

42.2

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The relative mean viability of the test item treated tissues was 42.2% after the 15-min exposure period.
The test item meets therefore the criteria to be classified as irritant according to EU labelling regulations Commission Directive 2001/59/EC. The symbol “I”, the indication of danger “Irritant” and the risk phrase R 38 “IRRITATING TO SKIN” are therefore required.

- The MTT solution containing the test material did not turn blue/purple in the pre-test. This was taken to indicate the test material did not reduce MTT.

- The relative mean tissue viability for the positive control treated tissues was 12.0% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 5.0%. The positive control acceptance criterion was therefore satisfied.

- The mean OD540 for the negative control treated tissues was 0.706 and the standard deviation value of the percentage viability was 8.1%. The negative control acceptance criterion was therefore satisfied.

- The standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues was 9.7%. The test item acceptance criterion was therefore satisfied.

 

Tab. 2: Mean OD540 values and percentage viabilities for the negative control, positive control material and test material

Item	OD540 of tissues	Mean OD540 of triplicate tissues	± SD of OD540	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	0.647	0.706	0.057	91.6	100*	8.1
	0.711			100.7
	0.761			107.8
Test Item	0.366	0.298	0.069	51.8	42.2	9.7
	0.299			42.4
	0.299			32.4
Positive Control Item	0.125	0.084	0.036	17.7	12.0	5.0
	0.069			9.8
	0.059			8.4

* The mean viability of the negative control tissues was set at 100%.

SD = Standard deviation

Negative control: Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca2+ and Mg2+

Positive control: Sodium Dodecyl Sulphate (SDS) 5% w/v

Interpretation of results:

other: Skin irrit. 2, H315. Classification according to Regulation (EC) No. 1272/2008 (CLP/EU GHS).

Reference 3

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

02 Oct 2012 - 05 Oct 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

(adopted 13 April 2004)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

(adopted 2008)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Species:

human

Strain:

other: reconstructed human epidermis

Details on test animals or test system and environmental conditions:

Not applicable.

Type of coverage:

other: Not applicable.

Preparation of test site:

other: intact reconstructed human epidermis

Vehicle:

unchanged (no vehicle)

Controls:

other: concurrent negative control tissue treated with 0.9% NaCl, and concurrent positive control treated with glacial acid

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 20 mg of the solid test material was applied topically to the corresponding tissues ensuring uniform coverage of the tissues. 100 µl of 0.9% w/v sodium chloride solution was added for wetting of the test material.

Duration of treatment / exposure:

3, 60 and 240 min

Observation period:

Not applicable.

Number of animals:

Not applicable. Tests were performed in duplicates for each treatment duration.

Details on study design:

PRE-TEST
- MTT Dye Metabolism, Cell Viability Assay
The MTT assay, a colourimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a purple formazan dye by mitochondrial succinate dehydrogenase in viable cells.

- Assessment of Direct Test Material Reduction of MTT
One limitation of the assay is possible interference of the test material with MTT. A test material may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test material is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test material present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified by the procedure described below.

- Test for Direct MTT Reduction
As specified, a test material may interfere with the MTT endpoint, if it was able to directly reduce MTT and at the same time was present on or in the tissues when the MTT viability test was performed. To identify this possible interference, the test material was checked for the ability to directly reduce MTT according to the procedure below:
20 mg of the test material was added to 2.2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at room temperature for 3 h. Untreated MTT solution was used as a control.
If the MTT solution containing the test material turns blue/purple relative to the control, the test material was presumed to have reduced the MTT.

PRE-INCUBATION (Day 0: tissue arrival)
2.2 mL of maintenance medium, warmed to approximately 37ºC, was pipetted into two wells of the first column of a pre-labelled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (2 units per plate). A different 12‑well plate was used for each test material, control and time point. The tissues were incubated at 37ºC, 5% CO2in air for at least 24 h. To avoid concurrent testing with other chemicals and potential cross contamination the tissues were incubated for a further 24 h following refreshment of the medium. 

MAIN TEST
- Application of Test Material and Rinsing (Day 2)
2.2 ml of assay medium, warmed to approximately 37ºC, was pipetted into 2 wells of the second and third columns of the 12-well plate. The tissues were transferred into the second column.
Duplicate tissues were treated with the test material for exposure periods of 3, 60 and 240 min. Duplicate tissues were treated with the positive and negative control materials for an exposure period of 240 min. 20 mg of the solid test material was applied topically to the corresponding tissues ensuring uniform coverage of the tissues. 100 µL of 0.9% w/v sodium chloride solution was added for wetting of the test material. Duplicate tissues, treated with 50 µL of 0.9% w/v sodium chloride solution served as negative controls. Duplicate tissues, treated with 50 µL of Glacial Acetic acid served as positive controls. The treated tissues were kept in the biological safety cabinet at room temperature for the appropriate exposure period.
At the end of each exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing Phosphate Buffered Saline Dulbeccos (PBS) with Ca2+and Mg2+. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of PBS to gently remove any residual test material. Each rinsed tissue was placed into the third column of the 12-well plate until all tissues were rinsed.
2.2 mL of 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into 2 wells of the fourth column of each 12 well plate. The tissues were transferred into the MTT filled wells. The tissues were incubated for 3 h±5 min at room temperature in a biological safety cabinet ensuring that the plates were protected from light. At the end of the 3‑h incubation period each tissue was placed onto absorbent paper to dry. The tissues were examined and the degree of MTT staining evaluated (qualitative evaluation of cell viability). Following qualitative evaluation of tissue viability a total biopsy of the epidermis was taken using the EPISKIN biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) were placed into labelled 1.5 mL micro tubes containing 850 µL of acidified isopropanol. Each tube was plugged, mixed thoroughly and stored overnight at room temperature, protected from light, to extract formazan crystals out of the MTT‑loaded tissues.

- Absorbance/Optical Density Measurements (Day 3)
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous coloured solution.
For each tissue, duplicate 200 µL samples were transferred to the appropriate wells of a pre-labelled 96‑well plate. 200 µL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 540 nm (without a reference filter) using the Anthos 2001 microplate reader.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min

Value:

91.6

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 min

Value:

65.8

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

240 min

Value:

67.7

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The relative mean viability of the test item treated tissues was:
- 240 min exposure : 67.7%
- 60 min exposure : 65.8%
- 3 min exposure : 91.6%
The test item was therefore considered to be non-corrosive to the skin.

- The MTT solution containing the test material did not turn blue/purple in the pre-test. This was taken to indicate the test material did not reduce MTT.

-The relative mean tissue viability for the positive control treated tissues was 18.7% relative to the negative control treated tissues following the 240- min exposure period. The positive control acceptance criterion was therefore satisfied.

- The mean OD540 for the negative control treated tissues was 0.155. The negative control acceptance criterion was therefore satisfied.

Tab. 2: Mean OD540 values and viabilities for the negative control, positive control material and test material

Item	Exposure Period (min)	Mean OD540 of duplicate tissues	Relative viability (%)
Negative control item	240	0.155	100*
Test item	240	0.105	67.7
	60	0.102	65.8
	3	0.142	91.6
Positive control item	240	0.029	18.7

* The mean viability of the negative control tissues was set at 100%.

Negative control: 0.9% NaCl

Positive control: glacial acid

Interpretation of results:

other: non-corrosive

Remarks:

Criteria used for interpretation of results: expert judgment

Conclusions:

Under the conditions of this in vitro study, the test material is non-corrosive to the reconstructed human epidermis used. However, this study does not provide sufficient and adequate information on skin irritation for classification purposes.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

05 Apr – 12 Apr 1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

yes

Remarks:

limited data on test substance

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Ace Animals. Inc., Boyertown, PA, USA
- Age at study initiation: young adults
- Weight at study initiation: 2-3 kg
- Housing: animals were housed individually in wire mesh bottom cages.
- Diet: NIH 09 Rabbit Ration (Zeigler Brothers, Inc., Gardners, PA, USA), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Environment-controlled rooms per "Guide for the Care and Use of Laboratory Animals"DHEW Publication No. (NIH) 85-23.
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

unchanged (no vehicle)

Controls:

other: the untreated eye served as control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL

Duration of treatment / exposure:

single application without washing

Observation period (in vivo):

7 days
Reading time points: 24, 48 and 72 h and 7 days

Number of animals or in vitro replicates:

6

Details on study design:

SCORING SYSTEM: Draize scoring system

TOOL USED TO ASSESS SCORE: fluorescein

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

out of all 6 animals

Time point:

24/48/72 h

Score:

2.06

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Irritation parameter:

iris score

Basis:

mean

Remarks:

out of all 6 animals

Time point:

24/48/72 h

Score:

0.72

Max. score:

2

Reversibility:

not fully reversible within: 7 days

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

out of all 6 animals

Time point:

24/48/72 h

Score:

2.83

Max. score:

3

Reversibility:

not fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

out of all 6 animals

Time point:

24/48/72 h

Score:

2.33

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Irritant / corrosive response data:

All animals showed positive ocular response following test article instillation. The untreated eyes of all animals showed no effects except of one animal which exhibited diffuse areas of corneal opacity at the 24 and 48 h reading.
After 7 days, effects were still apparent in 4/6, 2/6, 6/6 and 5/6 animals on cornea, iris, conjunctivae redness and concjunctivae, respectively.

 Table 1. Results of eye irritation study.

Rabbit #	Time [h]	conjunctivae		iris	cornea
		redness	swelling
1	24	2	1	0	2
	48	3	2	1	2
	72	2	2	1	2
	7 days	1	1	0	1
	average	2.3	1.7	0.7	2.0
2	24	3	3	1	2
	48	3	3	1	2
	72	3	3	0	2
	7 days	3	1	0	2
	average	3.0	3.0	0.7	2.0
3	24	3	3	1	2
	48	3	3	1	2
	72	2	2	1	2
	7 days	1	1	0	0
	average	2.7	2.7	1.0	2.0
4	24	3	2	1	2
	48	3	2	0	2
	72	3	2	1	2
	7 days	3	1	1	2
	average	3.0	2.0	0.7	2.0
5	24	3	2	1	2
	48	3	3	1	2
	72	3	3	1	3
	7 days	3	2	2	4
	average	3.0	2.7	1.0	2.3
6	24	3	2	0	2
	48	3	2	0	2
	72	3	2	1	2
	7 days	1	0	0	0
	average	3.0	2.0	0.3	2.0
average score	Time [h]	conjunctivae		iris	cornea
		redness	swelling
	24	2.83	2.17	0.67	2.00
	48	3.00	2.50	0.67	2.00
	72	2.67	2.33	0.83	2.17
	24+48+72	2.83	2.33	0.72	2.06

 

Interpretation of results:

other: Eye damage 1, H318. Classification according to Regulation (EC) No. 1272/2008 (CLP/EU GHS).

Conclusions:

In the present study irreversible effects within a shorter observation period were observed in all animals. Classification as Eye Damage Category 1 was based on an expert judgement considering a worst case assumption.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

02 Nov 2012

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)

Version / remarks:

adopted in 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Species:

other: bovine eyes from young cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source of eyes: by-product from an abattoir from freshly slaughtered animals
- Transportation: the eyes were excised by an abattoir employee and placed in Hanks’ Balanced Salt Solution (HBSS), supplemented with Penicillin/Streptomycin, and transported to the laboratory on ice packs.

Vehicle:

physiological saline

Remarks:

0.9% w/v sodium chloride solution

Controls:

other: negative controls: 0.9% NaCl; positive controls: imidazole 20% in physiol. saline (0.9% NaCl)

Amount / concentration applied:

TEST MATERIAL
- Concentration (if solution): 20% in vehicle

Duration of treatment / exposure:

240 min at 32 ± 1 °C

Observation period (in vivo):

not applicable, post-exposure incubation: 90 min

Number of animals or in vitro replicates:

Number of corneas for the test item: 3

Details on study design:

PREPARATION OF CORNEAS
The corneas were prepared immediately on arrival. All eyes were macroscopically examined before and after dissection. Only corneas free of damagee were used. The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed (epithelial side uppermost) in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders. The anterior and posterior chambers of each BCOP holder were filled with complete minimum essential medium (MEM) and plugged. The holders were incubated at 32±1ºC for 60 min. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

SELECTION OF CORNEAS AND OPACITY READING
The medium from both chambers of each holder was replaced with fresh complete MEM. A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated. Three corneas were numerically allocated to the test item. Three corneas were also numerically allocated to the negative control item and three corneas to the positive control item.

TREATMENT OF CORNEAS
The MEM was removed from the anterior chamber of the BCOP holder and the test item preparation or control items were applied to the cornea. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32±1ºC for 240 min. At the end of the exposure period the test item preparation and control items were removed from the anterior chamber and the cornea was rinsed three times with fresh complete MEM containing phenol red before a final rinse with complete MEM. The anterior and posterior chambers were refilled with fresh complete MEM. A post-treatment opacity reading was taken and each cornea was visually observed.

APPLICATION OF SODIUM FLUORESCEIN
Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32±1ºC for 90 min.

PERMEABILITY DETERMINATIONS
After incubation the medium in the posterior chamber of each holder was decanted and retained. 360 μL of medium representing each cornea was applied to a designated well on a 96-well plate and the optical density at 492nm (OD492) was measured. If values greater than 1.500 OD492 were obtained a 1 in 5 dilution of the medium in complete MEM was performed and the measurement repeated. The modified value was multiplied by 5 to reflect the 1 in 5 dilution.

HISTOPATHOLOGY
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labelled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

EVALUATION OF RESULTS
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.
The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.
The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.
The following formula was used to determine the In Vitro Irritancy Score:

In Vitro Irritancy Score = mean opacity value + (15 x mean OD492 value)

Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.
The condition of the cornea was visually assessed immediately after rinsing and at the final opacity measurement.

Irritation parameter:

cornea opacity score

Run / experiment:

240 min

Value:

9.7

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

other: permeability

Run / experiment:

240 min

Value:

4.498

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

in vitro irritation score

Run / experiment:

240 min

Value:

77.1

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The corneas treated with the test item were slightly cloudy post incubation. The corneas treated with the negative control item were clear post incubation. The corneas treated with the positive control item were cloudy post incubation.
The test item was considered to be an ocular corrosive or severe irritant. The test item meets therefore the criteria to classified as Eye irrit 1, H318 according to Regulation (EC) 1272/2008 and Xi, R41 according to Directive 67/548/EEC.

Tab. 1: Individual and Mean Corneal Opacity and Permeability Measurements

Treatment	Opacity					Permeability (OD)		In vitro Irritancy Score
	Cornea Number	Pre-Treatment	Post-Treatment Score	Post-Treatment - Pre-Treatment	Corrected Value		Corrected Value
Negative Control	1	3	6	3		0.033
	2	2	6	4		0.039
	3	5	4	-1		0.039
				2.0*		0.037***		2.6
Positive Control	4	1	63	62	60.0	1.895	1.858
	5	4	83	79	77.0	1.210	1.173
	6	3	77	74	72.0	1.640	1.603
					69.7**		1.545**	92.8
Test Item	7	2	18	16	14.0	3.935	3.898
	8	5	13	8	6.0	4.860	4.823
	9	4	15	11	9.0	4.810	4.773
					9.7**		4.498**	77.1

* Mean of the post incubation - pre-treatment values

** Mean corrected value

*** Mean permeability

OD = Optical density

Negative Control = 0.9% NaCl

Positive Control = imidazole 20% in 0.9% NaCl

Test Item = 20% in 0.9% NaCl

Tab. 2: Corneal Epithelium Condition Post Incubation

Treatment	Cornea Number	Observation: Post Treatment
Negative Control	1	clear
	2	clear
	3	clear
Positive Control	4	cloudy
	5	cloudy
	6	cloudy
Test Item	7	slightly cloudy
	8	slightly cloudy
	9	slightly cloudy

Interpretation of results:

other: Eye damage 1, H318. Classification according to Regulation (EC) No. 1272/2008 (CLP/EU GHS).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

An in vitro skin corrosion test, conducted according to OECD TG 431, and in compliance with GLP (Harlan, 2013b) is available for Sodium N-methyl-N-(1-oxotetradecyl)aminoacetate (CAS 30364-51-3). The test was performed on a total of 6 intact reconstructed human epidermis tissues per test group (negative control, positive control, and test substance), 2 replicates for each treatment. Corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after 3, 60, and 240 min treatment compared to the negative control tissues concurrently treated with 0.9% NaCl (= 100%). A test item is classified as corrosive in any case if the relative tissue viability after 3 min treatment is decreased below 35%. In addition, those test items classified as non-corrosive after 3 min (viability ≥ 35%) are classified as corrosive if the relative mean issue viability after 60 or 240 min treatment compared to the concurrent negative control tissues is decreased below 35%. In this study, neat Sodium N-methyl-N-(1-oxotetradecyl)aminoacetate showed no corrosive effects. The mean relative tissue viability scores (% negative control) were 91.6, 65.8 and 67.7% after 3, 60 and 240 min, respectively. Thus, under the conditions of this in vitro test, neat Sodium N-methyl-N-(1-oxotetradecyl)aminoacetate does not meet the criteria to be classified as skin corrosive.

Since the OECD TG 431 study cannot yield any data regarding skin irriation, a further skin irritation in vitro study using reconstructed human epidermis is also available for Sodium N-methyl-N-(1-oxotetradecyl)aminoacetate (CAS 30364-51-3). The study was conducted according to OECD TG 439 and in compliance with GLP (Harlan, 2013c). The test was performed in triplicate each for negative control, positive control and test substance, respectively. Irritating properties of the neat test item were predicted from the relative mean tissue viabilities obtained after 15 min treatment compared to the negative control tissues concurrently treated with Dulbecco’s Phosphate Buffered Saline (= 100%). A test item is classified as irritating if the relative tissue viability after 15 min treatment is ≤ 50%. In this study, Sodium N-methyl-N-(1-oxotetradecyl)aminoacetate showed irritating effects; the mean relative tissue viability (% negative control) was < 50% (42.2%) after 15 min. Based on the outcome of this in vitro study, Sodium N-methyl-N-(1-oxotetradecyl)aminoacetate meets the criteria to be classified as skin irritant Cat. 2.

Finally, the skin irritation properties of Sodium N-methyl-N-(1-oxotetradecyl)aminoacetate (CAS 30364-51-3) have been investigated in an in vivo study similar to OECD TG 404 in compliance with GLP (CIR panel, 2001). In the study, 6 New Zealand White rabbits were exposed to a 30% aqueous solution of the test substance onto the shaved skin for 4 h using an occlusive dressing. The treated skin was observed and evaluated at 0.5, 24 and 72 h after application. The animals showed very slight to well defined erythema being not fully reversible in 4/6 animals after 72 h (calculated mean erythema score out of all 6 animals over 24, 48 (assumed to be the same as 24 h score) and 72 h = 0.78). However, a decreasing tendency of the irritating effects after 72 h was apparent. In addition, the animals showed very slight to slight oedema resulting in a mean oedema score of 0.44 (mean out of all 6 animals over 24, 48 (assumed to be the same as 24 h score) and 72 h) being fully reversible within 72 h in all animals. Thus, due to the short observation period in this study and in combination with the decreasing tendency of the irritating effects, the result of the study was considered to be non-irritating at 30% (v/v).

In summary, neat Sodium N-methyl-N-(1-oxotetradecyl)aminoacetate (CAS 30364-51-3) has to be classified as Skin Irrit. 2 (H315). However, data indicate that below the specific concentration limit of 30%, the substance is considered to be non-irritating to skin.

Eye irritation

Eye irritation of Sodium N-methyl-N-(1-oxotetradecyl)aminoacetate (CAS 30364-51-3) was investigated in an in vitro bovine corneal opacity and permeability test, conducted according to OECD TG 437, and in compliance with GLP (Harlan, 2013d). Mean irritancy scores calculated were 2.6, 77.1, and 92.8 for the negative control, the test item, and the positive control, respectively. The positive control data were within the two standard deviations of the current historical control mean and therefore this assay was considered to be valid. Thus, Sodium N-methyl-N-(1-oxotetradecyl)aminoacetate is concluded to be corrosive to the bovine eye in the in vitro bovine corneal opacity and permeability test.

In addition, the eye irritation properties of Sodium N-methyl-N-(1-oxotetradecyl)aminoacetate (CAS 30364-51-3) were assessed in a study similar to OECD TG 405 in compliance with GLP (CIR panel, 2001). In a group of 6 New Zealand White rabbits, 0.1 mL of the unchanged test substance was instilled into one eye in a single application without washing. The eyes were observed and reactions were evaluated 24, 48 and 72 h and 7 days after instillation. All animals showed positive ocular response following test article instillation resulting in mean scores of 2.06/0.72/2.83/2.33 for cornea score/iris score/conjunctivae score/chemosis score, respectively, for all 6 animals over 24, 48 and 72 h. After 7 days, effects on cornea, iris, conjunctivae redness and chemosis were not fully reversible in 4/6, 2/6, 6/6 and 5/6 animals, respectively.

In summary, Sodium N-methyl-N-(1-oxotetradecyl)aminoacetate (CAS 30364-51-3) was shown to cause severe damages to the eyes both in vitro and in vivo. Based on the outcome of the in vitro study and considering a worst case assumption for the in vivo study, the substance meets the criteria to be classified for inducing serious damage to eyes (Eye Damage 1, H318).

Justification for classification or non-classification

The available in vivo data on skin irritation / corrosion of Sodium N-methyl-N-(1-oxotetradecyl)aminoacetate (CAS 30364-51-3) which was derived with a 30% (v/v) aqueous solution do not meet the criteria for classification according to Regulation (EC) No. 1272/2008 (CLP). However, in vitro studies performed with the neat substance indicate an irritating potential. Therefore, the neat substance is considered to be irritating to the skin (Skin Irrit. 2, H315) whereas it is considered to be not irritating at concentrations < 30% (v/v).

The available in vitro and in vivo data on eye irritation / severe eye damage of Sodium N-methyl-N-(1-oxotetradecyl)aminoacetate (CAS 30364-51-3) meet the criteria for classification for Eye Damage, Category 1 (H318) according to Regulation (EC) No. 1272/2008 (CLP).