2-morpholinoethanesulphonic acid

Endpoint summary

Description of key information

In an experimental study according to OECD TG 439 under GLP conditions, the test substance did not reduce cell viability. Therefore, it is considered to be not skin irritating and does not require to be classified (reference 7.3.1 -1).

In an experimental study according to OECD TG 438 under GLP conditions, the test substance scored into ICE Class I for corneal opacity and swelling and for fluorescein retention. With the overall ICE score of 3xI, the substance is classified as "No Category" for eye irritation (reference 7.3.2 -1).

Both studies have been performed with the hydrate form of the substance (CAS 1266615-59-1). However, same results are expected for the anhydrous form (CAS 4432-31-9).

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:

2016-06-29 to 2016-07-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

TISSUE
- Model used: EPISKIN(TM) Small Model (SM)
- Tissue batch number: 16-EKIN-026
- Date of initiation of testing: 29.06.2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37°C for 42 hours

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 mL PBS solution used to thoroughly rinse the tissue once
- Observable damage in the tissue due to washing: none
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 mL of 0.3 mg/mL MTT per well
- Incubation time: 3 hours +/- 5 min
- Spectrophotometer: not specified
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Barrier function: IC50=2.6 mg/mL
- Morphology: weel-differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum
- Contamination: absence of bacteria, fungus and mycoplasma
- Reproducibility:

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: no direct MTT interference

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be non-irritating to skin if the viability is greater than or equal to 50%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg (to moistened skin)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration: 5% aq.

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

103

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: demonstrated in a separate study (study no. 392.554.2938)

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: YES (mean OD of 0.762 in the range from 0.6 to 1.5; SD of 10.23 < 18)
- Acceptance criteria met for positive control: YES (mean viability of 18% in the range from 0 to 40%; SD of 7.55 < 18)
- Acceptance criteria met for variability between replicate measurements: YES (test substance: SD of 12.86 < 18)
- Range of historical values if different from the ones specified in the test guideline: no

Interpretation of results:

GHS criteria not met

Conclusions:

In a study according to OECD TG 439 , the test substance did not reduce cell viability. Therefore, it is considered not irritating to skin.

Executive summary:

A study according OECD TG 439 has been performed to predict the irritation potential of the test item by measurement of its cytotoxic effect, as reflected in the MTT assay. Triplicates of the human skin model EPISKIN (Reconstructed Human Epidermis, SkinEthic Laboratories) were treated with test item and incubated for 15 minutes at room temperature. Exposure of the reconstructed human epidermis skin units with test item was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in 5% CO2 protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically.

SDS (5% aq.) and 1×PBS treated epidermis (three units / positive and negative control) were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control. A test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control.In thisin vitroskin irritation test using the EPISKIN model, the test item did not show significantly reduced cell viability in comparison to the negative control (mean value: 103 %). All obtained test item viability results were far above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-06-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

2013

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni út 129. Hungary
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):
The heads were transported to Toxi-Coop ZRT. at the earliest convenience for use approximately within 2 hours from collection. The ambient temperature was optimal (19.3 ºC to 20.2 ºC) during the transport. All eyes used in the assay were from the same groups of eyes collected on one specific day. After collection, the heads were inspected for appropriate quality and wrapped with paper moistened with saline, then placed in a plastic box that can be closed (4-5 heads/box).
- Time interval prior to initiating testing: If the selected eyes were appropriate for the test, acclimatization started and was conducted for approximately 45 to 60 minutes.
- indication of any existing defects or lesions in ocular tissue samples:
After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of fluorescein solution 2 (w/v) % was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL isotonic saline. Then the fluorescein-treated cornea was examined with hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit. The cornea thickness was measured using the depth measuring device on the slit lamp microscope (Haag-Streit BQ 900) with the slit-width set at 9½, equalling 0.095 mm. Any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced.
- Indication of any antibiotics used: none

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.03 g (per eye)

CONTROLS
- Amount(s) applied (volume or weight with unit): 0.03g imidazole; 30 µL NaCl (in saline)

Duration of treatment / exposure:

10 sec

Duration of post- treatment incubation (in vitro):

4 hours

Number of animals or in vitro replicates:

3 eyes (test substance and positive control)
1 eye (negative control)

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES:
With the eyes still in the chicken heads, eyelids were carefully cut away with scissors from the chicken eyes. One small drop of fluorescein solution 2% (w/v) was applied onto the cornea surface for a few seconds and then rinsed off with 20 mL isotonic saline. The fluorescein-treated cornea was examined with a hand-held slit lamp or a slit lamp microscope. If the cornea was in good condition, the eyeball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors without cutting off the optical nerve too short. The procedure avoided pressure on the eye, which may lead to corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box to maintain the appropriate humidity.

EQUILIBRATION AND BASELINE RECORDINGS
The prepared eye was placed in a clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head), again avoiding too much pressure on the eye. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was applied to fix the eye properly. The clamp was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with saline solution dripping from a stainless steel tube, at a rate of approx. 3 to 5 drops/min. The door of the chamber was closed, except for manipulations and examination, to maintain temperature and humidity.
Eyes selected for testing were again examined with the slit lamp microscope. Eyes with a high baseline fluorescein staining (i.e. > 0.5) or corneal opacity score (i.e. > 0.5) were rejected.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: NaCl in saline (9 g/L) (n=1)

SOLVENT CONTROL USED: not applicable

POSITIVE CONTROL USED: Imidazole (n=3)

APPLICATION DOSE AND EXPOSURE TIME: 0.03 g per eye for 10 seconds

OBSERVATION PERIOD: 4 hours

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: Thorough rinsing with approx. 20 mL saline solution at ambient temperature
- Indicate any deviation from test procedure in the Guideline: none

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacity scores ranging from 0 (no opacity) to 4 (complete opacity; iris invisible) according to the test guideline were measured at all time points
- Damage to epithelium based on fluorescein retention: Fluorescein retention scores ranging from 0 - 3 according to the test guideline were measured at 0 and 30 min.
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: Cornea thickness was measured using the depth measuring device on the slit lamp microscope (Haag-Strei BQ 900) with the slit-width set at 9.5, i.e. 0.095mm.
- Macroscopic morphological damage to the surface: not applicable
- Others (e.g, histopathology): not performed

SCORING SYSTEM:
- Mean corneal swelling (%): according to TG
- Mean maximum opacity score: according to TG
- Mean fluorescein retention score at 30 minutes post-treatment: according to TG

DECISION CRITERIA: Decision criteria as indicated in the TG were used.

Irritation parameter:

percent corneal swelling

Remarks:

240 min

Run / experiment:

1st experiment

Value:

2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

2

Positive controls validity:

valid

Remarks:

30

Irritation parameter:

cornea opacity score

Run / experiment:

1st experiment

Value:

0.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0.5

Positive controls validity:

valid

Remarks:

4

Irritation parameter:

fluorescein leakage

Run / experiment:

1st experiment

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0

Positive controls validity:

valid

Remarks:

3

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none

DEMONSTRATION OF TECHNICAL PROFICIENCY: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline: not applicable

Interpretation of results:

GHS criteria not met

Conclusions:

In this in vitro eye corrosive and severe irritant study, using the Isolated Chicken Eye model, no ocular corrosion or severe irritation potential was observed. The overall ICE score was 3xI.

Executive summary:

A study according OECD TG 438 was performed to evaluate the potential ocular corrosivity and irritancy of the test item by its ability to induce toxicity in enucleated chicken eyes. This test identifies chemicals either inducing "serious eye damage", or not requiring classification for eye irritation or serious eye damage according to the GHS. The test item was applied in a single dose (30 mg/eye) onto the cornea of isolated chicken eyes and rinsed after 10 seconds with saline. Tested corneas were evaluated pre-treatment and at approximately 30, 75, 120, 180, and 240 minutes after the post-treatment rinse. The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects. All of the endpoints, with the exception of fluorescein retention (which was determined only at pre-treatment and 30 minutes after test substance exposure) were determined at each of the above time points.

The test item and Imidazole (positive control) were ground before use in the study. Test item and positive control were applied in an amount of 30 mg/eye by powdering the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance or positive control. Three test item treated eyes and three positive control eyes were used in this study.

One negative control eye was treated with 30μL saline solution.

After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with approximately 20 mL saline solution at ambient temperature and this procedure was repeated for each eye.

In this ICET, the test item did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. The overall ICE class was 3xI.

Positive and negative controls showed the expected results. The experiment was considered to be valid.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

A study according OECD TG 439 has been performed to predict the irritation potential of the test item by measurement of its cytotoxic effect, as reflected in the MTT assay. Triplicates of the human skin model EPISKIN (Reconstructed Human Epidermis, SkinEthic Laboratories) were treated with test item and incubated for 15 minutes at room temperature. Exposure of the reconstructed human epidermis skin units with test item was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in 5% CO2 protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically.

SDS (5% aq.) and 1×PBS treated epidermis (three units / positive and negative control) were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control. A test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control.In this in vitro skin irritation test using the EPISKIN model, the test item did not show significantly reduced cell viability in comparison to the negative control (mean value: 103 %). All obtained test item viability results were far above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

Eye irritation

A study according OECD TG 438 was performed to evaluate the potential ocular corrosivity and irritancy of the test item by its ability to induce toxicity in enucleated chicken eyes. This test identifies chemicals either inducing "serious eye damage", or not requiring classification for eye irritation or serious eye damage according to the GHS. The test item was applied in a single dose (30 mg/eye) onto the cornea of isolated chicken eyes and rinsed after 10 seconds with saline. Tested corneas were evaluated pre-treatment and at approximately 30, 75, 120, 180, and 240 minutes after the post-treatment rinse. The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects. All of the endpoints, with the exception of fluorescein retention (which was determined only at pre-treatment and 30 minutes after test substance exposure) were determined at each of the above time points.

The test item and Imidazole (positive control) were ground before use in the study. Test item and positive control were applied in an amount of 30 mg/eye by powdering the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance or positive control. Three test item treated eyes and three positive control eyes were used in this study.

One negative control eye was treated with 30 μL saline solution.

After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with approximately 20 mL saline solution at ambient temperature and this procedure was repeated for each eye.

In this ICET, the test item did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. The overall ICE class was 3xI.

Positive and negative controls showed the expected results. The experiment was considered to be valid.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on this data, the substance is not considered to be classified for skin or eye irritation under Regulation (EC) No 1272/2008, as amended for the fifteenth time in Regulation (EU) 2020/1182.