Morpholinium sulphamate

Endpoint summary

Description of key information

In vitro skin irritation:
The results obtained from an in vitro skin irritation test, using the EPISKIN model, indicated that the test item Morpholinium Sulphamate revealed no skin irritantion potential under the utilised testing conditions
In vivo eye irritation:
The test item morpholinium sulphamate applied to the rabbit's eye mucosa, caused slight to severe irritation effects on the conjunctiva within one hour after test item administration. The condition of the conjunctiva improved within 24 hours (slight to moderate effects) and 72 hours (slight effects) and the observed effects were fully reversible within 1 week. Thus, the test item was judged to be not irritating to the eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-12-22 to 2011-12-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline No. 439, In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method, adopted 22 July 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU Method B.46 In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test, adopted 23 July 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Cell type:

other: highly differentiated and stratified epidermis model

Cell source:

other: EpiSkinTMSM, EPISKIN Laboratories Lyon, France

Source strain:

other: not applicable

Details on test system:

The EPISKIN Model (EPISKIN SNC Lyon, France, Supplier: SKINETHIC Laboratories; 4, rue Alexandre Fleming, 69366 – LYON, France) is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-day culture period comprising the main basal, supra basal, spinous and granular layers and
a functional stratum corneum. Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

Details on Study design

Pre-incubation (day -1):
The “maintenance medium” was pre-warmed to 37°C. The appropriate number of assay plate wells were filled with the pre-warmed medium (2 mL per well). The epidermis units were placed, with the media below them in contact with the epidermis, into each prepared well and then incubated overnight at 37°C in an incubator with 5 % CO2.

Application (day 0):
Epidermal surface was first moistened with 10 µL deionised water (in order to improve further contact between powder and epidermis) and then 10 mg of the test item was applied evenly onto the skin. The test item was spread gently with a curved flat spatula in order to cover evenly all the skin surface if necessary.

A volume of 20 µL positive control (SDS 5 % aq.) or negative control (1x PBS) was applied to the skin surface by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary.

Exposure (day 0)
The plates with the treated epidermis units were incubated for the exposure time of 15 minutes at room temperature.

Rinsing (day 0):
After the incubation time the EPISKIN units were removed and rinsed thoroughly with PBS 1x solution to remove all of the test material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with a suitable pipette tip linked to a vacuum source (care was taken to avoid damaging the epidermis).

Post-incubation (day 0-2):
After rinsing the units were placed into the plate wells filled with fresh pre-warmed “maintenance medium” (2 mL/well) below them and then incubated for 42 hours at 37°C in an incubator with 5% CO2.

MTT test after 42 hours incubation (day 2):
After the 42 hours incubation the EPISKIN-SM units were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well) and then incubated for 3 hours at 37°C in an incubator with 5 % CO2 protected from light.

Formazan extraction (day 2):
At the end of incubation with MTT a formazan extraction step was undertaken:
A defined disk of epidermis from each replicate was cut from the unit (this involves the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube of 500 µL acidified isopropanol (one tube corresponding to one well of the tissue culture plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material to the acidified isopropanol then incubated for about two hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.

Cell viability measurements (day 2):
Following the formazan extraction, 2×200 µL samples from each tube were placed into the wells of a 96-well plate (labelled appropriately). The optical densities of each well were recorded using a 96-well plate spectrophotometer at a wavelength of 570 nm while using an acidified isopropanol solution blank (6×200 µL).

Control samples:

yes, concurrent no treatment

Amount/concentration applied:

The test item was applied in its original form, no formulation was required. 10 mg of the test item was applied to each EpiSkin epidermis units.

Duration of treatment / exposure:

15 min

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

98

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Interpretation of results:

not irritating

Conclusions:

In conclusion, the results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item Morpholinium Sulphamate revealed no skin irritantion potential under the utilised testing conditions

Executive summary:

In this in vitro skin irritation test using the EPISKIN model, the test item Morpholinium Sulphamate did not show a significantly reduced cell viability in comparison to the negative control. All obtained test item viability results were above 50% when compared to the viability values obtained from the negative control. Positive and negative controls showed the expected cell viability values within acceptable limits, thus the experiment was considered to be valid. The results indicated that the test item Morpholinium Sulphamate revealed no skin irritantion potential under the utilised testing conditions. According to the current OECD Guideline No. 439, Morpholinium Sulphamate is considered a non-irritant to skin and is therefore not classified.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2012-10-09 until 2012-10-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

24 April 2002

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: S & K LAP Kft. 2173 Kartal, Császár út 135, Hungary
- Age at study initiation: young adult male rabbits
- Weight at study initiation: 2834-3195 g
- Weight at end of study: 3077-3195
- Housing: Animals were housed individually in metal cages.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 18 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 ± 3°C
- Humidity: 30 - 70 %
- Air changes (per hr): 9-12
- Photoperiod (hrs dark / hrs light): 12 hours artificial light, from 6 a.m. to 6 p.m.

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

A weight of 0.1 g of the undiluted and well grinded test item morpholinium sulphamate was used for the study in a single dose.

Duration of treatment / exposure:

The eyes of the test animals were not washed out after the application of test item.

Observation period (in vivo):

The eyes were examined at 1, 24, 48 and 72 hour(s) and then at 1 and 2 weeks after the application.

Number of animals or in vitro replicates:

3 male animals

Details on study design:

SCORING SYSTEM: The eye irritation scores were evaluated according to the scoring system by Draize (1979) and OECD 405 (24 April 2002)

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 1 week

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.33

Max. score:

4

Reversibility:

fully reversible within: 1 week

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 1 week

Irritation parameter:

conjunctivae score

Remarks:

discharge

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 1 week

Irritation parameter:

conjunctivae score

Remarks:

discharge

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 1 week

Irritation parameter:

conjunctivae score

Remarks:

discharge

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 1 week

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 1 week

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 1 week

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.33

Max. score:

4

Reversibility:

fully reversible within: 1 week

Irritant / corrosive response data:

The eyes were examined at 1, 24, 48 and 72 hours then 1 week after the application.

One hour after treatment, some hyperemic blood vessels (score 1) were observed in two animals and diffuse, crimson coloured conjunctivae with not easily discernible individual vessels (score 2) occurred in one animal. The obvious swelling with partial eversion of lids (score 2) was detected in one animal and the conjunctival swelling with lids about half closed (score 3) was detected in two animals. The discharge with moistening of the lids and hairs just adjacent to lids (score 2) were observed in two animals and the discharge moistened lids and hairs on considerable area around the treated eye (score 3) was recorded in one animal.

24 hours after treatment, some hyperemic blood vessels (score 1) were observed in two animals and diffuse, crimson coloured conjunctivae with not easily discernible individual vessels (score 2) occurred in one animal. The obvious swelling with partial eversion of lids (score 2) was detected in all animals. The discharge with moistening of the lids and hairs just adjacent to lids (score 2) were observed in two animals and the amount of the discharge was different from normal (score 1) in one animal.

48 hours after treatment, some hyperemic blood vessels (score 1) were observed in all animals. The swelling was different from normal (score 1) in all animals and the amount of the discharge was different from normal (score 1) in two animals.

72 hours after the treatment, some hyperemic blood vessels (score 1) were observed in all animals and the swelling was different from normal (score 1) in one animal.

1 week after the treatment the study was terminated, since no primary irritation symptoms occurred.

During the study the control eyes of the animals were symptom-free.

General state and the behaviour of the animals were normal throughout the study period. There were no effects on body weight attributable to the treatment with the test item during the contact and observation period.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

In conclusion, test item morpholinium sulphamate applied to the rabbits' eye mucosa, caused slight to severe irritation effects on the conjunctiva within one hour after test item administration. The condition of the conjunctiva improved within 24 hours (slight to moderate effects) and 72 hours (slight effects) and the observed effects were fully reversible within 1 week.

Executive summary:

The acute eye irritation study of the test item morpholinium sulphamate was performed in three New Zealand White rabbits. The irritation effect of the test item was evaluated according to the method by Draize (1979) and OECD No.: 405, 2002. The test item was placed into the conjunctival sac of the left eye of each animal. The untreated right eye served as control. A weight of 0.1 g of the test item was used in well grinded state, as a single dose. The eyes of the test animals were not washed out after the application of test item. The eyes were examined at 1, 24, 48 and 72 hours then 1 week after the application. One hour after the treatment, slight to moderate conjunctival redness, moderate to severe chemosis and moderate to severe discharge were observed. 48 hours after the treatment, slight redness, chemosis and discharge were recorded. 1 week after the treatment, all animals became free of symptoms. 1 week after treatment the study was terminated, as all animals were free of symptoms of irritation.

The animals’ mean scores (considering readings at 24, 48 and 72 hours after the treatment) were as follows:

cornea opacity: 0.00, 0.00, 0.00

iris: 0.00, 0.00, 0.00

redness: 1.00, 1.33, 1.00

chemosis: 1.00, 1.00, 1.33

discharge: 1.00, 1.00, 0.33

In conclusion, test item morpholinium sulphamate applied to the rabbits' eye mucosa, caused slight to severe irritation effects on the conjunctiva within one hour after test item administration. The condition of the conjunctiva improved within 24 hours (slight to moderate effects) and 72 hours (slight effects) and the observed effects were fully reversible within 1 week. According to CLP criteria for classification and labelling requirements for dangerous substances and preparations, the test item does not have to be classified and has no obligatory labelling requirement for eye irritation. According to Regulation (EC) No 1272/2008, the test item has not been classified into any category.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro skin irritation:

In an in vitroskin irritation test using the EPISKIN model, the test item Morpholinium Sulphamate did not show a significantly reduced cell viability in comparison to the negative control. All obtained test item viability results were above 50% when compared to the viability values obtained from the negative control. Positive and negative controls showed the expected cell viability values within acceptable limits, thus the experiment was considered to be valid. The results indicated that the test item Morpholinium Sulphamate revealed no skin irritantion potential under the utilised testing conditions. According to the current OECD Guideline No. 439, Morpholinium Sulphamate is considered a non-irritant to skin and is therefore not classified.

 

In vivo eye irritation:

In an in vitro eye corrosives and severe irritants study, using the Isolated Chicken Eye model with morpholinium sulphamate, no ocular corrosion or severe irritation potential was observed. Thus, according to the guideline OECD 438, the test item cannot be classified as an ocular corrosive or severe eye irritant. Furthermore, the results suggest that the test item was slightly irritating. However, to obtain a definitive classification in relation to the irritation potential, a further in vivo rabbit study is required.

 

The acute eye irritation study of the test item morpholinium sulphamate was performed in three New Zealand White rabbits. The irritation effect of the test item was evaluated according to the method by Draize (1979) and OECD No.: 405, 2002. The test item was placed into the conjunctival sac of the left eye of each animal. The untreated right eye served as control. A weight of 0.1 g of the test item was used in well grinded state, as a single dose. The eyes of the test animals were not washed out after the application of test item. The eyes were examined at 1, 24, 48 and 72 hours then 1 week after the application. One hour after the treatment, slight to moderate conjunctival redness, moderate to severe chemosis and moderate to severe discharge were observed. 48 hours after the treatment, slight redness, chemosis and discharge were recorded. 1 week after the treatment, all animals became free of symptoms. 1 week after treatment the study was terminated, as all animals were free of symptoms of irritation.

The animals’ mean scores (considering readings at 24, 48 and 72 hours after the treatment) were as follows:

cornea opacity: 0.00, 0.00, 0.00

iris: 0.00, 0.00, 0.00

redness: 1.00, 1.33, 1.00

chemosis: 1.00, 1.00, 1.33

discharge: 1.00, 1.00, 0.33

 

In conclusion, test item morpholinium sulphamate applied to the rabbits' eye mucosa, caused slight to severe irritation effects on the conjunctiva within one hour after test item administration. The condition of the conjunctiva improved within 24 hours (slight to moderate effects) and 72 hours (slight effects) and the observed effects were fully reversible within 1 week. According to CLP criteria for classification and labelling requirements for dangerous substances and preparations, the test item does not have to be classified and has no obligatory labelling requirement for eye irritation.

Justification for selection of skin irritation / corrosion endpoint:
Guideline and CLP compliant in vitro skin irrtation study

Justification for selection of eye irritation endpoint:
Guideline and CLP compliant in vivo eye irrtation study

Justification for classification or non-classification

Based on the available experimental data the test item is not classified with regards to skin and eye irritation/corrosion according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighteenth time in Regulation (EU) No 2022/692.