Sodium 4-(2-hydroxyethyl)piperazin-1-ylethanesulphonate

Endpoint summary

Description of key information

The test item was determined to have no skin irritation potential in an in vitro study according to OECD 439 (reference 7.3.1-1). 

The test item was determined to have no eye irritation potential in an in vitro study according to OECD 438 (reference 7.3.2-1).

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:

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

Version / remarks:

06 July 2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: adult donors, not further specified

Justification for test system used:

The EPISKIN model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and no- skin irritating test substances (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM Small Model (EpiSkinTMSM) (manufactured by EPISKIN SNC Lyon, France)
- Tissue batch number: 16-EKIN-026
- Expiry date: 04 July 2016
- Certification and release date: 28 June 2016
- Date of initiation of testing: 29 June 2016

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: approximately 25 mL PBS 1x solution, 1 washing step
- 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: 0.3 mg/mL
- Incubation time: 3 hours (± 5 min)
- Spectrophotometer: 96-well plate spectrophotometer
- Wavelength: 570 nm
- Filter: not specified
- Filter bandwidth: not specified
- Linear OD range of spectrophotometer: not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Histology scoring: Historical control value: >= 19.5, 22.7 +/- 0.5 for batch used
- IC50: Historical control value: 1.5 mg/mL <= IC50 <= 3 mg/mL, 2.6 mg/mL for batch used
- Morphology: Well-differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum was satisfactory in the batch used.
- Contamination: Blood of donor: absence of HIV 1+2, hepatitis C antibodies and hepatitis B antigen HB; epidermal cells of donor: absence of bacteria, fungus and mycoplasma

NUMBER OF REPLICATE TISSUES:
3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test material did not interact with the MTT, therefore additional controls and data calculations were not necessary.

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 corrosive or irritating to skin if the viability after exposure is <= 50 %.
- The test substance is considered to be non-corrosive to skin if the viability after exposure is greater than 50 %.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 10 mg (to moistened skin)

NEGATIVE CONTROL
- Amount applied: 10 µL

POSITIVE CONTROL
- Amount applied: 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:

104

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100

Positive controls validity:

valid

Remarks:

18

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: No colour change was observed after three hours of incubation. The test material did not interact with the MTT, therefore additional controls and data calculations were not necessary. A false estimation of viability can be precluded.
- Colour interference with MTT: The test item showed no ability to become coloured in contact with water. The intrinsic colour of test item is white and therefore considered to be not able to significantly stain the tissues and lead false estimate of viability. Furthermore, the test item was completely removed from the epidermal surface at rinsing period. Additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use of the method the laboratory demonstrated the technical proficiency in a separate study using the ten Proficiency Chemicals according to OECD Test Guideline No. 439.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the mean OD value of the three negative control tissues was 0.762.
- Acceptance criteria met for positive control: yes, the mean OD value obtained for the positive control was 0.139 and this result corresponds to 18 % viability when compared to the results obtained from the negative control.
- Acceptance criteria met for variability between replicate measurements: Each calculated standard deviation value (SD) for the % viability was below 18.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was determined to have no skin irritation potential.

Executive summary:

EpiSkinTM SM test using the test item has been performed to predict its irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 439. Disks of EPISKIN (three units) were treated with test item and incubated for 15 minutes at room temperature. Exposure of test material 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 (three units / positive and negative control) epidermis were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to 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: 104 %). 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:

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

Version / remarks:

08 December 2010

Deviations:

no

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:

26 July 2013

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: slaughter house, TARAVIS KFT. 9600 Sárvár, Rábasömjéni út 129. Hungary
- Number of animals: not specified
- Characteristics of donor animals: not specified
- Storage, temperature and transport conditions of ocular tissue: heads were transported approximately within 2 hours from collection, 19.3 to 20.2 ºC, heads were wrapped with paper moistened with saline, placed in a plastic box that can be closed (4-5 heads/box)
- Indication of any existing defects or lesions in ocular tissue samples: Eyes with defects before treatment were rejected.
- Indication of any antibiotics used: not specified
- Selection and preparation of corneas: Non-damaged eyes were removed from the heads. The nictitating membrane was cut away with other connective tissue.
- Quality check of the isolated corneas: Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. 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.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.03 g (per eye)

CONTROLS
- Amounts applied: 0.03 g imidazole; 30 µL NaCl (in saline)

Duration of treatment / exposure:

10 seconds

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
At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than ±5-7 % within approximately 45 to 60 minutes before the start of application. Changes in thickness were not observed in the eyes. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effects after treatment. The location of any minor findings was marked on the record sheet as a drawing, if applicable. If any eye was considered to be unsuitable following baseline assessment, it was discarded.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: After 10 seconds the cornea surface was rinsed thoroughly with approx. 20 mL saline solution at ambient temperature. Imidazole was stuck on the corneas’ surface in all eyes at 30 minutes after the post-treatment rinse. The gentle rinsing with 20 mL saline was performed in all Imidazole treated eyes after the 30, 75, 120 and 180 minutes of observation, but cornea surfaces were not totally cleared at 240 minutes after the post-treatment rinse.

OBSERVATION PERIOD
30, 75, 120, 180 and 240 minutes after post-treatment rinse (minor variations +/- 5 minutes were considered acceptable)
Cornea thickness and opacity were measured at all time points. Flurescein retention was determined at baseline (t=0) and at 30 min.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: yes, using a slit lamp microscope
- Damage to epithelium based on fluorescein retention: yes, using a slit lamp microscope
- Swelling: depth measuring device on the slit lamp microscope (Haag-Streit BQ 900) with the slit-width set at 9½, equalling 0.095 mm
- Macroscopic morphological damage to the surface: yes, using a slit lamp microscope

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

Run / experiment:

mean maximum up to 240 min

Value:

2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

2

Positive controls validity:

valid

Remarks:

30

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Run / experiment:

mean maximum

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0.5

Positive controls validity:

valid

Remarks:

4.0

Irritation parameter:

fluorescein leakage

Run / experiment:

mean maximum

Value:

1

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

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:

The test item was determined to have no eye irritation potential.

Executive summary:

The purpose of this Isolated Chicken Eye Test (ICET) according to OECD 438 was to evaluate the potential ocular corrosivity and irritancy of the test item by its ability to induce toxicity in enucleated chicken eyes. The test compound was applied in a single dose (30 mg/eye) onto the cornea of isolated chicken. 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. Imidazole (positive control) was ground before use in the study. The test item and positive control were applied in an amount of 0.03 g/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 2xI, 1xII. 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

EpiSkin^TM SM test using the test item has been performed to predict its irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 439. Disks of EPISKIN (three units) were treated with test item and incubated for 15 minutes at room temperature. Exposure of test material 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 (three units / positive and negative control) epidermis were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to 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: 104 %). 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

The purpose of this Isolated Chicken Eye Test (ICET) according to OECD 438 was to evaluate the potential ocular corrosivity and irritancy of the test item by its ability to induce toxicity in enucleated chicken eyes. The test compound was applied in a single dose (30 mg/eye) onto the cornea of isolated chicken. 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. Imidazole (positive control) was ground before use in the study. The test item and positive control were applied in an amount of 0.03 g/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 2xI, 1xII. Positive and negative controls showed the expected results. The experiment was considered to be valid.

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on skin irritation, the test item does not require classification for skin irritation or corrosion according to Regulation (EC) No 1272/2008 (CLP), as amended for seventeenth time in Regulation (EU) No 2021/849.

 

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on the potential to cause damage to the eye, the test item does not require classification for eye irritation or damage according to Regulation (EC) No 1272/2008 (CLP), as amended for seventeenth time in Regulation (EU) No 2021/849.