N,N-dimethyldecylamine N-oxide

Endpoint summary

Description of key information

C10 AO was non-irritating to rabbit skin when tested in a GLP OECD TG 404 study [Leuschner J (2012)]. This result is supported by negative findings in an in vitro corrosivity study performed according to OECD TG 431 under GLP [Flugge C (2012)] and an in vitro skin irritation study  performed according to OECD TG 439 under GLP [Flugge C (2012)].

C10 AO was found to be non-corrosive/irritant in an in vitro BCOP test performed according to OECD 437 under GLP [Leuschner J (2012)]. However it was found to be a strong irritant in an in vitro HET-CAM test performed according to the ICCVAM Recommended Test Method Protocol [Haferkorn J (2012)].

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:

key study

Study period:

2011-09-30 to 2012-01-20

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)

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EST-1000
- Tissue batch number(s): EST-111205-001

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: not reported

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Single washing step with Dulbecco's phosphate buffered saline (D-PBS). Volume not reported.
- Observable damage in the tissue due to washing: None reported.
- Modifications to validated SOP: None reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours at 37°C
- Spectrophotometer: not specified
- Wavelength: 540 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability/barrier function: Each batch of the epidermal model used meets defined production release criteria, set by the supplier, among which those for viability and for barrier function are the most relevant (MTT, 2 hours Triton X-100: target > 50%). The barrier properties of the tissues were verified by the supplier.
- Morphology: No. of cornified layers - 5; No. of vital cell layers - 4.
- Contamination: The skin model was free of contamination with bacteria (including mycoplasma) or fungi.

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 123.5 μL
- Concentration (if solution): 40.5 %

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 123.5 μL
- Concentration (if solution): 100% (deionised water)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration (if solution): 8N

Duration of treatment / exposure:

3 minutes, 1 hour

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes

Value:

78.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: mean of 3 replicates

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour

Value:

65.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: mean of 3 replicates

Other effects / acceptance of results:

In comparison to the negative controls, the mean viability of cells exposed to the test item was 78.8% after a 3-minute exposure period and 65.9% after a 1-hour exposure. The OD540 values were well above the cut-off percentage cell viability values distinguishing corrosive from non-corrosive test items of <50% or <15% for a 3-minute or 1-hour treatment, respectively. Therefore, the test item was non-corrosive in this skin model and is predicted to be non-corrosive to human skin.
The mean viability of cells treated with the positive reference item 8 N KOH were 5.0% (3-minute incubation) and 1.2% (1-hour incubation) of the negative controls and were below the cut-off values. Hence, 8 N KOH caused pronounced corrosion in this skin model and is predicted to be corrosive to human skin.
All quality criteria required were fulfilled

Interpretation of results:

GHS criteria not met

Conclusions:

In comparison to the negative controls, the mean viability of cells exposed to the test item was 78.8% after a 3-minute exposure period and 65.9% after a 1-hour exposure. The OD540 values were well above the cut-off percentage cell viability values distinguishing corrosive from non-corrosive test items of <50% or <15% for a 3-minute or 1-hour treatment, respectively. Therefore, the test item was non-corrosive in this skin model and is predicted to be non-corrosive to human skin.

Executive summary:

In an in vitro skin corrosivity study performed in accordance with OECD Guideline 431 using the EST-100 model the test item, N,N-dimethyldecylamine-N-oxide (solution), was applied to the skin surface. De-ionised water was used as the negative control. 8 N KOH was used as the positive reference item. Two exposure times of 3 minutes or 1 hour were employed.

In comparison to the negative controls, the mean viability of cells exposed to the test item was 78.8% after a 3-minute exposure period and 65.9% after a 1-hour exposure. The OD₅₄₀values were well above the cut-off percentage cell viability values distinguishing corrosive from non-corrosive test items of <50% or<15% for a 3-minute or 1-hour treatment, respectively. Therefore, the test item was non-corrosive in this skin model and is predicted to be non-corrosive to human skin.

The mean viability of cells treated with the positive reference item 8 N KOH were 5.0% (3-minute incubation) and 1.2% (1-hour incubation) of the negative controls and were below the cut-off values. Hence, 8 N KOH caused pronounced corrosion in this skin model and is predicted to be corrosive to human skin.

Under the present test conditions N,N-dimethyldecylamine-N-oxide (solution) tested at two exposure times of 3 minutes or 1 hour was non-corrosive to skin in vitro.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-09-28 to 2012-01-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline No. 439

Qualifier:

according to guideline

Guideline:

other: EC method B46

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EST-1000
- Tissue batch number(s): EST-111205-001

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 21°C
- Temperature of post-treatment incubation (if applicable): not reported - 42 hours incubation

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Single washing step with Dulbecco's phosphate buffered saline (D-PBS). Volume not reported.
- Observable damage in the tissue due to washing: None reported
- Modifications to validated SOP: None reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours at 37°C
- Spectrophotometer: not specified
- Wavelength: 540 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability/Barrier function: Each batch of the epidermal model used meets defined production release criteria, set by the supplier, among which those for viability and for barrier function are the most relevant (MTT, 2 hours Triton X-100: target > 50%). The barrier properties of the tissues were verified by the supplier.
- Morphology: No. of cornified layers - 5; No. of vital cell layers - 4.
- Contamination: The skin model was free of contamination with bacteria (including mycoplasma) or fungi.

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after exposure and post-treatment incubation is less than 50%
- The test substance is considered to have no category if the viability ater exposure and post-treatment incubation is greater than 50%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 74.1 μL
- Concentration (if solution): 40.5%

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 74.1 μL
- Concentration (if solution): 100% (deionised water)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): 5%

Duration of treatment / exposure:

20 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

20 minutes exposure

Value:

74

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: mean of 3 replicates

Other effects / acceptance of results:

The mean viability of the cells exposed to the test item was 74.0% of the mean negative control value. The OD540 values were well above the cut-off percentage cell viability value that distinguishes irritant from non-irritant test items of >50% for a 20-minute exposure.
The test item was considered to be non-cytotoxic and predicted to be not irritant to skin.
The viability of cells treated with the positive reference item 5% SDS, was 8.3% of the negative controls and was below the cut-off value. Hence, 5% SDS is predicted to cause pronounced skin irritation.
All quality criteria required were fulfilled.

Interpretation of results:

GHS criteria not met

Conclusions:

The mean viability of the cells exposed to the test item was 74.0% of the mean negative control value. The OD540 values were well above the cut-off percentage cell viability value that distinguishes irritant from non-irritant test items of >50% for a 20-minute exposure.
The test item was considered to be non-cytotoxic and predicted to be not irritant to skin.

Executive summary:

In an in vitro skin irritation study performed in accordance with OECD Guideline 439 and EC method B46, using the EST-100 model the test item, N,N-dimethyldecylamine-N-oxide (solution), was applied to the model skin surface. De-ionised water was used as the negative control. 5% aqueous sodium dodecyl sulphate (SDS) was used as the positive reference item. The cell viability was measured by determining the optical density (OD) at a wavelength of 540 nm. An exposure time of 20 minutes was employed.

The mean viability of cells exposed to the test item was 74.0% of the negative controls. The mean OD₅₄₀value was well above the cut-off percentage cell viability value of > 50% that distinguishes irritant from non-irritant test items after a 20-minute exposure. The test item was considered to be non-cytotoxic and is predicted to be not irritant to skin.

The viability of cells treated with the positive reference item, 5% SDS, was 8.3% of the negative controls and below the 50% cut-off value. Hence, 5% SDS is predicted to cause pronounced skin irritation.

Under the present test conditions N,N-dimethyldecylamine-N-oxide (solution), tested at an exposure time of 20 minutes, was non-cytotoxic and not irritant to skin in vitro.

 

Reference 3

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 May 2012-01 August 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

Himalayan

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: LPT Laboratory of Pharmacology and Toxicology GmbH & Co. KG, Branch Löhndorf, 24601 Löhndorf/Post Wankendorf, Germany
- Age at study initiation:4.5-6.5 months
- Weight at study initiation: 2.6-3.0 kg
- Housing: The animals were kept singly in cages measuring 380 mm x 425 mm x 600 mm (manufacturer: Dipl. Ing. W. EHRET GmbH, 16352 Schön¬walde, Germany)
- Diet: Commercial diet, ssniff® K-H V2333 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany, served as food. The food was available ad libitum before and after the exposure period.
- Water: Drinking water was offered ad libitum before and after the exposure period.
- Acclimation period: At least 20 adaptation days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): No information
- Photoperiod (hrs dark / hrs light): 12h/12h

IN-LIFE DATES: From: To: 01 June - 19 June, 2012

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 mL
- Concentration (if solution): 40.5 % w/w

Duration of treatment / exposure:

4 hours

Observation period:

7 days

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: approx 6 cm2
- Type of wrap if used: Gauze

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Not applicable
- Time after start of exposure: Not applicable

SCORING SYSTEM: Draize

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 4 days

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 4 days

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritant / corrosive response data:

Time after removal of patch Animal #1 Animal #2 Animal #3
Erythema/Oedema Erythema/Oedema Erythema/Oedema
60 min 1/0 1/0 1/0
24 h 1/0 1/0 1/0
48 h 1/0 1/0 1/0
72 h 1/0 1/0 1/0
4 d 0/0 1/0 0/0
5 d -/- 1/0 -/-
6 d -/- 1/0 -/-
7 d -/- -/- -/-

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results of this study, C10 AO is not corrosive or irritant to the skin of rabbits.

Executive summary:

Under the present test conditions, three rabbits exposed for 4 hours to 0.5 mL N,N-dimethyldecylamine-N-oxide/patch (semi-occlusive conditions) showed the following effect:

Erythema (grade 1) was observed in all animals 60 minutes to 72 hours after patch removal, and in animal no. 2 until 6 days after patch removal.

According to EC Regulation 1272/2008 and subsequent regulations, the test item is non-irritating and no labelling is required.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

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:

supporting study

Study period:

31 August - 22 November, 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)

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Details on test animals or tissues and environmental conditions:

Bovine eyes from cattle in the age range of 6 to 12 months were obtained from a slaughterhouse . To minimize deterioration and bacterial contamination, on collection the eyes were completely submerged in Hanks’ Balanced Salt Solution (HBSS) containing 1% Penicillin/Streptomycin . Upon arrival at the laboratory the eyes were examined for defects such as but not limited to increased opacity, scratches and neovascularization. Only corneas from eyes free of defects were used.
The corneas were dissected from the eyes so as to leave a 2 to 3 mm rim of sclera and mounted in corneal holders with anterior (epithelium) and posterior (endothelium) chambers. Beginning with the posterior chambers, the chambers were filled to excess with pre-warmed Eagle’s Minimum Essential Medium (EMEM), while preventing bubble formation. The corneal holder was equilibrated at 32±1°C for at least one hour.
After the equilibration period, fresh pre-warmed EMEM was added to both chambers and baseline opacity readings were taken for each cornea. Corneas exhibiting macroscopic tissue damage (e.g. scratches, pigmentation, neovascularisation) or an opacity >7 opacity units were discarded. The mean opacity of all equilibrated corneas was calculated. A minimum of three corneas with opacity values close to the median value for all corneas were selected as negative (or solvent) control corneas. The remaining corneas were then distributed into treatment and positive control groups.

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 uL
- Concentration (if solution): 10%

VEHICLE
- Concentration (if solution): 0.9% saline

Duration of treatment / exposure:

10 minutes

Observation period (in vivo):

2 hours

Number of animals or in vitro replicates:

Three corneas were used for each treatment group (test item, negative and positive controls).

Details on study design:

Test groups and treatment:
Three corneas were used for each treatment group (test item, negative and positive controls).

Negative control item: 0.9% NaCl solution
Positive control item: 10% NaOH solution
Test item: 10% solution in 0.9% NaCl solution
Exposure period: 10 minutes

A volume of 750 µL of the test or control items was added to completely cover the cornea’s epithelium in the anterior chamber.
After the exposure period of 10 minutes the exposure solution was removed from each chamber and the epithelium was washed with EMEM containing phenol red (an indicator of pH), at least three times. Washing was repeated until no test item or discolouration (yellow (acid) or purple (alkaline)) of phenol red was visible. The corneas were rinsed a final time with EMEM to remove any remaining phenol red from the chamber. The chamber was then filled with EMEM without phenol red and the corneas were then incubated at 32±1°C for two hours. After this post-exposure incubation period, the corneas were examined.

Examination of the corneas
Corneal injury was assessed by evaluating the opacity and permeability of the cornea. Corneal opacity was determined by the amount of light transmission through the cornea measured quantitatively with the aid of an opacitometer resulting in opacity values measured on a continuous scale.
To determine the corneal permeability 1 mL sodium fluorescein solution (4 mg/mL) was added to the anterior chamber (epithelial surface) and the posterior chamber (endothelial surface) was refilled with fresh EMEM. The holder was incubated in a horizontal position at 32±1°C for 90±5 minutes. The amount of sodium fluorescein that crossed from the anterior to the posterior chamber was measured quantitatively using UV/VIS spectophotometry. Measurements at 492 nm were recorded as optical density (OD492) or absorbance values. The fluorescein permeability values were determined using OD492 values based upon a visible light spectrophotometer using a standard 1 cm path length.

EVALUATION
After correcting the opacity and mean permeability (OD492) values for background opacity and the negative control permeability OD492 values, the mean opacity and permeability OD492 values for each treatment group were combined in an empirically-derived formula to calculate an in vitro irritancy score (IVIS) for each treatment group as follows:
IVIS = mean opacity value + (15 x mean permeability OD492 value)
The opacity and permeability values were also evaluated independently to determine whether the test item induced corrosivity or severe irritation through only one of the two endpoints.

Decision criteria
A test item that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant.
As stated in the OECD guideline, if the test substance is not identified as an ocular corrosive or severe irritant, additional testing should be conducted for classification and labeling purposes. The BCOP test method has an overall accuracy of 79% (113/143) to 81% (119/147), a false positive rate of 19% (20/103) to 21% (22/103), and a false negative rate of 16% (7/43) to 25% (10/40), when compared to in vivo rabbit eye test method data classified according to the EPA, EU, or GHS classification systems. When substances within certain chemical (i.e., alcohols, ketones) or physical (i.e., solids) classes are excluded from the database, the accuracy of BCOP across the EU, EPA, and GHS classification systems ranges from 87% (72/83) to 92% (78/85), the false positive rates range from 12% (7/58) to 16% (9/56), and the false negative rates range from 0% (0/27) to 12% (3/26).

Irritation parameter:

in vitro irritation score

Run / experiment:

mean value

Value:

35.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Range = 31.2-40.1

Table 1: Opacity values

 

	Opacity [Opacity Value]	Mean NC	Corrected Opacity	Mean of group	SD
NaCl 0.9 %	0.7	0.8	-	-	-
	0.6		-
	1		-
NaOH 10 %	232.9		232.1	260.3	41.9
	309.2		308.4
	241.2		240.4
Test item 1:10#	25.3		24.5	26.6	4.3
	24.6		23.8
	32.3		31.5

 

NC: negative control

SD: standard deviation

# tested as 10% concentration in 0.9% NaCl   solution

 

Table 2: Permeability OD Values (492 nm)

 

 

	Well 1	Well 2	Mean of Duplicates	Mean NC	Corrected OD	Mean of Group	SD
NaCl 0.9 %	0.049	0.052	0.051	0.045	-	-	-
	0.038	0.038	0.038		-
	0.046	0.046	0.046		-
NaOH 10 %	0.522	0.525	0.524		0.479	0.477	0.0
	0.507	0.509	0.508		0.463
	0.54	0.528	0.534		0.489
Test item 1:10#	0.441	0.439	0.440		0.395	0.511	0.1
	0.669	0.661	0.665		0.620
	0.563	0.563	0.563		0.518

 

NC: negative control

SD: standard deviation

# tested as 10% concentration in 0.9% NaCl   solution

 

Table 3: In vitro irritancy score (IVIS)

 

	NaOH 10 %	Test item 1:10#
Holder 1	240.1	31.2
Holder 2	316.1	33.9
Holder 3	248.5	40.1
Mean	268.3	35.1
SD	41.7	4.5

 

 # tested as 10% concentration in 0.9% NaCl solution

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Based on the results of this study, C10AO is not corrosive to eyes or a severe eye irritant.

Executive summary:

In this study, performed according to OECD 437 under GLP, C10 AO (10% solution in 0.9% NaCl solution) was applied to the epithelial surface of three bovine corneas by addition to the anterior chamber of the corneal holder for an exposure time of ten minutes. Two further groups of three corneas were similarly treated with 0.9% NaCl solution (negative control) or 10% NaOH solution (positive control). Corneal opacity was measured quantitatively as the amount of light transmission through the cornea at 492 nm. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passed across the full thickness of the cornea, as detected in the medium in the posterior chamber. The measurements were used to calculate an in vitro irritancy score (IVIS) which was used to assign an in vitro hazard classification category prediction of the in vivo occular irritation potential of the test item. An opacity value of 26.6 and a permeability value of 0.511 compared to the negative control were determined. An IVIS of 35.1 was calculated. Hence C10 AO (solution) was considered to be non-cytotoxic and not irritant according to the IVIS value, which was <55.1. The test item is not classified as a severe irritant and not corrosive, based on the results of this test.The corneas treated with the positive control (10% NaOH solution) revealed an opacity value of 260.3 and a permeability value of 0.477 compared to the negative control. The IVIS value of 268.3 was well above the cut-off value of 55.1 and hence the acceptance criteria for the test were fulfilled.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 November - 28 November 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: ICCVAM-Recommended Test Method Protocol: Hen's Egg Test - Chorioallantoic Membrane (HET-CAM) Test Method (2010)

GLP compliance:

yes (incl. QA statement)

Species:

chicken

Strain:

other: White Leghorn

Details on test animals or tissues and environmental conditions:

Fertile white Leghorn chicken eggs
Weight: 48 – 52 g
Age: ≤7 days

Vehicle:

unchanged (no vehicle)

Controls:

yes

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.3 mL/egg
- Concentration (if solution): 40.5% (aqueous solution used without further dilution)

Duration of treatment / exposure:

20 seconds

Observation period (in vivo):

5 minutes

Number of animals or in vitro replicates:

3 eggs/group

Details on study design:

Test item: 3 eggs treated with the test item
Negative control item: 3 eggs treated with physiological saline
Positive control items:3 eggs treated with NaOH (0.1 N); 3 eggs treated with SDS (1%)

Application: on the chorioallantoic membrane of fertilized hens' eggs

SCORING SYSTEM: see tables below

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean of 3 tests

Value:

16.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: range 15-17

Table 1: HET-CAM Mean Irritation Scores (IS)

	Effect according to scoring scheme for irritation testing
	Haemorrhage	Lysis	Coagulation	Irritation Score (IS)
C10 AO       Mean SD	7	3	7	17
	7	3	7	17
	7	3	5	15
	7.0 0.0	3.0 0.0	6.3 1.2	16.3 1.2
Negative control (0.9% NaCl soln)   Mean SD	0	0	0	0
	0	0	0	0
	0	0	0	0
	0.0 0.0	0.0 0.0	0.0 0.0	0.0 0.0
Positive control (0.1 N NaOH)   Mean SD	7	3	9	19
	7	3	7	17
	7	3	7	17
	7.0 0.0	3.0 0.0	7.7 1.2	17.7 1.2
Positive control (1% SDS)   Mean SD	7	3	0	10
	7	3	0	10
	7	3	0	10
	7.0 0.0	3.0 0.0	7.0 0.0	10.0 0.0

 

SD: standard deviation

1% SDS: 1% aqueous sodium dodecyl sulfate

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

Based on the results of this test, C10 AO is considered to be strongly irritating to eyes

Executive summary:

Three eggs each were treated with 0.3 mL C10 AO solution/egg and with the control items. 0.9% NaCl solution was used as the negative control item. 0.1 N Sodium hydroxide (NaOH) or 1% aqueous sodium dodecyl sulphate (SDS) were used as the positive control items. The administration volume for the control items was 0.3 mL per egg. After administration of the test item blood vessels including the capillary system and the albumen were examined and scored for irritant effects (haemorrhage, coagulation and lysis) during 5 minutes. The eggs treated with C10 AO solution revealed an effect with an irritation score (IS) of 16.3. The test item was considered to be a strong irritant.

The positive control items 0.1 N NaOH or 1% SDS caused the expected effect with irritation scores (IS) of 17.7 or 10.0, respectively and, hence, were well within the historical data-range.No effects were observed in the negative control 0.9% NaCl solution.Hence,the HET-CAM assay is considered to be valid.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

In an in vitro skin corrosivity study [Flugge C (2012)] performed in accordance with OECD TG 431 under GLP using the EST-100 model, C10 AO (123.5 µL, 40.5 %w/w aqueous solution) was applied to the model skin surface. De-ionised water was used as the negative control and 8 N KOH was used as the positive control.Two exposure times of 3 minutes or 1 hour were employed. The cell viability was measured by determining the optical density (OD) at a wavelength of 540 nm. In comparison to the negative controls, the mean viability of cells exposed to C10 AO was 78.8% after a 3-minute exposure period and 65.9% after a 1-hour exposure.The OD₅₄₀values were well above the cut-off percentage cell viability values distinguishing corrosive from non-corrosive test items of <50% or <15% for a 3-minute or 1-hour treatment, respectively. The mean viability of cells treated with the positive control 8 N KOH were 5.0% (3-minute incubation) and 1.2% (1-hour incubation) of the negative controls and were below the cut-off values. It is concluded that C10 AO was non-corrosive in this skin model and is predicted to be non-corrosive to human skin.

In an in vitro skin irritation study [Flugge C (2012)] performed in accordance with OECD TG 439 and EC method B46, using the EST-100 model C10 AO (74.1 µL, 40.5 %w/w aqueous solution) was applied to the model skin surface. De-ionised water was used as the negative control and 5% aqueous sodium dodecyl sulphate (SDS) was used as the positive control. The cell viability was measured by determining the optical density (OD) at a wavelength of 540 nm. An exposure time of 20 minutes was employed.The mean viability of cells exposed to the test item was 74.0% of the negative controls. The mean OD₅₄₀value was well above the cut-off percentage cell viability value of > 50% that distinguishes irritant from non-irritant test items after a 20-minute exposure. The viability of cells treated with the positive control, 5% SDS, was 8.3% of the negative controls and below the 50% cut-off value. It is concluded that C10 AO was non-cytotoxic in this skin model and is predicted to be not irritant to skin.

The results of the in vitro studies were not in accordance with the results of in vivo studies performed with other, higher chain length, amine oxides such as C12-14 AO and C12-18 AO. Because of this, and in order to ensure the protection of workers in particular, an in vivo study was performed. In this study performed according to OECD TG 404 under GLP [Leuschner J (2012)] C10 AO (0.5 mL, 40.5 %w/w aqueous solution) was applied to the shaved backs of a total of three Himalayan rabbits for four hours under semi-occlusive conditions. After exposure, the patches were removed. Erythema (grade 1) was observed in all animals from 60 minutes to 72 hours after patch removal and in one animal until six days after patch removal. No oedema was observed in any animal at any time point. It is concluded that C10 AO is not irritant to rabbit skin based on the results of this study.

Eye irritation

Two in vitro studies are available for C10 AO.

In the first study [Leuschner J (2012)] performed according to OECD 437 under GLP, C10 AO (10% solution in 0.9% NaCl solution) was applied to the epithelial surface of three bovine corneas by addition to the anterior chamber of the corneal holder for an exposure time of ten minutes. Two further groups of three corneas were similarly treated with 0.9% NaCl solution (negative control) or 10% NaOH solution (positive control). Corneal opacity was measured quantitatively as the amount of light transmission through the cornea at 492 nm. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passed across the full thickness of the cornea, as detected in the medium in the posterior chamber. The measurements were used to calculate an in vitro irritancy score (IVIS) which was used to assign an in vitro hazard classification category prediction of the in vivo occular irritation potential of the test item.An opacity value of 26.6 and a permeability value of 0.511 compared to the negative control were determined. An IVIS of 35.1 was calculated which is below the cut-off value of 55.1.The corneas treated with the positive control (10% NaOH solution) revealed an opacity value of 260.3 and a permeability value of 0.477 compared to the negative control. The IVIS value of 268.3 was well above the cut-off value of 55.1 and hence the acceptance criteria for the test were fulfilled. On the basis of this study C10 AO is considered to be non-cytotoxic and non-irritant to eyes.

It is stated in paragraph 8 of OECD TG 437 that a limitation of the test method is that it does not consider conjunctival and iridal injuries. As part of an integrated testing strategy it was decided to perform a second in vitro test that does consider these endpoints, namely a Hen’s Egg Test – Chorioallantoic Membrane (HET-CAM).

In this test [Haferkorn J (2012)] performed according to the ICCVAM-Recommended Test Method Protocol under GLP, groups of three eggs were treated with either C10 AO (0.3 mL/egg, 40.5% aqueous solution) 0.9% NaCl solution (0.3 mL/egg, negative control) 0.1 N Sodium hydroxide (NaOH; 0.3 mL/egg , positive control) or 1% aqueous sodium dodecyl sulphate (SDS; 0.3 mL/egg , positive control). The appropriate substance was placed directly onto the chorioallantoic membrane of each egg, ensuring at least 50 % of the surface area was covered. After administration, the membranes were observed over a period of 300 seconds and the blood vessels, including the capillary system and the albumen were examined and scored for irritant effects (haemorrhage, coagulation and lysis). The eggs treated with C10 AO caused effects with an irritation score (IS) of 16.3. The positive controls (0.1 N NaOH or 1% SDS) both caused the expected effect with irritation scores (IS) of 17.7 or 10.0, respectively and, hence, were well within the historical data-range. No effects were observed in the negative control, 0.9% NaCl solution. Hence, the HET-CAM assay is considered to be valid. Based on the IS classification scheme C10 AO is considered to be a strong irritant to eyes (score range 9-21).

According to the Guidance on the Application of the CLP Criteria (version 2.0 – April 2012) section 3.3.2.1.2.4 there is regulatory acceptance in the EU that a substance can be considered a severe eye irritant (Serious eye damage Category 1) based on positive results in the HET-CAM test without the need for in vivo testing.

Justification for classification or non-classification

C10 AO is not classified for skin irritation as it was found to be non-irritating to rabbit skin when tested in a GLP OECD TG 404 study [Leuschner J (2012)]. This result is supported by negative findings in an in vitro corrosivity study performed according to OECD TG 431 under GLP [Flugge C (2012)] and an in vitro skin irritation study performed according to OECD TG 439 under GLP [Flugge C (2012)].

The substance is classified as Eye Damage Category 1 based on the strong irritant effects found in an in vitro HET-CAM test performed according to the ICCVAM Recommended Test Method Protocol [Haferkorn J (2012)].