1-Octanamine, N,N-dimethyl-, N-oxide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 January 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Principles of method if other than guideline:

End points studied: Corneal opacity, corneal thickness and condition and fluorescein uptake.

GLP compliance:

yes (incl. QA statement)

Remarks:

Schedule 1 (Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by 2004/0994))

Test material

Test material information

Constituent 1

Reference substance name:

N,N-dimethyl-N-octylhydroxylamine

EC Number:

607-854-9

Cas Number:

2605-78-9

Molecular formula:

CH3(CH2)7N(CH3)2O

IUPAC Name:

N,N-dimethyl-N-octylhydroxylamine

impurity 1

Reference substance name:

Dimethyl(octyl)amine

EC Number:

230-939-3

EC Name:

Dimethyl(octyl)amine

Cas Number:

7378-99-6

Molecular formula:

C10H23N

IUPAC Name:

N,N-dimethyloctan-1-amine

impurity 2

Reference substance name:

Methanol

EC Number:

200-659-6

EC Name:

Methanol

Cas Number:

67-56-1

Molecular formula:

CH4O

IUPAC Name:

Methanol

impurity 3

Test material form:

liquid: viscous

Specific details on test material used for the study:

- Analytical purity: 82.3%
- Purity test date: Not available
- Lot/batch No.: GN-8
- Expiration date of the lot/batch: Not available
- Appearance: Amber coloured, extremely viscous liquid
- Storage: In the dark at room temperature
- Additional information on test material used for the study: It was attempted to purify the substance to a purity of >80% by using an alternative synthetic route, as opposed to the usual commercial route of synthesis which results in a purity of the substance of approximately 40% in water. This alternative synthetic route generated the substance at 82.3% purity, however it also generated an impurity (methanol at 4.0%) which is not present in the commercial substance. Based on the classification and labelling of methanol, it is considered that this additional impurity would not have an influence on any of the endpoints discussed in this dossier other than the acute oral toxicity study. Therefore all studies (including the one covering this endpoint) contained in this dossier (other than the acute oral toxicity study) were conducted on the 82.3% pure substance containing the 4.0% methanol impurity. The acute oral toxicity study has been conducted on the commercially generated substance at a purity of approximately 40% in water.

Test animals / tissue source

Species:

rabbit

Strain:

not specified

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.1ml undiluted

Duration of treatment / exposure:

10 seconds

Observation period (in vivo):

60, 120, 180 and 240 minutes for all tests except Fluorescein uptake, which was measured at 240 minutes.

Number of animals or in vitro replicates:

3

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): 20ml saline at approximately 32°C
- Time after start of exposure: ten seconds


SCORING SYSTEM:
McDonald- Shadduck Score Systen

CORNEA: Measurement of cloudiness grading:

0 = Normal cornea. Appears with the slit-lamp as having a bright grey line on the epithelial surface and a bright grey appearance of the stoma
1 = Some loss of transparency. Only the anterior half of the strom is involved as observed with an optical section of the slit-lamp. The underlying structures are clearly visible with diffuse illumination, although some cloudinedd can be readily apparent with diffuse illumination.
2 = Moderate loss of transparency. In addition to involving the anterior stroma, the cloudiness extends all the way to the endothelium. The stroma has lost its mable-like appearance and is homogeneously white. With diffues illumination, underlying structures are just visible.
3= Involvement of the entire thickness of the stroma. With the optical section cannot clearly visualise the endothelium. With the diffuse illumination, the underlying structures cannot be seen.

The surface of the cornea relative to the area of cloudiness is divided into five grades from 0 to 4.

0 = Normal cornea with no area of cloudiness
1 = 1 to 25% area of stromal cloudiness
2 = 26% to 50% area of stromal cloudiness
3 = 51% to 75% area of stromal cloudiness
4 = 76 to 100% area of stromal cloudiness

FlOURESCEIN
0 = Absence of flourescein staining
1 = Slight flourescein staining confined to a small focus. With diffuse illumination the underlying structures are clearly visible, although there is some loss of detail
2 = Moderate flourescein staining confined to a small focus. With diffuse illumination the underlying structures are clearly visible, although there is some loss of detail.
3 = Marked flourescein staining. Staining may involve a larger portion of the cornea. With diffuse illumination underlying structures are barely visible but are not completely obliterated.
4 = Extreme flourescein staining. With diffuse illumination the underlying structures cannot be seen.


TOOL USED TO ASSESS SCORE: hand-slit lamp, biomicroscope and fluorescein


-Test material administration:

Eye held in perspex clamp, stored in superfusion apparatus irrigated with a saline drip. Clamp and eye removed from superfusion chamber then placed in petri dish Test material applied as evenly as possible over cornea. Washed of after 10 seconds with 20ml of saline solution 32°C. Returned to superfusion chamber still in clamp and a saline drip repositioned to irrigate the eye.

-Observations
Corneal cloudiness was observed pre-enucleation, post equilibration and at 60, 120, 180 and 240 minutes following treatment.
Examination of the eye via a slit-lamp biomicroscope. Thickness of cornea was measured by an ultrasonic pachymeter. A measurement was made at the optical centre and at four other locations at the apex of the cornea. A mean value was calculated. These observations were taken pre-enucleation, post equilibration and at 60, 120, 180 and 240 minutes following treatment.

Condition of the cornea was assessed at 60, 120, 180 and 240 minutes following treatment with a slit-lamp biomicroscope.

The uptake of flourescein was assessed pre-enucleation, post equilibration and 240 minutes following treatment. This was carried out using a cobalt blue filter of the slit-lamp biomicroscope after the addition of Flourescein Sodium drops.

These observations were graded with the above stated criteria.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

1h to 4h post exposure

Value:

>= 1 - <= 2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not applicable

Remarks on result:

positive indication of irritation

Results 2

Irritation parameter:

percent corneal swelling

Run / experiment:

1h post exposure

Value:

ca. 68.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not applicable

Remarks on result:

positive indication of irritation

Results 3

Irritation parameter:

percent corneal swelling

Run / experiment:

2h post exposure

Value:

ca. 101.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not applicable

Remarks on result:

positive indication of irritation

Results 4

Irritation parameter:

percent corneal swelling

Run / experiment:

4h post exposure

Value:

ca. 137.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not applicable

Remarks on result:

positive indication of irritation

Results 5

Irritation parameter:

fluorescein retention score

Run / experiment:

1h to 4h post exposure

Value:

ca. 2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not applicable

Remarks on result:

positive indication of irritation

Any other information on results incl. tables

Individual Scores for Corneal Opacity

	Test eyes												Control eyes
Chamber Number	6A				8A				10A				7A				9A
Time After Treatment (mins)	60	120	180	240	60	120	180	240	60	120	180	240	60	120	180	240	60	120	180	240
Degree of Corneal Opacity	1+	1+	2+	2+	1+	1+	2+	2+	1+	1+	2+	2+	0	0	0	0	0	0	0	0
Area of Corneal Opacity	4	4	4	4	4	4	4	4	4	4	4	4	0	0	0	0	0	0	0	0

+ = Meets or exceeds cut-off value indicating a severe ocular irritant.

Individual Measurements for Corneal thickness (µm)

Test Eyes
Time After Treatment (mins)		60						120						180						240
Corneal position		oc	1	2	3	4	Mean	oc	1	2	3	4	Mean	oc	1	2	3	4	Mean		oc	1	2	3	4	Mean
Chamber Number	6A	698	648	650	649	654	659.8	799	788	771	747	688	758.6	894	864	884	914	789	869.2		953	926	951	899	827	911.2
	8A	533	549	608	645	622	591.4	679	711	743	735	705	714.6	805	814	844	802	788	810.6		840	851	900	861	816	853.6
	10A	583	590	643	615	631	6112.4	792	702	768	791	701	750.8	865	722	842	855	743	815.4		888	835	918	868	798	861.4

Control Eyes
Time After Treatment (mins)		60						120						180						240
Corneal position		oc	1	2	3	4	Mean	oc	1	2	3	4	Mean	oc	1	2	3	4	Mean		oc	1	2	3	4	Mean
Chamber Number	7A	384	376	372	377	373	376.4	374	369	368	365	370	369.2	378	374	369	372	369	372.4		386	388	370	385	378	381.4
	9A	404	398	377	368	371	383.6	404	401	387	385	383	392.0	410	401	381	376	380	389.6		406	398	383	375	374	387.2

oc = optical centre

Determination of Corneal Swelling (%)

Test eyes
Chamber Number	Observation Period (mins)	Mean Corneal Thickness (µm)	Corneal Swelling (%)a	Chamber Number	Observation Period (mins)	Mean Corneal Thickness (µm)	Corneal Swelling (%)a	Chamber Number	Observation Period (mins)	Mean Corneal Thickness (µm)	Corneal Swelling (%)a
6A	Post equilibriation	367.6	N/A	8A	Post equilibriation	360.6	N/A	10A	Post equilibriation	376.2	N/A
	60 Post treatment	659.8	79.5		60 Post treatment	591.4	64.0		60 Post treatment	612.4	62.8
	120 Post treatment	758.6	106.4		120 Post treatment	714.6	98.2		120 Post treatment	750.8	99.6
	180 Post treatment	869.2	136.5		180 Post treatment	810.6	124.8		180 Post treatment	815.4	116.7
	240 Post treatment	911.2	147.9		240 Post treatment	853.6	136.7		240 Post treatment	861.4	129.0

Control eyes
Chamber Number	Observation Period (mins)	Mean Corneal Thickness (µm)	Corneal Swelling (%)a	Chamber Number	Observation Period (mins)	Mean Corneal Thickness (µm)	Corneal Swelling (%)a
7A	Post equilibriation	354.8	N/A	9A	Post equilibriation	366.6	N/A
	60 Post treatment	376.4	6.1		60 Post treatment	383.6	4.6
	120 Post treatment	369.2	4.1		120 Post treatment	392.0	6.9
	180 Post treatment	372.4	5.0		180 Post treatment	389.6	6.3
	240 Post treatment	381.4	7.5		240 Post treatment	387.2	5.6

a = % corneal swelling = (((mean corneal thicknes post-treatment)-(mean corneal thickness pre-treatment))/mean corneal thicknes pre-treatment)x100

N/A = Not applicable

+ = Exceeds cut-off value and therefore indicates a severe eye irritant.

Summary of tablulated corneal swelling results:

Test eyes:

Mean corneal swelling 1 hours post-treatment = 68.8%+

Mean corneal swelling 2 hours post-treatment = 101.4%+

Mean corneal swelling 4 hours post-treatment = 137.9%+

Control eyes:

Mean corneal swelling 1 hour post-treatment = 5.4%

Mean corneal swelling 2 hours post-treatment = 5.5%

Mean corneal swelling 4 hours post-treatment = 6.6%

Corneal Epithelium Condition:

Test Eyes
Chamber Number	Time After Treatment (mins)
	60	120	180	240
6A	Sloughing +	Sloughing +	Sloughing +	Sloughing +
8A
10A			Sloughing +	Sloughing +

Control Eyes
Chamber Number	Time After Treatment (mins)
	60	120	180	240
7A
9A

+ = Meets cut-off value indicating a severe ocular irritant

Individual Scores for Fluorescein Uptake (240 Minutes Post Dosing)

	Test eyes			Control eyes
Chamber Number	6A	8A	10A	7A	9A
Intensity of Flourescein Uptake	2+	2+	2+	0	0
Area of Flourescein Uptake	4	4	4	0	0

+ = Meets or exceeds cut-off value indicatin a severe ocular irritant

Applicant's summary and conclusion

Interpretation of results:

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

Remarks:

Migrated information

Conclusions:

From the in vitro tests, the test substance has proved to have a high ocular irritancy.

Executive summary:

Introduction:       It is a legal and ethical duty under the animals (scientific Procedure) Act 1986 that, in the interest of animal welfare, the unnecessary use of animals is avoided, and that any testing which is likely to produce severe effects in a rabbit eye and to confirm this initial assessment, a Rabbit Enucleated Eye Test (REET) was performed.

This step-wise procedure is in accordance with OECD Test guideline 405, UK Home Office regulations and Safepharm Laboratories Ltd. Ethical Testing strategy.

A study was performed to assess the ocular irritancy potential of the test material in the rabbit following application onto the cornea of the enucleated eye. The reults of the study are believed to be of value in predicting the occular irritation potential of the test material in man.

Methods:       0.1ml of the test material was applied onto the cornea of each of three enucleated eyes which had been maintained at a temperature of 32 deg C +/- 1.5 degC within the superfision chamber. A further two enucleated eyes were treated, for control purposes, with saline solution ( 0.9% Sodium Chloride)

Conclusion:      Following assessment of the data for all endpoints the test material was considered to have the potential to cause severe ocular irritancy in vivo. The in vivo eye irritation study is therefore not required.