2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol

Endpoint summary

Description of key information

In an experimental study according to OECD test guideline 439 under GLP conditions, the test substance did not reduce cell viability (96 % with an SD of 14.52) and is considered not irritating to skin (reference 7.3.1-1).

 

In an in vitro eye corrosive and severe irritant study, using the Isolated Chicken Eye model (OECD 438) with the test substance, no ocular corrosion potential was observed. The overall ICE score was 1xI, 2xII (reference 7.3.2-1).

In an experimental study according to OECD test guideline 492 under GLP conditions, the test substance reduced the cell viability in two experiments (55.8 % and 29.0 %) (reference 7.3.2-2).

Based on the results of both studies the test substance is considered to have potential to cause eye irritation but not serious damage to the eye.

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 donors

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:

TISSUE
- Model used: EPISKIN(TM) Small Model, manufactured by EPISKIN SNC Lyon, France
- Tissue batch number: 16-EKIN-026
- Production date: not specified
- Shipping date: not specified
- Delivery date: not specified
- 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
- Filter: not specified
- Filter bandwidth: not specified
- Linear OD range of spectrophotometer: not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA: secured to batch release quality control

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
- The test substance is considered to be non-irritating to skin if the viability is greater than 50 %.
- The test substance is considered to be corrosive or irritating to skin if the viability is below or equal to 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:

mean

Value:

96

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.
- 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.

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
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes, the mean OD value of the three negative control tissues was 0.762. 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. Each calculated standard deviation value (SD) for the % viability was below 18.

Table 1: Results

Substance	Optical Density (OD)		Viability (%)
Negative Control 1x PBS	1	0.672	88
	2	0.798	105
	3	0.815	107
	mean	0.762	100
	standard deviation (SD)		10.23
Positive Control SDS (5 % aq.)	1	0.077	10
	2	0.151	20
	3	0.19	25
	mean	0.139	18
	standard deviation (SD)		7.55
Test item	1	0.746	98
	2	0.619	81
	3	0.839	110
	mean	0.735	96
	standard deviation (SD)		14.52

Interpretation of results:

GHS criteria not met

Conclusions:

In an experimental study according to OECD test guideline 439 under GLP conditions, the test substance did not reduce cell viability (96 % with an SD of 14.52). Therefore, it is considered not irritating to skin.

Executive summary:

An EpiSkinTMSM 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 (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 % CO2protected 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: 96 %). 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.

The results obtained from thisin vitroskin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. The test item BIS-TRIS is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category).

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
- Storage, temperature and transport conditions of ocular tissue: Head collection was performed by a slaughter house technician. Heads were removed immediately after sedation of the chickens (sedation was happened by electric current). The heads were transported at the earliest convenience for use approximately within 2 hours from collection. The ambient temperature was optimal (19.4 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.
- Time interval prior to initiating testing: 2 h
- Indication of any existing defects or lesions in ocular tissue samples: 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).

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 (in saline)

Duration of treatment / exposure:

10 seconds

Observation period (in vivo):

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 +/- 5 minutes were considered acceptable).
Cornea thickness and opacity were measured at all time points. Fluorescein retention was determined at baseline (t=0) and at 30 min.

Duration of post- treatment incubation (in vitro):

4 hours as observation period

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 stell 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 to ensure that they were in good condition. 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.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: Thorough rinsing with approx. 20 mL saline solution at ambient temperature after 10 seconds of treatment. Imidazole and test item were 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. The gentle rinsing with 20 mL saline was performed in all (three eyes) test item treated eyes after the 30, 75 and 120 of observation. All test item treated eyes were totally cleared at 180 minutes after the post-treatment rinse.
- 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: 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:
Following scores were measured accoring to test guideline
- Mean corneal swelling (%)
- Mean maximum opacity score
- Mean fluorescein retention score at 30 minutes post-treatment

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

Irritation parameter:

percent corneal swelling

Run / experiment:

mean (240 min)

Value:

3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0

Positive controls validity:

valid

Remarks:

28

Irritation parameter:

cornea opacity score

Run / experiment:

mean

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0

Positive controls validity:

valid

Remarks:

4

Irritation parameter:

fluorescein leakage

Run / experiment:

mean

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: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
Positive and negative control values were within the corresponding historical control data ranges.

Table 1: Results for the test item

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	1%	I
Mean maximum corneal swelling at up to 240 min	3%	I
Mean maximum corneal opacity	1.0	II
Mean fluoresin retention	1.0	II
Other Observations	None
Overall ICE class	1xI, 2xII

 

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:

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, 2xII.

Executive summary:

A Isolated Chicken Eye Test (ICET) according to OECD 438 was conducted 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. 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, 2xII.

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

According to the guideline OECD 438, the test items 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, the test item has been categorized as “No prediction can be made”.

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 2016-12-01

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: commercially available EpiOcularTM tissue kit (tissue consist of normal, human-derived keratinocytes) obtained from MatTek In Vitro Life Science Laboratories (Bratislava, Slovakia) (Kit designation: 1st Experiment: OCL-212-EIT, delivered 26.10.2016, Batch no.: 23742; 2nd Experiment: OCL-212-EIT, delivered 29.11.2016, Batch no.: 23754), 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: 50.5 and 51.9 mg/tissue (1st experiment) and 51.1 and 50.3 mg/tissue (2nd experiment)

Duration of treatment / exposure:

1st experiment: 6 h
2nd experiment: 6 h and 3 minutes

Duration of post- treatment incubation (in vitro):

1st experiment: 17 h and 49 minutes
2nd: experiment: 18 h

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 OED TG 492.
- RhCE tissue construct used, including batch number: EpiOcularTM tissue (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia) Kit designation: OCL-212-EIT, Batch no.: 1st Experiment: 23742; 2nd Experiment: 23754
- Doses of control substances used: 50 μL/tissue
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: Exposure of ca. 6h at 37 ± 1 °C; 25 min post soak at room temperature; ca. 18 h post-incubation at 37 ± 1 °C
- 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. Therefore, no such controls were used in the main test.
- Number of tissue replicates used per test chemical and controls: 2 for each experiment
- Wavelength and band pass used for quantifying MTT formazan, and linearity range of measuring device: plate spectrophotometer at 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 controls were within the range of the historical data.
- Complete supporting information for the specific RhCE tissue construct used: see above
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: not reported
- 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: 1st exp.: 0.1 % < 20 %, 2nd exp.: 9.7 % < 20 %; positive control: 1st exp.: 7.5 % < 20 %, 2nd exp.: 2.9 % < 20 %)
- Acceptable variability between tissue replicates for the test chemical: Yes (1st exp.: 10.9 % < 20 %, 2nd exp.: 0.3 % < 20 %)

As the first experiment resulted in duplicate values above and below the classification threshold of 60 %, a second experiment was conducted.

Irritation parameter:

other: % viability

Run / experiment:

Experiment 1 (mean)

Value:

55.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

39.5

Irritation parameter:

other: % viability

Run / experiment:

Experiment 2 (mean)

Value:

29

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

32.4

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

Table 1: Results

Designation	Positive Control	Test item
Experiment 1
% Viability (Tissue 1)	35.8	61.3
% Viability (Tissue 2)	43.3	50.4
% Viability Mean	39.5	55.8
Experiment 2
% Viability (Tissue 1)	30.9	29.1
% Viability (Tissue 2)	33.8	28.9
% Viability Mean	32.4	29

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

In an experimental study according to OECD test guideline 492 under GLP conditions, the test substance substantially reduced the cell viability in two experiments (55.8 % and 29.0%). Therefore, it is considered as eye irritant/inducing serious eye damage.

Executive summary:

A study according to OECD 492 was conducted to evaluate the eye irritation potential of the test item. Two experiments were performed. The first experiment was not sufficient to determine the properties of the test item due to non-concordant replicates. The result of the second experiment was unequivocal and led to the same classification as the mean value of the first experiment.

The solid test item was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of at least 6 hours. After treatment, the 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 and methyl acetate was used as positive control. The controls showed the following results: 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 (1st experiment) and 1.8 (2nd experiment). The positive control showed clear eye irritating effects (< 50 %), the relative tissue viability was reduced to 39.5 % (1st experiment) and 32.4 % (2nd experiment).

Variation within tissue replicates was acceptable in both experiments (range ≤ 20 %).

After treatment with the test item, the mean value of relative tissue viability was 55.8 % (1st experiment) and 29.0 % (2nd experiment). Both values are below the threshold for eye irritation potential (≤ 60 %).

Under the conditions of the test system, the test item is considered as eye irritant / inducing serious eye damage in the EpiOcularTM Eye Irritation Test.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

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 (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: 96 %). 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.

The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. The test item BIS-TRIS is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category).

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 (ICET) according to OECD 438 (version 2013) was conducted resulting in “No prediction can be made” and therefore, neither classification in Category 1 (UN GHS) nor no classification (UN GHS: No Category) was triggered. Therefore an eye irritation study using Reconstructed human Cornea-like Epithelium according to OECD 492 (version 2015) 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).

 

OECD 438

An Isolated Chicken Eye Test (ICET) according to OECD 438 (version 2013) was conducted 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. 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, 2xII.

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

According to the guideline OECD 438 (version 2013), the test items 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 (version 2013), the test item has been categorized as “No prediction can be made”.

 

OECD 492

A study according to OECD 492 (version 2015) was conducted to evaluate the eye irritation potential of the test item. Two experiments were performed. The first experiment was not sufficient to determine the properties of the test item due to non-concordant replicates. The result of the second experiment was unequivocal and led to the same classification as the mean value of the first experiment.

The solid test item was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of at least 6 hours. After treatment, the 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 and methyl acetate was used as positive control. The controls showed the following results: 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 (1st experiment) and 1.8 (2nd experiment). The positive control showed clear eye irritating effects (< 50 %), the relative tissue viability was reduced to 39.5 % (1st experiment) and 32.4 % (2nd experiment).

Variation within tissue replicates was acceptable in both experiments (range ≤ 20 %).

After treatment with the test item, the mean value of relative tissue viability was 55.8 % (1st experiment) and 29.0 % (2nd experiment). Both values are below the threshold for eye irritation potential (≤ 60 %).

Under the conditions of the test system, the test item is considered as eye irritant / inducing serious eye damage in the EpiOcularTM Eye Irritation Test according to OECD 492 (version 2015).

 

Conclusion

Based on the result from the Isolated Chicken Eye Test according to OECD 438 (version 2013) 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 (version 2015), determined that the test item induces eye damage, but that no prediction regarding classification as Category 1 or Category 2 of the test item can be made.

Based on the conducted studies under the respective guideline versions a classification as “No category” was not indicated. While the result of the OECD 438 (version 2013) study pointed toward a possibility that Category 1 was not required, it could not clearly be eliminated due to the result “No predication can be made”. OECD guideline 492 (version 2015) determined that a classification of either Category 1 or 2 is required. Therefore, the worst-case was assumed for the registration for REACH deadline in May 2018 and the substance was classified into Category 1 for inducing serious eye damage.

In the meantime new guideline versions for both OECD test guidelines were published and the impact of the changes of the guidelines were checked for the update (Summer 2020). The current guideline versions are OECD TG 438 of 27th June 2018 and OECD TG 492 of 18th June 2019. In both guidelines the procedures for the conduction of the described experiments remained the same, as did the criteria for evaluation of the results (e.g. ICE class for OECD 438). However, in both guidelines the prediction models according to the UN GHS classification changed due to new scientific knowledge. In OECD 438 version 2018 the overall ICE class of 1xI, 2xII is considered as “No category” (previously “No predication can be made”). For OECD 492 the predicted classification besides “No category” changed to “No predication can be made” (from previously “potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1)”.

Considering the experimental results of the studies under the new guideline versions the following is determined:

According to the guideline OECD 438 (version 2018), the test item has been categorized as “No category”.

Under the conditions of the test system, the test item is considered as “No predication can be made” in the EpiOcularTM Eye Irritation Test according to OECD 492 (version 2019).

 

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 based on the updated guidelines. The weight of evidence with the two conducted studies determined that the test substance is not to be classified as UN GHS Category 1. With the results of “No Category” and “No predication can be made”, it was decided to assume the worst-case classification in order to avoid an in vivo study. Therefore, the test item is considered to be eye irritating and classified into Category 2 (UN GHS).

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 the eighteenth time in Regulation (EU) 2022/692.

 

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 requires classification for causing eye irritation into Category 2 (H319: Causes serious eye irritation) according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighteenth time in Regulation (EU) 2022/692.