Morpholine, 4-C12-14-alkyl derivs.

Endpoint summary

Description of key information

The available data suggest that C12-14 alkylmorpholine is moderately irritating to the skin and only slightly irritating to eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 26 March 2013 to 15 May 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to the OECD Guideline and EU Method in compliance with GLP.

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)

Species:

rabbit

Strain:

other: New Zealand White (Hsdlf:NZW)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Leicestershire, UK
- Age at study initiation: 12-20 weeks old
- Weight at study initiation: 2.63 to 2.85 kg
- Housing: The animals were individually housed in suspended cages.
- Diet: 2930C Teklad global rabbit diet supplied by Harlan Laboratories UK Ltd., Oxon UK (ad libitum)
- Water: Free access to mains drinking water
- Acclimation period: 5 d

ENVIRONMENTAL CONDITIONS
- Temperature: 17 to 23 °C
- Humidity: 30 to 70%
- Air changes: 15 changes/h
- Photoperiod: The lighting was controlled by a time switch to give 12 h continuous light (06:00 to 18:00) and 12 h darkness

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

0.5 mL

Duration of treatment / exposure:

3-minute, 1 and 4 h

Observation period:

14 d

Number of animals:

3

Details on study design:

TEST SITE
On the day before the test each of a group of three rabbits was 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 substance 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.

REMOVAL OF TEST SUBSTANCE
One patch was removed at each of three time points: 3 minutes, 1 h and 4 h after application. Any residual test substance was removed by gentle swabbing with cotton wool soaked in distilled water.

SCORING SYSTEM: Draize

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

other: 24, 48 and 72 h

Score:

0.66

Max. score:

4

Reversibility:

fully reversible within: 14 d

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

other: 24, 48 and 72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 14 d

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

other: 24, 48 and 72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 14 d

Irritation parameter:

edema score

Basis:

animal #1

Time point:

other: 24, 48 and 72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 7 d

Irritation parameter:

edema score

Basis:

animal #2

Time point:

other: 24, 48 and 72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 14 d

Irritation parameter:

edema score

Basis:

animal #3

Time point:

other: 24, 48 and 72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 14 d

Irritation parameter:

primary dermal irritation index (PDII)

Basis:

mean

Time point:

other: 24, 48 and 72 h

Score:

2.8

Max. score:

8

Reversibility:

fully reversible within: 14 d

Irritant / corrosive response data:

3-minute exposure period: Moderate desquamation was noted at the treated skin site at the Day 7 observation. No other evidence of skin irritation was noted during the study. The treated skin site appeared normal at the 14 d observation.
1-h exposure period: Well-defined erythema and very slight edema were noted at the treated skin site at the 72 h observation. Very slight erythema and moderate desquamation were noted at the treated skin site at the 7 d observation. The treated skin site appeared normal at the 14-d observation.
4-h exposure period: Very slight erythema and very slight or slight edema was noted at two treated skin sites immediately after patch removal. Very slight or well-defined erythema and very slight or slight edema were noted at these two treated skin sites one hour after patch removal with well-defined erythema and very slight or slight edema noted at the 24 and 48-h observations. Well-defined erythema and very slight or slight edema were noted at all treated skin sites at the 72 h observations. Well-defined erythema and very slight edema were noted at one treated skin site with very slight erythema, with or without very slight edema, noted at the other two treated skin sites at the 7 d observation. The erythema extended approx 2 cm beyond two treated skin sites at the 24, 48 and 72 h observations and at one of these treated skin sites at the 7 d observation. Edema extended ventrally below one treated skin site at the 24, 48 and 72 h observations. Loss of skin elasticity was noted at one treated skin site at the 48 and 72 h observations. Moderate desquamation was noted at all treated skin sites with superficial cracking of the epidermis also noted at one treated skin site at the 7 d observation. Glossy skin was noted at two treated skin sites at the 7 and 14 d observations. Reduced regrowth of fur was also noted at these two treated skin sites at the 14 d observation.

Other effects:

All animals showed expected gain in body weight during the study.

TABLES:

Table 1: Individual skin reactions following 3 -minute and 1-h exposures:

Skin Reaction	Observation time (following patch removal)	Individual Scores -Rabbit number and sex
		73069 Male
		3 -minute exposure	1 -h exposure
	Immediately	0	0
	1 h	0	0
	24 h	0	0
Erythema/Eschar Formation	48 h	0	0
	72 h	0	2
	7 d	0D	1D
	14 d	0	0
	Immediately	0	0
	1 h	0	0
	24 h	0	0
Edema Formation	48 h	0	0
	72 h	0	1
	7 d	0	0
	14 d	0	0

D = Moderate desquamation,

Table 2. Individual skin reactions following 4-h exposure:

Skin reaction	Observation time (following patch removal)	Individual scores – Rabbit number and sex					Total
		73069 Male		73110 Male		73160 Male
Erythema/Eschar Formation	Immediately	0	1		1		( 2 )
	1 h	0	1		2		( 3 )
	24 h	0	2R		2R		4
	48 h	0	2R		2RLe		( 4 )
	72 h	2	2R		2RLe		6
	7 d	1D	1DG		2RDGCr		( 4 )
	14 d	0	0GFr		0GFr		( 0 )
Edema Formation	Immediately	0	1		2		( 3 )
	1 h	0	1		2		( 3 )
	24 h	0	1		2Oe		3
	48 h	0	1		2Oe		( 3 )
	72 h	1	1		2Oe		4
	7 d	0	1		1		( 2 )
	14 d	0	0		0		( 0 )
Sum of 24 and 72-h readings (S) : 17
Primary Irritation Index (S/6) : 17/6 = 2.8
Classification : MODERATE IRRITANT

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

D = Moderate desquamation

Le = Loss of skin elasticity

G = Glossy skin

Cr = Superficial cracking of the epidermis

Fr = Reduced regrowth of fur

R = Reaction extends approximately 2 cm beyond treatment site

Oe = Edema extends ventrally below treatment site

Interpretation of results:

Category 2 (irritant)

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the study conditions, the test substance produced moderate skin irritation in rabbits.

Executive summary:

The study was performed to assess the irritancy potential of C12-14 alkylmorpholine following single, 3-minute, 1 and 4-h, semi-occluded applications to the intact rabbit skin according to the OECD Guideline 404 and EU Method B.4 in compliance with GLP.

0.5 mL of the test substance was applied directly to the skin under a 2.5 cm x 2.5 cm cotton gauze patch.

3-minute and 1 h semi-occluded applications of the test substance to the intact skin of one rabbit produced no corrosive effects. A single 4-h, semi occluded application of the test substance to the intact skin of three rabbits produced well-defined erythema and very slight to slight edema. Other skin reactions noted were moderate desquamation, loss of skin elasticity, glossy skin, superficial cracking of the epidermis, reduced regrowth of fur and reactions extending beyond the treatment site. No corrosive effects were noted.

Under the study conditions, the test substance produced moderate skin irritation with a primary irritation index of 2.8.

The average of the reactions at 24, 48 and 72 hrs were below 2.3 for oedema and erythema/eschar formation for all three animals, and the scoring at the end of the 14-day observation was 0 for all animals. However, considering observations as moderate desquamation in all three animals, and the involvement of reactions extending beyond treatment site, loss of skin elasticity, superficial cracking of the epidermis in two animals resulting to glossy skin and reduced regrowth of fur still observed at the end of the 14-day observation period, classification for Cat.2 skin irritation seems justified.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 11 April, 2013 to 19 April, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to the OECD Guideline and EU Method in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

other: New Zealand White (Hsdlf:NZW)

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Leicestershire, UK
- Age at study initiation: 12 to 20 weeks old.
- Weight at study initiation: 2.49 or 2.83 kg
- Housing: The animals were individually housed in suspended cages.
- Diet: 2930C Teklad global rabbit diet supplied by Harlan Laboratories UK Ltd., Oxon UK (ad libitum)
- Water: Free access to mains drinking water
- Acclimation period: 5 d

ENVIRONMENTAL CONDITIONS
- Temperature: 17 to 23 °C
- Humidity: 30 to 70%
- Air changes: 15 changes/h
- Photoperiod: The lighting was controlled by a time switch to give 12 h continuous light (06:00 to 18:00) and 12 h darkness

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

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

Duration of treatment / exposure:

The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test substance, and then released.

Observation period (in vivo):

Assessment of ocular damage/irritation was made approx 1 h and 24, 48 and 72 h following treatment.

Number of animals or in vitro replicates:

2

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): No

SCORING SYSTEM: Draize score

TOOL USED TO ASSESS SCORE: Light source from a standard ophthalmoscope

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

other: 24, 48 and 72 h

Score:

0

Max. score:

4

Reversibility:

other: No adverse effects were observed.

Irritation parameter:

iris score

Basis:

mean

Time point:

other: 24, 48 and 72 h

Score:

0

Max. score:

2

Reversibility:

other: No adverse effects were observed.

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

other: 24, 48 and 72 h

Score:

0.66

Max. score:

3

Reversibility:

fully reversible within: 72 h

Irritation parameter:

chemosis score

Basis:

mean

Time point:

other: 24, 48 and 72 h

Score:

0.66

Max. score:

4

Reversibility:

fully reversible within: 72 h

Irritant / corrosive response data:

No corneal or iridial effects were noted during the study. Moderate conjunctival irritation was noted in one treated eye and minimal conjunctival irritation was noted in the other treated eye one and 24 h after treatment. Minimal conjunctival irritation was noted in both treated eyes at the 48 h observation. Both treated eyes appeared normal at the 72 h observation.

Other effects:

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

Table 1. individual scores and individual total scores for ocular irritation:

Rabbit number and sex	73111 Male				73124 Male
	IPR = 2				IPR = 2
Time after treatment	1 h	24 h	48 h	72 h	1 h	24 h	48 h	72 h
Cornea
E = Degree of Opacity	0	0	0	0	0	0	0	0
F = Area of Cornea Involved	0	0	0	0	0	0	0	0
Score (E x F) x 5	0	0	0	0	0	0	0	0
Iris
Score (D x 5)	0	0	0	0	0	0	0	0
Conjunctivae
A = Redness	2	1	1	0	2	2	1	0
B = Chemosis	1	1	1	0	2	1	1	0
C = Discharge	0	1	0	0	1	1	0	0
Score (A + B + C) x 2	6	6	4	0	10	8	4	0
Total Score	6	6	4	0	10	8	4	0

 

Table 2. Individual total scores and group mean scores for ocular irritation:

Rabbit number and sex	Individual total scores at:
	1 h	24 h	48 h	72 h
73111 Male	6	6	4	0
73124 Male	10	8	4	0
Group total	16	14	8	0
Group mean score	8	7	4	0

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the study conditions, the test substance produced mild irritation which was fully reversible with 72 h after treatment.

Executive summary:

The study was performed to assess the irritancy potential of C12-14 alkylmorpholine to the eye of the New Zealand White rabbit in accordance with the OECD Guideline 405 and EU Method B.5 in compliance with GLP.

A volume of 0.1 mL of the test substance was placed into the conjunctival sac of the right eye. The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test substance, and then released. The left eye remained untreated and was used for control purposes. Theassessment of ocular damage/irritation was made approx 1 h and 24, 48 and 72 h following treatment.

No corneal or iridial effects were noted during the study. Moderate conjunctival irritation was noted in one treated eye and minimal conjunctival irritation was noted in the other treated eye one and 24 h after treatment. Minimal conjunctival irritation was noted in both treated eyes at the 48 h observation. Both treated eyes appeared normal at the 72 h observation. Both animals showed expected gain in body weight during the study. The maximum group mean score was calculated to be 8.0.

Under the study conditions, the test substance produced mild irritation which was fully reversible with 72 h after treatment.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin:

An in vitro study was performed to assess the corrosive potential of C12-14 alkylmorpholine by means of the Human Skin Model Test with EpiDerm™ tissues models according to the OECD Guideline 431 in compliance with GLP. Independent duplicate tissues of EpiDerm™ were exposed to either the test substance, the negative control (deionised water) or the positive control (8.0 N KOH) for 3 min and 1 h, respectively. After exposure to the negative control the absorbance values met the required acceptability criterion of mean OD570 ≥ 0.8 for both treatment intervals thereby confirming the acceptable quality of the tissues. After the 42 h incubation period was completed for all tissues of each time point cell culture inserts were transferred from the holding plates to the MTT-plates. After a 3 h incubation period (37 ± 1 °C, 5 ± 0.5 % CO2) MTT solution was aspirated from the wells and wells were rinsed three times with DPBS. Inserts were transferred into new 24 well plates. The inserts were immersed into extractant solution by gently pipetting 2 mL extractant solution (isopropanol) into each insert. The level rose above the upper edge of the insert, thus the tissue was completely covered from both sides. The 24 well plate was sealed to inhibit isopropanol evaporation. The formazan salt was extracted for about 19 h at room temperature. Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 min exposure period (28.6%) and for the 1 h exposure period (9.7%) thus confirming the validity of the test system and the specific batch of tissue models. After 3 min and 1 h exposure of the test substance to the tissues the relative absorbance values decreased to 74.8% and to 87.8%, respectively. Both values did not exceed the threshold for corrosivity which is defined to be 50% after the 3 min exposure and 15% after the 1 h exposure. Therefore, the test substance was not considered to be corrosive.

 

An in vitro study was conducted to evaluate the irritation potential of C12-14 alkylmorpholine by means of the human skin model test according to the OECD Guideline 439, in compliance with GLP. Each three tissues of the human skin model EpiDerm™ were treated with the test substance, the negative or the positive control for 60 min. 30 µL of the liquid test substance were applied to each tissue and spread to match the surface of the tissue. 30 µL of either the negative control (Dulbecco's phosphate buffered saline) or the positive control (5% Sodium lauryl sulfate) were applied to each tissue. The test substance and the positive and negative controls were washed off the skin tissues after 60 min treatment. After further incubation for about 40.5 h the tissues were treated with the MTT solution for 3 h following nearly 70 h extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm. After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD≥1.0 and ≤2.5 for 60 min treatment interval thus showing the quality of the tissues. Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 5.5% thus ensuring the validity of the test system. The standard deviations between the % variabilities of the test substance, the positive and negative controls were below 4% (threshold of the OECD Guideline 439: 18%), thus ensuring the validity of the study. Compared to the relative absorbance value of the negative control the mean relative absorbance value decreased to 95.5% after exposure of the test substance to the skin tissues. This value is well above the threshold for irritancy of ≤ 50%. Therefore, the test substance is not considered to possess an irritant potential.

 

To confirm the obtained in vitro results, a study was performed to assess the irritancy potential of C12-14 alkylmorpholine following single, 3-minute, 1 and 4-h, semi-occluded applications to the intact rabbit skin according to the OECD Guideline 404 and EU Method B.4 in compliance with GLP. 0.5 mL of the test substance was applied directly to the skin under a 2.5 cm x 2.5 cm cotton gauze patch. 3-minute and 1 h semi-occluded applications of the test substance to the intact skin of one rabbit produced no corrosive effects. A single 4-h, semi occluded application of the test substance to the intact skin of three rabbits produced well-defined erythema and very slight to slight oedema. Other skin reactions noted were moderate desquamation, loss of skin elasticity, glossy skin, superficial cracking of the epidermis, reduced regrowth of fur and reactions extending beyond the treatment site. No corrosive effects were noted. Under the study conditions, the test substance produced moderate skin irritation with a primary irritation index of 2.8.

The average of the reactions at 24, 48 and 72 hrs were below 2.3 for oedema and erythema/eschar formation for all three animals, and the scoring at the end of the 14-day observation was 0 for all animals. However, considering observations as moderate desquamation in all three animals, and the involvement of reactions extending beyond treatment site, loss of skin elasticity, superficial cracking of the epidermis in two animals resulting in both animals to glossy skin and reduced regrowth of fur still observed at the end of the 14-day observation period, classification for Cat.2 skin irritation seems justified.

 

Eye:

An in vitro study was performed to assess the corneal irritation and damage potential of C12-14 alkylmorpholine by means of the BCOP assay using fresh bovine corneae according to the OECD Guideline 437 and EU Method B.47 in compliance with GLP. The test substance was tested undiluted. The positive control 2-ethoxyethanol was tested neat. Saline was used as negative control substance. After a first opacity measurement of the fresh bovine corneae (t0), the neat test substance, the positive, and the negative controls were applied to corneae and incubated for 10 min at 32 ± 1 °C. After the incubation phase the test substance, the positive, and the negative controls were each rinsed from the corneae. Further, the corneae were incubated for another 120 min at 32 ± 1 °C in complete medium, and opacity was measured a second time (t130). After the opacity measurements permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 min at 32 ± 1°C.With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean in vitro irritation score 1.38). Relative to the negative control, the test substance did not induce an increase of the corneal opacity or permeability. The calculated mean in vitro irritation score was 0.00 (threshold for corrosivity / severe irritancy: ≥ 55.1). Under the test conditions, the test substance is classified as not corrosive / not severe irritant to the eye.

 

In order to derive a definitive classification a study was performed to assess the irritancy potential of C12-14 alkylmorpholine to the eye of the New Zealand White rabbit in accordance with the OECD Guideline 405 and EU Method B.5 in compliance with GLP. A volume of 0.1 mL of the test substance was placed into the conjunctival sac of the right eye. The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test substance, and then released. The left eye remained untreated and was used for control purposes. The assessment of ocular damage/irritation was made approx.. 1 h and 24, 48 and 72 h following treatment. No corneal or iridial effects were noted during the study. Moderate conjunctival irritation was noted in one treated eye and minimal conjunctival irritation was noted in the other treated eye one and 24 h after treatment. Minimal conjunctival irritation was noted in both treated eyes at the 48 h observation. Both treated eyes appeared normal at the 72 h observation. Both animals showed expected gain in body weight during the study. Under the study conditions, the test substance produced mild irritation which was fully reversible with 72 h after treatment.

Justification for selection of skin irritation / corrosion endpoint:
In vivo study is preferred over in vitro study.

Justification for selection of eye irritation endpoint:
In vivo study is preferred over in vitro study.

Effects on skin irritation/corrosion: moderately irritating

Justification for classification or non-classification

Although twoin vitroskin irritation and corrosion studies did not reveal any signs of skin irritation, an in vivo study showed that C12-14 alkylmorpholine can produce moderate skin irritation in rabbits. Hence, the data justifies classification as skin irritation category 2; H315-causes skin irritation (hazard statement) according to CLP criteria (EC 1272/2008). Further, based on the available reliablein vivoeye irritation study, an instillation of 0.1 mL of the test substance resulted in mild irritation which had completely resolved within 72 h. Hence, the data does not qualify for eye irritation classification according to CLP (EC 1272/2008) criteria.

No data is available regarding possible respiratory irritation. However, considering that no classification is needed for eye irritation, and that following the low vp the likelihood of exposure is limited, respiratory irritation is not likely to occur.