2,2'-(piperazine-1,4-diyl)bis(ethanesulphonic) acid

Endpoint summary

Description of key information

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

 

In an experimental study according to OECD test guideline 438 under GLP conditions, the test substance did not cause ocular corrosion or severe irritation in the enucleated chicken eyes and scored into ICE Class II for corneal swelling and into ICE Class III for corneal opacity and into ICE class I fluorescein retention (reference 7.3.2-1).

 

In an experimental study according to OECD test guideline 492 under GLP conditions, the test substance did not affect cell viability and thus it is considered as not irritating (reference 7.3.2-2).

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:

28 July 2015

Deviations:

no

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

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Adult donor 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: EPISKIN(TM) Small Model (SM)
- Tissue batch number: 16-EKIN-026
- Production date: not specified
- Shipping date: not specified
- Delivery date: not specified
- Certification and release date: 28.06.2016
- Expiry date: 04.07.2016
- 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

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: 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
- Barrier function: IC50=2.6 mg/mL (HC: 1.5<= IC50 <= 3 gm/mL)
- Morphology: well-differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum
- Contamination: absence of bacteria, fungus and mycoplasma, blood of donor without: HIV1 +2 and hepatitis C antibodies and hepatitis B antigen HB

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 chemical 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.
- The test chemical can be considered as non-irritant to skin in accordance with UN GHS No Category if the tissue viability after exposure and post-treatment incubation is more than (>) 50 %.
- Justification for the selection of the cut-off point(s) if different than recommended in 439: Not different.

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 (± 0.5 min)

Duration of post-treatment incubation (if applicable):

42 hours (± 1h)

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

106

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100

Positive controls validity:

valid

Remarks:

18

Remarks on result:

no indication of irritation

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.
- 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.

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 (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: (test substance: SD of 9.36 < 18)
- Range of historical values if different from the ones specified in the test guideline: no

Interpretation of results:

GHS criteria not met

Conclusions:

The test item showed no skin irritation potential.

Executive summary:

An EpiSkinTM SM test with 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. Triplicates of the human skin model EPISKIN 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.

The test item did not show significantly reduced cell viability in comparison to the negative control (mean value: 106 %). 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

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016-06-22

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:

26 July 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:

08 December 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 (slaughter house)
- Number of animals: not specified
- Characteristics of donor animals: not specified
- Storage, temperature and transport conditions of ocular tissue: 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). The ambient temperature was optimal (19.4 ºC to 20.2 ºC) during the transport.
- Time interval prior to initiating testing: Heads were transported for use approximately within 2 hours from collection.
- Indication of any existing defects or lesions in ocular tissue samples: Eyes were evaluated before removal from heads (using fluorescein solution) and afterwards (see below). Only eyes in good condition were used for the experiment.
- Indication of any antibiotics used: not specified
- Selection and preparation of corneas: Please refer to details on study design.
- Quality check of the isolated corneas: Please refer to details on study design.

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
- Amount applied: 0.03 g imidazole; 30 µL NaCl solution

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
The eyeball was carefully removed from the orbit. The procedure avoided pressure on the eye in order to prevent distortion of the cornea and subsequent 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 so that the appropriate humidity was maintained. The prepared eye was placed in a steel 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 by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was applied to fix the eye properly. The clamp with the eyeball 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 approximately 3 to 5 drops/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity. The appropriate number of eyes was selected and after being placed in the superfusion apparatus, the selected eyes were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the isotonic saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. 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, equaling 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.

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.

OBSERVATION PERIOD
The control and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within ±5 minutes were considered acceptable.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: 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, while taking care not to damage the cornea but attempting to remove all the residual test item if possible.
- Indicate any deviation from test procedure in the Guideline: none

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The cornea opacity was measured at all time points.
- Damage to epithelium based on fluorescein retention: Fluorescein retention was determined at baseline (t=0) and 30 minutes after the post-treatment rinse.
- Swelling: 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.
- Macroscopic morphological damage to the surface: Damages eyes were discarded before experiment. Eyes were checked for damage due to the experiment.
- Others (e.g, histopathology): No histopathology evaluation was 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 was used.

Irritation parameter:

percent corneal swelling

Run / experiment:

Mean maximum, up to 240 min

Value:

7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0

Positive controls validity:

valid

Remarks:

28

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Run / experiment:

Mean maximum

Value:

2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0

Positive controls validity:

valid

Remarks:

4.0

Irritation parameter:

fluorescein retention score

Run / experiment:

Mean

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0

Positive controls validity:

valid

Remarks:

3.0

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: None reported.

DEMONSTRATION OF TECHNICAL PROFICIENCY: not specified

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

Table 1: Results for the test item

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	6 %	II
Mean maximum corneal swelling at up to 240 min	7 %	II
Mean maximum corneal opacity	2.0	III
Mean fluoresin retention	0.5	I
Other Observations	None
Overall ICE class	1xI, 1xII, 1xIII

 

Table 2: Results for positive control Imidazole

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	21 %	III
Mean maximum corneal swelling at up to 240 min	28 %	III
Mean maximum corneal opacity	4.0	IV
Mean fluoresin retention	3.0	IV
Other Observations	Cornea opacity score 4 was observed in three eyes at 30 minutes after the post-treatment rinse.
Overall ICE class	1xIII, 2xIV

The positive control Imidazole was classed as corrosive/severely irritating, UN GHS Classification: Category 1. 

 

Table 3: Results for the negative control NaCl (9 g/L saline)

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0 %	I
Mean maximum corneal swelling at up to 240 min	0 %	I
Mean maximum corneal opacity	0.0	I
Mean fluoresin retention	0.0	I
Other Observations	None
Overall ICE class	3xI

 

Based on the overall ICE Class the negative control NaCl (9 g/L saline) had no significant effects on the chicken eye in this study.

Positive and negative control values were within the corresponding historical control data ranges.

 

Table 4: Historical control data of Positive control (Period of 2011 - 2015)

IMIDAZOLE HISTORICAL CONTROL Dose level: 30 mg / eye

n=168	Relative obobservation time (m(min)	Corneal thickness					Corneal opacity score						Fluorescein retention
		30	75	120	180	240		30	75	120	180	240		∆FR
Maximium	swelling (%):	34	45	49	54	44	Max. OS:	4.0	4.0	4.0	4.0	4.0	Max. FR:	3.0
Minimum swelling (%):		3	9	12	14	15	Min. OS:	2.8	3.3	3.5	3.5	3.5	Min. FR:	2.7
Average:		19	27	31	35	37	Average:	3.7	3.9	3.9	3.9	3.9	Average:	3.0

 

Table 5: Historical control data of Negative control (Period of 2011 - 2015)

NaCl (9 g/L saline) SOLUTION HISTORICAL CONTROL Dose level: 30 µL / eye

n=120	Relative obobservation time (m (min)	Corneal thickness					Corneal opacity score						Fluorescein retention
		30	75	120	180	240		30	75	120	180	240		∆FR
Maximum	swelling (%):	3	3	3	4	3	Max. OS:	0.5	0.5	0.5	0.5	0.5	Max. FR:	0.5
Minimum swelling (%):		0	0	0	0	0	Min. OS:	0	0	0	0	0	Min. FR:	0.0
Average:		0.1	0.3	0.3	0.3	0.3	Average:	0.0	0.0	0.0	0.0	0.0	Average:	0.0

Remark:

n = number of examined eyes

∆FR = Difference between fluorescein retention and fluorescein retention reference value

OS = Opacity score

Interpretation of results:

study cannot be used for classification

Conclusions:

The test item did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. The overall ICE class was 1xI, 1xII, 1xIII.

Executive summary:

A study according to OECD 438 was conducted with the test item. The purpose of this Isolated Chicken Eye Test (ICET) 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 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 Imidazole (positive control) was ground before use in the study. The 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 1xI, 1xII, 1xIII. Positive and negative controls showed the expected results. The experiment was considered to be valid.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016-10-24 to 2016-10-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

28. Jul. 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability: The EpiOcularTM Eye Irritation Test (EIT) predicts the acute ocular irritation potential of chemicals by measurement of its irreversible tissue damage caused by cytotoxic effects in the human cornea model. Within a testing strategy, the EpiOcularTM EIT is used as a replacement of the Draize Eye Irritation Test. Solids are within the applicability domain of the test method.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: not applicable
- Cell line used, its source, passage number and confluence of cells used for testing: not applicable
- RhCE tissue used, including batch number: EpiOcularTM tissue kit obtained from MatTek In Vitro Life Science Laboratories (Bratislava, Slovakia) (Kit designation: OCL-212-EIT; delivered 26.10.2016; Batch no.: 23742), incl. a certificate of analysis and functionality test results

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: Tissue 1: 50.5 mg; Tissue 2: 50.9 mg

Controls
- Amount applied: 50 μL/tissue

Duration of treatment / exposure:

6 h

Duration of post- treatment incubation (in vitro):

17 h and 49 min

Number of animals or in vitro replicates:

2 tissues

Details on study design:

- Details of the test procedure used: The test was conducted according to the OCED TG 492.
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: exposure: 6 hours at 37 ± 1 °C, 5 ± 1 % CO2
and 80 – 100 % relative humidity; post soak: 25 minutes at room temperature; post-treatment incubation: 17 hours and 49 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity
- Description of any modifications to the test procedure: none
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: In a pre-test, no indication for direct MTT reduction or colouring by the test substance was observed.
- Number of tissue replicates used per test chemical and controls (positive control, negative control): 2
- Wavelength and band pass used for quantifying MTT formazan, and linearity range of measuring device: 570 nm
- Description of the method used to quantify MTT formazan: spectrophotometer
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: The evaluation criteria of the OECD TG 492, i.e. a threshold of 60 % to discriminate non eye irritants from eye irritants, were used.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: Both the responses of the negative and the positive control were within the range of the historical data.
- Complete supporting information for the specific RhCE tissue construct or hCE cells used
- Reference to historical data of the RhCE tissue construct: see above
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: not specified
- Positive and negative control means and acceptance ranges based on historical data: Acceptable OD range of negative control: 1.000 - 2.024; acceptable relative viability range of the positive control: 14.5 - 49.3 %
- Acceptable variability between tissue replicates for positive and negative controls: Yes (negative control: 0.1 % < 20 %; positive control: 7.5 % < 20 %)
- Acceptable variability between tissue replicates for the test chemical: Yes (1.3 % < 20 %)

Irritation parameter:

mean percent tissue viability 

Run / experiment:

Mean

Value:

102.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

39.5

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none reported.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The criterion for optical density of the negative control was fulfilled: The OD value was 1.5 (> 0.8 and < 2.5).
- Acceptance criteria met for positive control: The positive control induced a decrease in the relative absorbance as compared to the negative control to 39.5 %. Variation within the replicates was acceptable (≤20 %).
- Range of historical values if different from the ones specified in the test guideline: not applicable

Table 1: % Viability Positive Control and Test Item

Designation	Positive Control	Test item
% Viability (Tissue 1)	35.8 %	102.7 %
% Viability (Tissue 2)	43.3 %	101.4 %
% Viability Mean	39.5 %	102.1 %

 

Table 2: Validity

Criterion	Demanded	Found
OD of negative control	≥ 0.8 and ≤ 2.5	1.5
% viability of positive control	< 50 % of negative control	39.5 %
Range within replicates	≤ 20 %	0.1 % (negative control) 7.5 % (positive control) 1.3 % (test item)

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is considered as not eye irritant in the EpiOcular Eye Irritation Test.

Executive summary:

The eye irritation potential of the test substance was investigated in an experimental study according to OECD test guideline 492 under GLP conditions using the EpiOcularTM tissue. The solid test item was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 6 hours. After treatment, the respective substance was rinsed from the tissue; then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution. Demineralised water was used as negative control, Methyl acetate was used as positive control. After treatment with the negative control, the absorbance values were within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.5, OD was 1.5. The positive control showed clear eye irritating effects, the relative absorbance value was reduced to 39.5 % (< 50 %). Variation within tissue replicates was acceptable (range ≤ 20 %). After treatment with the test item, the relative absorbance values were increased to 102.1%. This value is above the threshold for eye irritation potential (≤ 60 %). Under the conditions of the test, the test item is considered as not eye irritant in the EpiOcular Eye Irritation Test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

An EpiSkinTM SM test with 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. Triplicates of the human skin model EPISKIN 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.

The test item did not show significantly reduced cell viability in comparison to the negative control (mean value: 106 %). 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

To determine the corrosion or irritation potential of the test substance for the eye a stepwise weight of evidence approach was done. An Isolated Chicken Eye Test according to OECD 438 was conducted resulting in “No prediction can be made” and therefore, the test item was neither required classification in Category 1 (UN GHS) nor required no classification (UN GHS: No Category). Therefore an eye irritation study using Reconstructed human Cornea-like Epithelium according to OECD 492 was conducted in accordance with the top down approach described in the Guidance on an integrated approach on testing and assessment (IATA) for serious eye damage and eye irritation (Series on testing and assessment No. 263; version 2017-07-20).

 

OECD 438 (reference 7.3.2-1)

A study according to OECD 438 was conducted with the test item. The purpose of this Isolated Chicken Eye Test (ICET) 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 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 Imidazole (positive control) was ground before use in the study. The 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 1xI, 1xII, 1xIII. Positive and negative controls showed the expected results. The experiment was considered to be valid. In this in vitro eye corrosive and severe irritant study, using the Isolated Chicken Eye model with the test item, no ocular corrosion or severe irritation potential was observed. The overall ICE score was 1xI, 1xII, 1xIII. According to the guideline OECD 438, the test item’s overall in vitro classification is neither UN GHS Classification Category I (an ocular corrosive or severe eye irritant) nor No Category. Thus, according to the guideline OECD 438, test item has been categorized as “No prediction can be made”.

 

OECD 492 (reference 7.3.2 -2)

The eye irritation potential of the test substance was investigated in an experimental study according to OECD test guideline 492 under GLP conditions using the EpiOcularTM tissue. The solid test item was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 6 hours. After treatment, the respective substance was rinsed from the tissue; then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution. Demineralised water was used as negative control, Methyl acetate was used as positive control. After treatment with the negative control, the absorbance values were within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.5, OD was 1.5. The positive control showed clear eye irritating effects, the relative absorbance value was reduced to 39.5 % (< 50 %). Variation within tissue replicates was acceptable (range ≤ 20 %). After treatment with the test item, the relative absorbance values were increased to 102.1%. This value is above the threshold for eye irritation potential (≤ 60 %). Under the conditions of the test, the test item is considered as not eye irritant in the EpiOcular Eye Irritation Test.

 

Conclusion

Based on the result from An Isolated Chicken Eye Test according to OECD 438 a categorization into Category 1 was excluded. A classification as No category according to UN GHS was not possible due to the result of “No predication can be made”. The second in vitro study, the eye irritation study using Reconstructed human Cornea-like Epithelium according to OECD 492, determined that no classification (UN GHS “No category”) of the test item is required. Using the top down approach described in the Guidance on IATA for serious eye damage and eye irritation an overall conclusion for the test item can be reached. The weight of evidence with the two conducted studies determined that the test substance is not to be classified as UN GHS Category 1 and Category 2. Therefore, the test item is considered to be not eye irritating or damaging to the eyes and requires no classification.

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 and eye irritation, the test item is not classified for causing skin irritation or corrosion and for causing damage to the eye or eye irritation according to Regulation (EC) No 1272/2008 (CLP), as amended for the fifteenth time in Regulation (EU) 2020/1182.