C16-18 (evennumbered, C18 unsaturated) alkyl bis(2-hydroxyethyl) amine oxide

Endpoint summary

Description of key information

There is an in-vitro skin corrosion, Episkin study and an in-vivo rabbit skin irritation/corrosion study for Ethanol, 2,2’-iminobis-N-tallow alkyl derivatives, N-oxides. There is no eye irritation study but there is an in-vivo eye irritation/corrosion study in rabbits available for read across for the source substance 2,2’-(octadec-9-enylimino)diethanol, which is structurally similar but considered unlikely to underestimate he eye irritation as it is more unsaturated and therefore more reactive.

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:

Experimental start date 16 August 2017 Experimental completion date 18 August 2017

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Method B.40bis of Commission Regulation (EC) No 440/2008, of 30 May 2008,

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: Tallow bis(2-hydroxyethyl)amineoxide CAS No 61791-46-6
Batch: 1548134
Purity: 50.66%
Physical state/Appearance: extremely pale yellow paste
Expiry Date: 17 March 2018
Storage Conditions: room temperature in the dark

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

EpiDerm™ Reconstructed Human Epidermis Model Kit
Supplier: MatTek
Date received: 15 August 2017
EpiDermTM Tissues (0.63cm2) lot number: 25837
Assay Medium lot number: 081017ALA
Upon receipt of the EpidermTM tissues, the sealed 24-well plate was stored in a refrigerator until use.

Vehicle:

unchanged (no vehicle)

Details on test system:

Assessment of Direct Test Item Reduction of MTT
MTT Dye Metabolism, Cell Viability Assay
The MTT assay, a colorimetric 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 blue formazan dye by mitochondrial succinate dehydrogenase in viable cells.
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 is still a sufficient amount 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 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 item was checked for the ability to directly reduce MTT according to the procedure below:
50 μL of the test item was added to 1 mL of a freshly prepared 1.0 mg/mL MTT solution. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 60 minutes. Untreated MTT solution was tested concurrently to act as a control.
If the MTT solution containing the test item turns blue/purple relative to the control, the test item was presumed to have reduced the MTT.

Assessment of Color Interference with the MTT Endpoint
A test item may interfere with the MTT endpoint if it is colored or if it becomes colored when in wet or aqueous conditions. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed.
50 μL of test item was added to 300 μL of sterile water. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 60 minutes. A visual assessment of the color was then made.

Main Test
Pre-Incubation
The assay medium was pre-warmed before use. 0.9 mL of this assay medium was pipetted into the appropriate wells of two pre-labeled 6-well plates for both the 3-Minute and 60-Minute exposure periods. EpiDerm™ tissues were transferred into the 6-well plates containing the assay medium. The 6-well plates containing the EpiDerm™ samples were pre-incubated (37 °C, 5% CO2) for approximately 1 hour before dosing.

Application of Test Item and Rinsing
Before pre-incubation was complete, a 24-well plate was prepared for use as a “holding plate” for both the 3-Minute and 60-Minute exposure periods. This plate was used to maintain the viability of the tissue inserts between rinsing following chemical exposure and MTT loading. Another 24-well plate was prepared for the MTT loading. 300 μL of either pre-warmed assay medium (holding plate) or MTT medium (MTT loading plate) was dispensed into each well. The two plates were placed into the incubator until required.
After pre-incubation of the EpiDerm™ tissues, the medium was aspirated and replaced with 0.9 mL of fresh assay medium. The 6-well plate for the 3-Minute exposure period was returned to the incubator, while the other was being dosed for the 60-Minute exposure. For the 60-Minute exposure period, 50 μL of sterile distilled water (negative control) was added to the first two tissues. The tissues were dosed at regular intervals to allow for the time taken to rinse each tissue following exposure and to ensure that each tissue gets an equal exposure time. 50 μL of the test item and 50 μL of 8.0 N Potassium Hydroxide (positive control) were also applied to the corresponding tissues in turn. The plate was returned to the incubator (37 °C, 5% CO2) for the 60-Minute exposure period.
When dosing for the 60-Minute exposure period was complete, the same procedure was repeated for the 3-Minute exposure period. Because the exposure time was so short, the tissues were dosed at regular intervals to ensure that each tissue received an equal exposure time and to allow for the time taken to rinse each tissue following exposure. Rinsing was achieved by filling and emptying each tissue under a constant soft stream of Dulbecco’s Phosphate Buffered Saline (DPBS) to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper. Each tissue was placed into the prepared holding plate until all tissues were rinsed. They were then blotted and transferred to the 24-well plate prepared for MTT loading. The plate was incubated Page 13
(37 °C, 5% CO2) for 3 hours. Once the 60-Minute exposure period was complete, the same rinsing and MTT loading procedure was repeated.
After the 3-Hour MTT incubation was complete, the inserts were blotted and transferred to labeled 24-well plates for MTT extraction. 2 mL of MTT extractant (isopropanol) was used to completely immerse each insert and the plate was covered with plate sealer to prevent Isopropanol evaporation. The plates stood overnight at room temperature, to allow extraction to proceed.

Absorbance/Optical Density Measurements
After extraction, each tissue was pierced with a pipette fitted with a 1000 μL tip and the extraction solution was forced vigorously up and down to form a homogenous solution. 3 x 200 μL aliquots of the extract were transferred to the appropriate wells of a pre-labeled 96-well plate. 200 μL of isopropanol alone was added to the three wells designated as blanks. Absorbency at 570nm (OD570) of each well was measured using the Labtech LT-4500 microplate reader.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 μL of test item. The test item was used as supplied.
50 μL of negative control. The negative control item was used as supplied.
50 μL of positive control. The positive control item was used as supplied.

Duration of treatment / exposure:

3 minutes and 60 minutes

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes

Value:

89.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes

Value:

96.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Direct MTT Reduction
The MTT solution containing the test item did not turn blue/purple. This was taken to indicate the test item did not reduce MTT.

Assessment of Color Interference with the MTT endpoint
The solution containing the test item did not become colored. This was taken to indicate the test item did not have the potential to cause color interference.

Quality Criteria
The mean OD570 for the negative control treated tissues was 1.506 for the 3-Minute exposure period and 1.478 for the 60-Minute exposure period. The negative control acceptance criteria were therefore satisfied.
The relative mean tissue viability for the positive control treated tissues was 3.0% relative to the negative control following the 60-Minute exposure period. The positive control acceptance criterion was therefore satisfied.
In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

Mean OD₅₇₀Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Tissue	Exposure Period	MeanOD570of individual tissues	Mean OD570of duplicate tissues	Standard Deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative Control	3 Minutes	1.590	1.506	0.119	7.9	100*
		1.422
	60 Minutes	1.498	1.478	0.029	2.0
		1.457
Positive Control	3 Minutes	0.055	0.052	0.004	na	3.5
		0.049
	60 Minutes	0.042	0.045	0.004	na	3.0
		0.047
Test Item	3 Minutes	1.330	1.348	0.025	1.8	89.5
		1.365
	60 Minutes	1.352	1.427	0.105	7.4	96.5
		1.501

OD = Optical density

* = The mean percentage viability of the negative control tissue is set at 100%

na = Not applicable

Relative mean % = (mean OD570 of test item/ mean OD570 of negative control) x 100

Coefficient of variation = (standard deviation/ mean OD570 of duplicate tissues) x 100

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was considered to be non-corrosive to the skin.

Executive summary:

Introduction

The purpose of this test is to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes.

Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.

Methods

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction.

At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density (OD) was measured at 570 nm (OD570).

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Results

The relative mean viabilities for each treatment group were as follows:

Exposure Period	Percentage Viability
	Negative Control	Positive Control	Test Item
3 minute	100*	3.5	89.5
60 minute	100*	3.0	96.5

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

Quality criteria: The quality criteria required for acceptance of results in the test were satisfied.

Conclusion

The test item was considered to be non-corrosive to the skin.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 07 November 2017 Experimental completion date 19 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: Tallow bis(2-hydroxyethyl)amineoxide CAS No 61791-46-6
Batch Number: 1548134
Purity: 50.66%
Physical state / Appearance: extremely pale yellow paste
Expiry Date: 27 March 2018
Storage Conditions: room temperature in the dark

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Animal Information
Two New Zealand White (Hsdlf:NZW) strain rabbits were supplied by Envigo RMS (UK) Limited, Leicestershire, UK. At the start of the study the animals weighed 3.13 or 3.70 kg and were 12 to 52 weeks old. After an acclimatization period of at least 5 days each animal was given a number unique within the study which was written with a black indelible marker-pen on the inner surface of the ear and on the cage label.

Animal Care and Husbandry
The animals were individually housed in suspended cages. Free access to mains drinking water and food (2930C Teklad Global Rabbit diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study. The diet and drinking water were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
The temperature and relative humidity were set to achieve limits of 17 to 23 °C and 30 to 70% respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

0.5 mL of the test item. For the purpose of the study the test item was used as supplied.

Duration of treatment / exposure:

3 minutes, 1 hour and 4 hours

Observation period:

72 hours

Number of animals:

2

Details on study design:

Study Design
On the day before the test two rabbits were clipped free of fur from the dorsal/flank area using veterinary clippers. Only animals with a healthy intact epidermis by gross observation were selected for the study.
One rabbit was initially treated. Three suitable sites were selected on the back of the rabbit. A quantity of 0.5 mL of the test item was applied directly to the skin under a 2.5 cm x 2.5 cm cotton gauze patch. Each patch was secured in position with a strip of surgical adhesive tape. To prevent the animal interfering with the patches, the trunk of the rabbit was wrapped in an elasticated corset for the duration of the exposure period.
One patch was removed at each of three time points: 3 minutes, 1 hour and 4 hours after application. Any residual test item was removed by gentle swabbing with cotton wool soaked in distilled water. Due to a technician error, the treatment site decontamination for the 4 hour exposure skin site in the initial animal was not recorded. Given that the all other treatment sites were decontaminated using distilled water, it is reasonable to assume that the same procedure was followed.
After consideration of the skin reactions produced in the first animal, an additional animal was treated with 0.5 mL of test item. One patch was applied to the back of the rabbit and was allowed to remain in contact with the skin for a period of 4 hours.
Immediately following removal of the patches and approximately 1, 24, 48 and 72 hours later, the test sites were examined for evidence of primary irritation and scored according to the following scale:

EVALUATION OF SKIN REACTIONS
Erythema and Eschar Formation Value
No erythema: 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beef redness) to eschar formation preventing grading of erythema 4

Edema Formation
No edema 0
Very slight edema (barely perceptible) 1
Slight edema (edges of area well-defined by definite raising) 2
Moderate edema (raised approximately 1 millimeter) 3
Severe edema (raised more than 1 millimeter and extending beyond the area of exposure) 4
Any other skin reactions and clinical signs of toxicity, if present, were also recorded.
Additional observations were made on Days 7 and 14 to assess the reversibility of skin reactions.
Individual body weights were recorded on Day 0 (the day of dosing) and at the end of the observation period.

Irritation parameter:

erythema score

Basis:

mean

Remarks:

3 minute exposure

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

mean

Remarks:

3 minute exposure

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

erythema score

Basis:

mean

Remarks:

1 hour exposure

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

mean

Remarks:

3 hour exposure

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

erythema score

Basis:

mean

Remarks:

4 hours exposure

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 14 days

Remarks:

slight desquamation

Remarks on result:

probability of mild irritation

Irritation parameter:

edema score

Basis:

mean

Remarks:

4 hours exposure

Time point:

24/48/72 h

Score:

1.5

Max. score:

4

Reversibility:

fully reversible within: 14 days

Remarks on result:

probability of mild irritation

Irritant / corrosive response data:

3-Minute Exposure Period
No evidence of skin irritation was noted during the study.

1-Hour Exposure Period
No evidence of skin irritation was noted during the study.

4-Hour Exposure Period
Very slight erythema and slight edema were noted at both treated skin sites immediately after patch removal. Very slight to well defined erythema and slight edema were noted at both treated skin sites at the one hour observation. Well defined erythema with very slight to slight edema was noted at both treated skin sites at the 24, 48 and 72 Hour observations. Very slight to well defined erythema and very slight edema were noted at both treated skin sites at the 7 Day observation.
Loss of skin elasticity was noted at one treated skin site at the 72-Hour observation with loss of skin elasticity and flexibility noted at both treated skin sites at the 7-Day observation. Slight desquamation was noted at one treated skin site at the 14-Day observation.
One treated skin site appeared normal at the 14-Day observation.

Other effects:

Body Weight
Both animals showed expected gain in body weight during the study.

Individual Skin Reactions Following 3-Minute and 1-Hour Exposures

Skin Reaction	Observation Time (following patch removal)	Individual Scores
		Rabbit Number and Sex
		75795 Female
		3-Minute Exposure	1-Hour Exposure
Erythema/Eschar Formation	Immediately	0	0
	1 Hour	0	0
	24 Hours	0	0
	48 Hours	0	0
	72 Hours	0	0
	7 Days	0	0
	14 Days	0	0
Edema Formation	Immediately	0	0
	1 Hour	0	0
	24 Hours	0	0
	48 Hours	0	0
	72 Hours	0	0
	7 Days	0	0
	14 Days	0	0

Individual Skin Reactions Following 4-Hour Exposure

Skin Reaction	Observation Time (following patch removal)	Individual Scores		Total
		Rabbit Number and Sex
		75795 Female	75868 Female
Erythema/Eschar Formation	Immediately	1	1	(2 )
	1 Hour	2	1	( 3 )
	24 Hours	2	2	4
	48 Hours	2	2	( 4 )
	72 Hours	2	2Le	4
	7 Days	1LeLf	2LeLf	( 3 )
	14 Days	0D	0	( 4 )
Edema Formation	Immediately	2	2	( 4 )
	1 Hour	2	2	( 4 )
	24 Hours	2	1	3
	48 Hours	2	1	( 3 )
	72 Hours	2	1	3
	7 Days	1	1	( 2 )
	14 Days	0	0	( 0 )
Sum of 24 and 72‑Hour Readings (S)             :              14
Primary Irritation Index (S/4)                      :              14/4 = 3.5
Classification                                         :              MODERATE IRRITANT

(   ) =   Total values not used for calculation of primary irritation index

D =       Slight desquamation

Le =      Loss of skin elasticity

Lf =       Loss of skin flexibility

Interpretation of results:

Category 3 (mild irritant) based on GHS criteria

Conclusions:

The test item produced a primary irritation index of 3.5 and was classified as a Moderate irritant to rabbit skin according to the Draize classification scheme.
The test item does not meet the criteria for classification according to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.
The test item meets the criteria for classification as a Category 3 (Mild irritation) according to the Globally Harmonized System of Classification and Labeling of Chemicals.

Executive summary:

Introduction

The study was performed to assess the irritancy potential of the test item following single, 3-Minute, 1 and 4-Hour, semi-occluded applications to the intact rabbit skin.

Results

3-Minute and 1-Hour semi-occluded applications of the test item to the intact skin of one rabbit produced no evidence of skin irritation.

A single 4-Hour, semi occluded application of the test item to the intact skin of two rabbits produced well defined erythema and slight edema. Loss of skin elasticity, loss of skin flexibility and slight desquamation were also noted. One treated skin site appeared normal at the 14 Day observation. No corrosive effects were noted.

Conclusion

The test item produced a primary irritation index of 3.5 and was classified as a Moderate irritant to rabbit skin according to the Draize classification scheme.

The test item does not meet the criteria for classification according to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

The test item meets the criteria for classification as a Category 3 (Mild irritation) according to the Globally Harmonized System of Classification and Labeling of Chemicals.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 March 1990 -23 April 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to EC/OECD guidelines and under GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Broekman Institute , Someren, The Netherlands
- Age at study initiation: 14 or 16 weeks
- Weight at study initiation: 2698-2851 gram
- Housing: Individually in cages with perforated floors and equipped with an automatic drinking system (ITL, Bergen, The Nether lands).
- Diet (e.g. ad libitum): 100g per day
- Water (e.g. ad libitum): ad linitum
- Acclimation period: At least five days under test conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 14-23
- Humidity (%): 46-75
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Temperature was approximately 4 hours below 18ºC , due to a technical failure of the heating system), this did not affect the outcoem of the study.

IN-LIFE DATES: From: 20 March 1990 -23 April 1990

Vehicle:

unchanged (no vehicle)

Controls:

other: the left eye of each animals served as control

Amount / concentration applied:

0.1 ml

Duration of treatment / exposure:

A single instillation of 0.1 ml in the right eye af the animals was observed for 21 days.

Observation period (in vivo):

The eyes of each animal were examined approximately 1, 24, 48 and 72 hours and 7, 14 and 21 days after instillation o f the test substance.

Immediately after the 24 hours observation, a solution o f 2% fluorescein in water (pH adjusted to 7.0) was applied to both eyes of each animal to
quantitatively examine the potential for corneal injury. The bright green stained area indicating epithelial damage was estimated as a percentage of the total corneal area. This procedure was repeated in all three animals on day 4 and in animal 3068 also on days 8, 15 and 22.

Number of animals or in vitro replicates:

3

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): not done

SCORING SYSTEM:
Scoring system eye irritation

Cornea: degree of density (area most dense taken for reading)
0 No ulceration or opacity
1 Scattered or diffuse areas of opacity (other than slight dulling of normal lustre), details of iris clearly visible
2 Easily discernible translucent areas, details of iris slightly obscured
3 Nacrous areas, no details of iris visible, size of pupil barely discernible
4 Opaque cornea, iris not discernible through the opacity

Area of Cornea Involved
1 One quarter (or less) but not zero
2 Greater than one quarter but less than half
3 Greater than half but less than three quarters
4 Greater than three quarters, up to whole area

Iris
0 Normal
1 Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia or injection, any of these or combination of any thereof, iris still reacting to light (sluggish reaction is positive)
2 No reaction to light, haemorrhage, gross destruction (any or all of these)

Conjunctivae
Redness (refers to palpebral and bulbar conjunctivae cornea and iris)

0 Blood vessels normal
1 Some blood vessels definitely hyperaemic
(injected)
2 Diffuse, crimson colour, individual vessels not easily discernible
3 Diffuse, beefy red

Chemosis (lids and/or nictitating membranes)
0 No swelling 0
1 Any swelling above normal (includes nictitating membranes)
2 Obvious swelling with partial eversion of lids
3 Swelling with lids about half-closed
4 Swelling with lids more than half-closed

Discharge
0 No discharge
1 Any amount different from normal (does not include small amounts observed in inner canthus of normal animals)
2 Discharge with moistening of the lids and hairs just adjacent to lids
3 Discharge with moistening of the lids and hairs a considerable area around the eye

TOOL USED TO ASSESS SCORE: fluorescein, In case standard lighting was considered inadequate for observing minor effects, eye examinations were performed using a pocket flash light.

Irritation parameter:

overall irritation score

Remarks:

corneal opacity/iritis/conjunctival redness/oedema

Basis:

mean

Remarks:

all animals

Time point:

other: 24-48-72 hours

Score:

1.03

Max. score:

4

Reversibility:

not fully reversible within: 21 dyas

Remarks on result:

other: One animal still showed slight conjunctival redness after 21 days.

Irritant / corrosive response data:

Instillation o f AROMOX 0/12 into one of the eyes of each of three albino rabbits affected the cornea and the conjunctivae in all three animals and the
iris in animal 3068. The opacity of the cornea was reversible within 48 hours in animals 3070 and 3084 and within 21 days in animal 3068. The injec tion of the iris in animal 3068 was reversible within 7 days. The irritation of the conjunctivae was reversible within 14 days in animal 3070, within 21 days in animal 3084, while slight conjunctival redness was still observed in animal 3068 on day 22. No corrosion was observed.
Treatment o f the eyes with fluorescein 2%, 24 hours a f t e r t e s t substance
i n s t i l l a t i o n revealed corneal e p i t h e l i a l damage i n a l l three animals and i n
animal 3 0 6 8 on days 4, 8 and 15.
C

Other effects:

No toxic symptoms were observed in the animals during the test period and no mortality occurred.

Rabbit No and sex	Region of the eye			Hours after instillation							Average 24-48 hours
				1	24	48	72	7(days)	14(days)	21(days
3068 f	Cornea	Degree of opacity		0	1	1	1	0	0	0	1
		Area of opacity		0	2	1	1	1	1	0
	Iris			0	0	1	1	0	0	0	0.7
	Conjunctivae		Redness	2	3	3	3	2	1	1	3
			Chemosis	2	2	1	1	0	1	0	1.7
			Discharge	0	2	1	1	0	0	0
3070 f	Cornea		Degree of opacity	0	0	0	0	0	0	0	0
			Area of opacity	0	1	0	0	0	0	0
	Iris			0	0	0	0	0	0	0	0
	Conjunctivae		Redness	1	2	2	2	1	0	0	2
			Chemosis	2	2	1	0	0	0	0	1
			Discharge	0	1	0	0	0	0	0
3084 f	Cornea		Degree of opacity	1	1	0	0	0	0	0	0.3
			Area of opacity	1	1	0	0	0	0	0
	Iris			0	0	0	0	0	0	0	0
	Conjunctivae		Redness	1	2	2	2	1	1	0	2
			Chemosis	2	1	1	0	0	0	0	0.7
			Discharge	0	1	0	0	0	0	0

Interpretation of results:

Category 2A (irritating to eyes) based on GHS criteria

Remarks:

Migrated information

Conclusions:

In the study report a draize calculation is used and the classification follows the DSD (83/467/EEC, Annex VI of the EEC council Directive 67/548/EEC) which leads to a non-classification. However three animals have a ≥ 2 an average score 24-48-72 hours after exposure for conjuntival redness. The degree of opacity is ≥ 1 in in one animal. All effects execpt for slight redness in the eye of one animal are fully reversible within 21 days or sooner. No effects of corrosion are observed. Therefore based on these results, although some slight effects are still seen after 21 days, the substance is classified according GHS in category 2A (irritating to eyes).

Executive summary:

The Purpose of this study was to assess the possible irritation or corrosion potential when a single dose of the test item was placed in the conjunctival sac of a rabbit eye. This study was carried out in accordance with OECD Guideline No. 405 and EC guideline B.5. The test item was instilled in one o f the eyes of each o f the three animals, followed by seven observations at approximately 1, 24, 48 and 72 hours and 7, 14 and 21 days after test article administration. Under the conditions of this study, the test item resulted in adverse effects on the cornea, iris and conjunctivae. The opacity of the cornea was reversible within 48 hours in two animals and within 21 days in the third animal. The injection of the iris, observed in one animal, was reversible within 7 days. The irritation of the conjunctivae was reversible within 21 days in two animals, while the third animal still showed slight conjunctival redness at termination of the study. No corrosion and no signs o f systemic intoxication were observed. In the study report a draize calculation is used and the classification follows the DSD (83/467/EEC, Annex VI of the EEC council Directive 67/548/EEC) which leads to a non-classification. However three animals have a ≥ 2 an average score 24-48-72 hours after exposure for conjuntival redness. The degree of opacity is ≥ 1 in in one animal. All effects except for slight redness in the eye of one animal are fully reversible within 21 days or sooner. No effects of corrosion are observed. Therefore based on these results, although some slight effects are still seen after 21 days, the substance is classified according GHS in category 2A (irritating to eyes).

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

20 March 1990 -23 April 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to EC/OECD guidelines and under GLP.

Justification for type of information:

There is no eye irritation data for Ethanol, 2, 2’-iminobis-N-tallow alkyl derivatives, N-oxides, but there is in-vivo eye irritation test data for read across from the source substance, the structural similar 2, 2’-(octadec-9-enylimino)diethanol. 2, 2’-(octadec-9-enylimino)diethanol has ca 80% C18 unsaturated, 6% C18 saturated and 9% C16 and ca 1.5% C14. This compares to tallow such as in the target Ethanol, 2, 2’-iminobis-N-tallow alkyl derivatives, N-oxides which typically has 35% C18 unsaturated, 26% C18 saturated, 35% C16 and 2.4% C14 and 1.3% C17. The higher degree of unsaturation in the 2, 2’-(octadec-9-enylimino)diethanol is expected to result in higher reactivity from the increased double bonds in the unsaturated C18 at 80% as opposed to 35% in the tallow. Therefore the potential for eye irritation/damage of the 2, 2’-(octadec-9-enylimino)diethanol is not expected to be an underestimate of the eye irritation/damage toxicity of the target Ethanol, 2,2’-iminobis-N-tallow alkyl derivatives, N-oxides.

Reason / purpose for cross-reference:

read-across source

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Broekman Institute , Someren, The Netherlands
- Age at study initiation: 14 or 16 weeks
- Weight at study initiation: 2698-2851 gram
- Housing: Individually in cages with perforated floors and equipped with an automatic drinking system (ITL, Bergen, The Nether lands).
- Diet (e.g. ad libitum): 100g per day
- Water (e.g. ad libitum): ad linitum
- Acclimation period: At least five days under test conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 14-23
- Humidity (%): 46-75
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Temperature was approximately 4 hours below 18ºC , due to a technical failure of the heating system), this did not affect the outcoem of the study.

IN-LIFE DATES: From: 20 March 1990 -23 April 1990

Vehicle:

unchanged (no vehicle)

Controls:

other: the left eye of each animals served as control

Amount / concentration applied:

0.1 ml

Duration of treatment / exposure:

A single instillation of 0.1 ml in the right eye af the animals was observed for 21 days.

Observation period (in vivo):

The eyes of each animal were examined approximately 1, 24, 48 and 72 hours and 7, 14 and 21 days after instillation o f the test substance.

Immediately after the 24 hours observation, a solution o f 2% fluorescein in water (pH adjusted to 7.0) was applied to both eyes of each animal to
quantitatively examine the potential for corneal injury. The bright green stained area indicating epithelial damage was estimated as a percentage of the total corneal area. This procedure was repeated in all three animals on day 4 and in animal 3068 also on days 8, 15 and 22.

Number of animals or in vitro replicates:

3

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): not done

SCORING SYSTEM:
Scoring system eye irritation

Cornea: degree of density (area most dense taken for reading)
0 No ulceration or opacity
1 Scattered or diffuse areas of opacity (other than slight dulling of normal lustre), details of iris clearly visible
2 Easily discernible translucent areas, details of iris slightly obscured
3 Nacrous areas, no details of iris visible, size of pupil barely discernible
4 Opaque cornea, iris not discernible through the opacity

Area of Cornea Involved
1 One quarter (or less) but not zero
2 Greater than one quarter but less than half
3 Greater than half but less than three quarters
4 Greater than three quarters, up to whole area

Iris
0 Normal
1 Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia or injection, any of these or combination of any thereof, iris still reacting to light (sluggish reaction is positive)
2 No reaction to light, haemorrhage, gross destruction (any or all of these)

Conjunctivae
Redness (refers to palpebral and bulbar conjunctivae cornea and iris)

0 Blood vessels normal
1 Some blood vessels definitely hyperaemic
(injected)
2 Diffuse, crimson colour, individual vessels not easily discernible
3 Diffuse, beefy red

Chemosis (lids and/or nictitating membranes)
0 No swelling 0
1 Any swelling above normal (includes nictitating membranes)
2 Obvious swelling with partial eversion of lids
3 Swelling with lids about half-closed
4 Swelling with lids more than half-closed

Discharge
0 No discharge
1 Any amount different from normal (does not include small amounts observed in inner canthus of normal animals)
2 Discharge with moistening of the lids and hairs just adjacent to lids
3 Discharge with moistening of the lids and hairs a considerable area around the eye

TOOL USED TO ASSESS SCORE: fluorescein, In case standard lighting was considered inadequate for observing minor effects, eye examinations were performed using a pocket flash light.

Irritation parameter:

overall irritation score

Remarks:

corneal opacity/iritis/conjunctival redness/oedema

Basis:

mean

Remarks:

all animals

Time point:

other: 24-48-72 hours

Score:

1.03

Max. score:

4

Reversibility:

not fully reversible within: 21 dyas

Remarks on result:

other: One animal still showed slight conjunctival redness after 21 days.

Irritant / corrosive response data:

Instillation o f AROMOX 0/12 into one of the eyes of each of three albino rabbits affected the cornea and the conjunctivae in all three animals and the
iris in animal 3068. The opacity of the cornea was reversible within 48 hours in animals 3070 and 3084 and within 21 days in animal 3068. The injec tion of the iris in animal 3068 was reversible within 7 days. The irritation of the conjunctivae was reversible within 14 days in animal 3070, within 21 days in animal 3084, while slight conjunctival redness was still observed in animal 3068 on day 22. No corrosion was observed.
Treatment o f the eyes with fluorescein 2%, 24 hours a f t e r t e s t substance
i n s t i l l a t i o n revealed corneal e p i t h e l i a l damage i n a l l three animals and i n
animal 3 0 6 8 on days 4, 8 and 15.
C

Other effects:

No toxic symptoms were observed in the animals during the test period and no mortality occurred.

Rabbit No and sex	Region of the eye			Hours after instillation							Average 24-48 hours
				1	24	48	72	7(days)	14(days)	21(days
3068 f	Cornea	Degree of opacity		0	1	1	1	0	0	0	1
		Area of opacity		0	2	1	1	1	1	0
	Iris			0	0	1	1	0	0	0	0.7
	Conjunctivae		Redness	2	3	3	3	2	1	1	3
			Chemosis	2	2	1	1	0	1	0	1.7
			Discharge	0	2	1	1	0	0	0
3070 f	Cornea		Degree of opacity	0	0	0	0	0	0	0	0
			Area of opacity	0	1	0	0	0	0	0
	Iris			0	0	0	0	0	0	0	0
	Conjunctivae		Redness	1	2	2	2	1	0	0	2
			Chemosis	2	2	1	0	0	0	0	1
			Discharge	0	1	0	0	0	0	0
3084 f	Cornea		Degree of opacity	1	1	0	0	0	0	0	0.3
			Area of opacity	1	1	0	0	0	0	0
	Iris			0	0	0	0	0	0	0	0
	Conjunctivae		Redness	1	2	2	2	1	1	0	2
			Chemosis	2	1	1	0	0	0	0	0.7
			Discharge	0	1	0	0	0	0	0

Interpretation of results:

Category 2A (irritating to eyes) based on GHS criteria

Remarks:

Migrated information

Conclusions:

In the study report a draize calculation is used and the classification follows the DSD (83/467/EEC, Annex VI of the EEC council Directive 67/548/EEC) which leads to a non-classification. However three animals have a ≥ 2 an average score 24-48-72 hours after exposure for conjuntival redness. The degree of opacity is ≥ 1 in in one animal. All effects execpt for slight redness in the eye of one animal are fully reversible within 21 days or sooner. No effects of corrosion are observed. Therefore based on these results, although some slight effecst are still seen after 21 days, the substance is classified according GHS in category 2A (irritaing to eyes).

Executive summary:

The Purpose of this study was to assess the possible irritation or corrosion potential when a single dose of the test item was placed in the conjunctival sac of a rabbit eye. This study was carried out in accordance with OECD Guideline No. 405 and EC guideline B.5. The test item was instilled in one o f the eyes of each o f the three animals, followed by seven observations at approximately 1, 24, 48 and 72 hours and 7, 14 and 21 days after test article administration. Under the conditions of this study, the test item resulted in adverse effects on the cornea, iris and conjunctivae. The opacity of the cornea was reversible within 48 hours in two animals and within 21 days in the third animal. The injection of the iris, observed in one animal, was reversible within 7 days. The irritation of the conjunctivae was reversible within 21 days in two animals, while the third animal still showed slight conjunctival redness at termination of the study. No corrosion and no signs o f systemic intoxication were observed. In the study report a draize calculation is used and the classification follows the DSD (83/467/EEC, Annex VI of the EEC council Directive 67/548/EEC) which leads to a non-classification. However three animals have a ≥ 2 an average score 24-48-72 hours after exposure for conjuntival redness. The degree of opacity is ≥ 1 in in one animal. All effects execpt for slight redness in the eye of one animal are fully reversible within 21 days or sooner. No effects of corrosion are observed. Therefore based on these results, although some slight effecst are still seen after 21 days, the substance is classified according GHS in category 2A (irritating to eyes).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The test item meets the criteria for classification as a Category 3 (Mild irritation) according to the Globally Harmonized System of Classification and Labelling of Chemicals. was not corrosive in the Episkin in-vitro skin corrosion assay. The ECHA guidance accepts that for certain fatty amines chemistry such as this, that they can give false negative results in these in-vitro tests. Therefore an in-vivo rabbit skin irritation study was performed.

The application of Ethanol, 2,2’-iminobis-N-tallow alkyl derivatives, N-oxides to the skin resulted in moderate signs of irritation.

These included erythema and slight oedema. No corrosive effects were noted on the treated skin of any animal at any of the measuring intervals and no clinical signs were observed.

Ethanol, 2,2’-iminobis-N-tallow alkyl derivatives, N-oxides does not meet the criteria for classification according to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

The test item meets the criteria for classification as a Category 3 (Mild irritation) according to the Globally Harmonized System of Classification and Labelling of Chemicals.

There is no eye irritation data for Ethanol, 2,2’-iminobis-N-tallow alkyl derivatives, N-oxides, but there is in-vivo eye irritation test data for read across from the source substance, the structural similar 2,2’-(octadec-9-enylimino)diethanol. 2,2’-(octadec-9-enylimino)diethanol has ca 80% C18 unsaturated, 6% C18 saturated and 9% C16 and ca 1.5% C14. This compares to tallow such as in the target Ethanol, 2,2’-iminobis-N-tallow alkyl derivatives, N-oxides which typically has 35% C18 unsaturated, 26% C18 saturated, 35% C16 and 2.4% C14 and 1.3% C17. The higher degree of unsaturation in the 2,2’-(octadec-9-enylimino)diethanol is expected to result in higher reactivity from the increased double bonds in the unsaturated C18 at 80% as opposed to 35% in the tallow. Therefore the potential for eye irritation/damage of the 2,2’-(octadec-9-enylimino)diethanol is not expected to be an underestimate of the eye irritation/damage toxicity of the target Ethanol, 2,2’-iminobis-N-tallow alkyl derivatives, N-oxides.The opacity of the cornea was reversible within 48 hours in two animals and within 21 days in the third animal. The injection of the iris, observed in one animal, was reversible within 7 days. The irritation of the conjunctivae was reversible within 21 days in two animals, while the third animal still showed slight conjunctival redness at termination of the study. No corrosion and no signs of systemic intoxication were observed. In the study report a draize calculation is used and the classification follows the DSD (83/467/EEC, Annex VI of the EEC council Directive 67/548/EEC) which leads to a non-classification. However three animals have a>2 an average score 24-48-72 hours after exposure for conjuntival redness. The degree of opacity is>1 in in one animal. All effects except for slight redness in the eye of one animal are fully reversible within 21 days or sooner. No irreversible effects are observed. Therefore based on these results, although some slight effects are still seen after 21 days, the substance is classified according GHS in category 2A (irritating to eyes) or EU CLP category 2 (Regulation EC No 1272/2008).

Justification for classification or non-classification

The test item does not meet the criteria for classification as a skin irritant according to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

The test item meets the criteria for classification as a Category 3 (Mild irritation) according to the Globally Harmonized System of Classification and Labelling of Chemicals. was not corrosive in the Episkin in-vitro skin corrosion assay. The ECHA guidance accepts that for certain fatty amines chemistry such as this, that they can give false negative results in these in-vitro tests. Therefore an in-vivo rabbit skin irritation study was performed.

The test item did not induce significant or irreversible damage to the rabbit eye. In the study report a draize calculation is used and the classification follows the DSD (83/467/EEC, Annex VI of the EEC council Directive 67/548/EEC) which leads to a non-classification. However three animals have a >2 an average score 24-48-72 hours after exposure for conjuntival redness. The degree of opacity is >1 in in one animal. All effects except for slight redness in the eye of one animal are fully reversible within 21 days or sooner. No effects of corrosion are observed. Therefore based on these results, although some slight effects are still seen after 21 days, the substance is classified according GHS in category 2A (irritating to eyes) or EU CLP category 2 (Regulation EC No 1272/2008).