Blue MGi 1037-Na (notification presscake)

Endpoint summary

Description of key information

Skin:

The read across substance Blue MGi 1037, a structural analogue of the target substance, is not irritating to the skin.

Eye:

In an ex vivo eye irritation assay according to OECD guideline 438 (ICE), an ICE Score of 1x I and 2x II was determined. Thus, no prediction could be made.

In an ex vivo eye irritation assay according to OECD guideline 437 (BCOP), an IVIS of 5.52 was determined. Thus, no prediction could be made.

In an in vitro eye irritation assay according to OECD guideline 492 (RhCE), a rel. cell viability of 3.3 % was determined. The test item showed eye irritating properties.

In a supporting study according to OECD TG 405 performed with the structurally similar read-across substance Blue MGi 1037 the eyes of animals showed slight blue colouration, which was not reversible until day 21. Based on this persistent discolouration the read-across substance is classified for eye damaging. Therefore, the substance Blue MGi 1037-Na is also classified for serious eye damage Cat.1 since persistent coloration of eyes is expected also for the substance Blue MGi 1037-Na.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 June 2000 - 8 June 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EEC Directive 2004/73, B.4

Version / remarks:

1992

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Elevage Scientifique des Dombes, France
- Age at study initiation: 14 weeks
- Weight at study initiation: 3-3.2 kg
- Housing: individually in stainless steel cages
- Diet: ad libitum, pelleted standard Provimi Kliba 3418 rabbit maintenance diet
- Water: ad libitum, tap water
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

water

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 500 mg

Duration of treatment / exposure:

4 h

Observation period:

1, 24, 48, 72 hours after patch removal

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: left flank
- coverage: 6 cm2
- Type of wrap: semi-occlusive dressing and tape

REMOVAL OF TEST SUBSTANCE
- Washing: water
- Time after start of exposure: 4 hours

OBSERVATION TIME POINTS
1, 24, 48, 72 hours

SCORING SYSTEM:
- Method of calculation: Draize

Irritation parameter:

erythema score

Basis:

mean

Remarks:

of all animals. No erythema formation were observed.

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

mean

Remarks:

of all animals. No edema formation were observed.

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritant / corrosive response data:

IRRITATION:
The test item did not elicit any skin reactions at the application site.

COLORATION/CORROSION:
Light blue staining was observed in all animals after removal of the dressing and during the whole study period.
Neither alterations of the treated skin were observed nor were corrosive effects evident on the skin.

Other effects:

VIABILITY/MORTALITY/CLINICAL SIGNS:
No clinical signs of systemic toxicity were noted and no mortality occurred.

BODY WEIGHTS:
The body weights of all rabbits were within the normal range of variability.

Interpretation of results:

GHS criteria not met

Conclusions:

The structual analogue BLUE MGi 1037 is considered to be not irritating to rabbit skin.

Executive summary:

The primary skin irritation potential of the structural analogue substance BLUE MGi 1037 was investigated by topical semi-occlusive application of 0.5 g to 6 cm2 intact left flank of each of three young adult New Zealand White rabbits. The duration of treatment was four hours. The scoring of skin reactions was performed 1, 24, 48 and 72 hours after removal of the dressing. The scores of each animal at the following reading times (24, 48, 72 hours) were used in calculating the respective mean values for each type of lesion.

The primary irritation score was calculated by totaling the individual cumulative scores at 24, 48 and 72 hours and then dividing by the number of data points. The primary irritation score was 0.00 (max. 8.0).

Very slight erythema was observed in one male animal (no. 91) one hour after treatment. Local signs (mean values from 24 to 72 hours) consisted of grade 0.00 erythema and grade 0.00 oedema.

Light blue staining produced by the test item of the treated skin was observed in all animals after removal of the dressing and during the whole study period. No corrosive effects were noted on the treated skin of any animal at any measuring interval.

Based upon the referred classification criteria (EEC Commission Directive 93/21 /EEC of April 27, 1993), BLUE MGi 1037 is considered to be "not irritating" to rabbit skin.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reason / purpose for cross-reference:

read-across source

Irritation parameter:

erythema score

Basis:

mean

Remarks:

of all animals. No erythema formation were observed.

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

mean

Remarks:

of all animals. No edema formation were observed.

Time point:

24/48/72 h

Score:

0

Max. score:

4

Interpretation of results:

GHS criteria not met

Reference 3

Endpoint:

skin irritation: in vitro / ex vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro skin irritation study does not need to be conducted because adequate data from an in vivo skin irritation study are available

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:

From 2017-05-31 to 2017-07-20

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:

28 April 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

chicken

Strain:

other: COBB 500

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni út 129. Hungary
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):
The heads were transported to Toxi-Coop ZRT. at the earliest convenience for use approximately within 2 hours from collection. The ambient temperature was optimal (19.4 – 20.3 ºC) during the transport. All eyes used in the assay were from the same groups of eyes collected on one specific day. After collection, the heads were inspected for appropriate quality and wrapped with paper moistened with saline, then placed in a plastic box that can be closed (4-5 heads/box).
After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of fluorescein solution 2 (w/v) % was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL isotonic saline. Then the fluorescein-treated cornea was examined with hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.
The eyeball was carefully removed from the orbit by holding the nictitating membrane with 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 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 4 drops/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.
The appropriate numbers of eyes were 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, equalling 0.095 mm.
- Indication of any existing defects or lesions in ocular tissue samples: 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.

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.03 g
- Negative control: physiological saline 30 µL, batch no.: PP/2015/05309
- Positive control: Imidazole, batch no.: SLBK9670V

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

4 hours

Number of animals or in vitro replicates:

Treatment, positive controls: 3 eyes
Negative control: 1 eye

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
After being placed in the superfusion apparatus, the selected eyes were examined 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. 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. If the selected eyes were appropriate for the test, acclimatization started and was conducted for approximately 45 to 60 minutes. The temperature was verified to be in the range of 32 ± 1.5 °C in all chambers during the acclimatization and treatment periods.

BASELINE DETERMINATION
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. Slight changes in thickness (0% to 2%) were observed in the eyes, a finding considered as normal when maintaining enucleated eyes. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effects after treatment.

TREATMENT
The eyes were held in horizontal position over a container to catch waste, while the test item was applied onto the centre of the cornea, by attempting to cover the cornea surface uniformly with the test substance, while taking care not to damage or touch the cornea with the application equipment. The exposure period was 10 seconds.
The three positive control eyes were treated in a similar way with 0.03 g Imidazole.
One negative control eye was treated with saline solution in a volume of 30 µL with a micropipette, in such a way that the entire surface of the cornea was covered, taking care not to damage or touch the cornea with the application equipment.

REMOVAL OF TEST SUBSTANCE
At the end of the application the cornea surface was rinsed thoroughly with 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. The eye in the holder was then returned to its chamber. The time while the eye was out of the chamber was limited to the minimum.
The 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 and test item 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.

OBSERVATIONS
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.
The cornea thickness and cornea opacity were measured at all time points. Fluorescein retention was determined at baseline (t=0) and 30 minutes after the post-treatment rinse.
At the end of the procedures, the corneas were carefully removed from the eyes and placed individually into labeled containers of preservative fluid (4% formaldehyde) for potential histopathology and stored.


SCORING SYSTEM
Calculation of corneal swelling, cornea opacity scores and fluorescein retention scores.
Evaluation of the test results based on to the criteria given in OECD 438 (July 2013).

TOOL USED TO ASSESS SCORE:
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.

Irritation parameter:

cornea opacity score

Run / experiment:

1

Value:

1

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

Sligt effects, ICE class II

Irritation parameter:

percent corneal swelling

Run / experiment:

1

Value:

>= 9 - <= 10

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

Slight effect, ICE class II

Irritation parameter:

fluorescein retention score

Run / experiment:

1

Value:

0.5

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

ICE class I

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.

Test Item: DRIMRLE / Blue MGi 1037-Na salt

 

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	9%	II
Mean maximum corneal swelling at up to 240 min	10%	II
Mean maximum corneal opacity	1.0	II
Mean fluorescein retention	0.5	I
Other Observations	None
Overall ICE Class	1 x I, 2 x II

 

 

Positive Control: Imidazole

 

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	36%	IV
Mean maximum corneal swelling at up to 240 min	46%	IV
Mean maximum corneal opacity	4.0	IV
Mean fluorescein retention	3.0	IV
Other Observations	Corneal opacity score 4 was observed in three eyes at 30 minutes after the post-treatment rinse.
Overall ICE Class	3 x IV

 

 

Negative Control: NaCl (9 g/L saline)

 

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	2%	I
Mean maximum corneal swelling at up to 240 min	2%	I
Mean maximum corneal opacity	0.0	I
Mean fluorescein retention	0.5	I
Other Observations	None
Overall ICE Class	3 x I

 

Interpretation of results:

other: “No prediction can be made”

Conclusions:

The test item has been categorized as “No prediction can be made”.

Executive summary:

In this in vitro eye irritation study using the Isolated Chicken Eye model, no ocular corrosion or severe irritation potential was observed after exposure to Blue MGi 1037-Na salt. The overall ICE score was 1xI, 2xII.

 According to the guideline OECD 438, the Blue MGi 1037-Na salt 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:

From 2017-09-19 to 2018-03-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

2013-07

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

2017-02-14

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: AB Schlachthof GmbH & Co. KG, 63739 Aschaffenburg, Germany
- Characteristics of donor animals: least 9 month old
- Storage, temperature and transport conditions of ocular tissue: The isolated eyes were stored in HBSS containing 1% (v/v) Penicillin/Streptomycin (100 units/mL penicillin and 100 μg/mL streptomycin) in the cooled slaughter-house until transportation on the same morning to the laboratory using a Styrofoam box.
- Time interval prior to initiating testing: The corneae were isolated on the same day after delivery. The corneae were directly used in the BCOP test within 1 hour after preparation on the same day.
- indication of any existing defects or lesions in ocular tissue samples: All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The basal opacity of all corneae was recorded. Each cornea with a value of the basal opacity > 7 was discarded.
- Indication of any antibiotics used: Yes

Vehicle:

physiological saline

Remarks:

20% suspension (w/v) in saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.75 mL
- Concentration: 20 %

Duration of treatment / exposure:

4 h

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea.

QUALITY CHECK OF THE ISOLATED CORNEAS
The basal opacity of all corneae was recorded. Each cornea with a value of the basal opacity > 7 was discarded.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
Saline (0.9% NaCl in deionised water)

POSITIVE CONTROL USED
10% (w/v) benzalkonium chloride in 0.9% (w/v) NaCl (saline)

APPLICATION DOSE AND EXPOSURE TIME
20% (w/v) for 4 h

TREATMENT METHOD
open chamber

POST-INCUBATION PERIOD
No

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: The test item or the control items, respectively, were each rinsed off from the according application sides with saline.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacitometer determines changes in the light transmission passing through the corneae, and displays a numerical opacity value. This value was recorded in a table. The opacitometer (OP_KiT opacitometer (Electro Design, 63-Riom, France)) was calibrated as described in the manual and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a microtiter plate reader at OD490 (Versamax® Molecular Devices).


SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: The decision criteria as indicated in the TG was used.

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

5.52

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No prediction can be made

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
- Range of historical values if different from the ones specified in the test guideline: Yes

Table 2: Summary of the Results

Test Group	Opacity value = Difference (t240-t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS	Proposedin vitroIrritancy Score	Standard deviation ofin vitroscore
		Mean		mean
Neg. Control	0	0.33	0.074	0.065	1.11	1.31	No Category	0.53
	1		0.061		1.92
	0		0.061		0.92
Pos. Control	118.67*		-0.011*		118.50	122.90	Category 1	4.48
	122.67*		0.005*		122.74
	127.67*		-0.014*		127.45
Test Item	4.67*		0.098*		6.13	5.52	No prediction can be made	1.33
	2.67*		0.089*		4.00
	4.67*		0.118*		643

* = corrected values

Interpretation of results:

other: The test item has been categorized as “No prediction can be made”.

Conclusions:

In an eye vivo eye irritation assay (BCOP) according to OECD guideline 437, the test item did not show serious eye damaging properties. However, a prediction for the damage hazard according to GHS cannot be made.

Executive summary:

The eye irritation/serious eye damaging potential of the test item was assessed in an ex vivo assay (BCOP) according to OECD guideline 437. The induced opacity and increased permeability was investigated in isolated bovine corneas after exposure to the test item as a 20% (w/v) solution in a 0.9% sodium chloride solution. As negative control 0.9% sodium chloride solution and as positive control 20% (w/v) Imidazole was used. Three corneas were used per group (negative control, positive control or test substance group). After a first opacity measurement of the untreated bovine corneas, 750 µL of the dissolved test item, positive or negative control were applied on the corneas and incubated for 240 minutes. After the incubation phase the test item, the positive, and the negative control were rinsed from the corneas and the opacity was measured again. After the opacity measurements, the permeability of the corneas was determined by application of a fluorescein solution for 90 minutes. The amount of fluorescein solution that crossed the cornea was measured spectrophotometrically. The opacity and permeability assessments were combined to determine an In Vitro Irritancy Score (IVIS). After treatment with the negative control the calculated IVIS was 1.31. Treatment with the positive control revealed an IVIS of 122.9. The study fulfilled the acceptance criteria and was considered as valid. The mean IVIS obtained after treatment with the test substance was 5.52 and, thus, above 3 but below 55, i.e. according to OECD 437 the test substance is not Category 1, but no further classification can be made.

Reference 3

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 2017-11-09 to 2017-11-30

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:

2015-07

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
According to the respective guideline.
- Description of the cell system used:
EpiOcular™ kits and MTT-100 kits are purchased from MatTek Corporation (82105 Bratislava, Slovakia). The EpiOcular™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. The EpiOcular™ tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELL, 10 mm diameter). EpiOcular™ tissues were shipped at 2 - 8 °C on medium-supplemented agarose gels in a 24-well plate on Tuesday. On day of receipt of the EpiOcular™ tissues, the equilibration step (15 minutes at room temperature in the 24-well shipping container) started. 1.0 mL of the medium was aliquoted into the appropriate wells of pre-labeled 6-well plates.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 mg

Duration of treatment / exposure:

6 h

Duration of post- treatment incubation (in vitro):

post soak incubation: 25 min
post treatment incubation: 18 h

Number of animals or in vitro replicates:

2 /treatment

Details on study design:

- Details of the test procedure used
MatTek Corporation Protocol: EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; for use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model; 29 June 2015.

- RhCE tissue construct used, including batch number
EpiOcular™ tissues, Lot No.: 27015
- Doses of test chemical and control substances used
Test chemical: 50 mg
Controls: 50 µL
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods
treatment: 6 h, standard culture conditions (37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH
post soak incubation: 25 min, room temerature
post treatment incubation: 18 h, standard culture conditions (37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH)

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals
Since the test item showed to reduce MTT, a functional check using freeze-killed tissue controls (killed controls = KC) was performed in at least one definitive assay to evaluate whether the test material is not binding to the tissue and leading to a false MTT reduction signal.
Since the test item was coloured, additional tests had to be performed to assess, if it changes colour after contact with water or isopropanol. For this purpose, approximately 50 mg each of the test item were added either to 1.0 mL of water or to 2 mL isopropanol in a glass tube. The water mixture was incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air for one hour, the isopropanol mixture for 3 hours at room temperature.
Since the test item became intensively coloured in water and isopropanol, it had to be considered as possibly interacting with the MTT measurement and an additional test on viable tissues (without MTT addition) had to be performed.
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled)
2
- Wavelength used for quantifying MTT formazan, and measuring device (e.g. spectrophotometer)
570 nm, plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro Enterprise, version 4.7.1)
- Description of the method used to quantify MTT formazan
Absorbance of MTT formazan at 570 nm
- Complete supporting information for the specific RhCE tissue construct used
see "Any other information on materials and methods"

Irritation parameter:

other: viability [%]

Value:

3.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Table 3: Summary of the Results

Treatment Group	OD 570 nm Well 1	OD 570 nm Well 2	Mean OD of 2 Wells	Mean OD of 2 Wells blank corrected	Mean OD of Treatment Group blank corrected	Rel. Viability [%] Tissue 1, 2 *	Absolute Value of the Difference of Rel. Viability  Tissue 1,2[%]	Mean Rel. Viability [%]**
Blank	0.035	0.038	0.036
Negative Control	1.709	1.709	1.709	1.672	1.655	101.1	2.1	100.0
	1.666	1.681	1.674	1.637		98.9
Positive Control	0.673	0.672	0.672	0.636	0.646	38.4	1.2	39.0
	0.694	0.690	0.692	0.656		39.6
Test Item	0.110	0.108	0.109	0.073	0.071	4.4	0.3	3.3***
	0.105	0.105	0.105	0.068		4.1
Blank	0.035	0.038	0.036
Negative Control Viable Tissues	0.043	0.043	0.043	0.007	0.009	0.4	0.3	0.5
	0.049	0.046	0.047	0.011		0.7
Test Item Viable Tissues	0.042	0.042	0.042	0.006	0.008	0.3	0.3	0.5
	0.047	0.048	0.047	0.011		0.7
Negative Control Freeze killed Tissues	0.101	0.101	0.101	0.064	0.060	3.9	0.5	3.6
	0.092	0.092	0.092	0.056		3.4
Test Item Freeze killed Tissues	0.101	0.100	0.101	0.064	0.067	3.9	0.3	4.0
	0.107	0.106	0.106	0.070		4.2
* Relative viability [rounded values]: 100 x (absorbance test item/po. control/neg. control) / (mean absorbance neg. control)  ** Mean relative viability [rounded values]: 100 x ((mean absorbance test item/pos. control/neg. control) / mean absorbance neg. control)  *** corrected value

The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water or isopropanol led to a change in colour. Therefore, an additional test with viable tissues without MTT addition was necessary.

Optical evaluation of the MTT-reducing capacity of the test item with MTT-reagent showed blue colour. Therefore, an additional test with freeze-killed tissues was necessary.

The mean relative absorbance value of the test item, corresponding to the cell viability,decreased to 3.3% (threshold for irritancy:≤60%), consequently the test item was irritant toeye.

Concerning acceptance criteria:

·        The negative control OD is > 0.8 and < 2.5 (1.666 and 1.709).

·        The mean relative viability of the positive control is below 50 % of the negative control viability (39.0 %).

·        The difference of viability between the two relating tissues of a single item is < 20 % (valuesbetween 0.3 % and 2.1 %) in the same run (for positive and negative controltissues and tissues of single test items).This applied also to the killed controls (items and negative control) and the additional viable tissues (without MTT addition) which were calculated as percent values related to the viability of the relating negative control.

Interpretation of results:

other: eye irritating, UN GHS Cat 1 or 2

Conclusions:

In an in vitro eye irritation study (RhCE) according to OECD guideline 492, the test item did show eye irritating properties.

Executive summary:

An in vitro study eye irritation study according to OECD guideline 492 was performed to assess the eye irritation potential of the test item by means of the Human Cornea Model Test.

The test item proved to be an MTT reducer in the MTT pre-test. Also, its intrinsic colour was intensive and it proved to dye water and isopropanol in the colour interference pre-test. Therefore, additional tests with freeze-killed and viable tissues had to be performed. The viability values resulted in these additional tests were used to correct the values gained in the normal tests.

Each 50 mg of the test item, were applied to each of duplicate tissue for 6 hours. Each 50 µL of the negative control (deionised water) and of the positive control (methyl acetate) were also applied to duplicate tissues each.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of OD > 0.8 and < 2.5, thus showing the quality of the tissues. Treatment with the positive control induced a decrease below 50 % viability compared with the negative control value in the relative absorbance, thus ensuring the validity of the test system. The difference of viability between the two relating tissues was < 20 % in the same run (for test item tissues, positive and negative control tissues). Irritating effects were observed following incubation with the test item. Compared with the value of the negative control, the relative mean absorption value corresponding to the viability of the tissues decreased below 60 % (determined value for the test item 3.3 %).

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item possesses an eye irritating potential.

Reference 4

Endpoint:

eye irritation: in vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reason / purpose for cross-reference:

read-across source

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: persistant staining in eyes

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

other: persistant staining in eyes

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.67

Max. score:

3

Reversibility:

fully reversible within: 7 d

Remarks on result:

other: persistant staining in eyes

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 7 d

Remarks on result:

other: persistant staining in eyes

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 7 d

Remarks on result:

other: persistant staining in eyes

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 7 d

Remarks on result:

other: persistant staining in eyes

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 7 d

Remarks on result:

other: persistant staining in eyes

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 7 d

Remarks on result:

other: persistant staining in eyes

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin:

The primary skin irritation potential of the structural analogue substance BLUE MGi 1037 was investigated by topical semi-occlusive application of 0.5 g to 6 cm2 intact left flank of each of three young adult New Zealand White rabbits. The duration of treatment was four hours. The scoring of skin reactions was performed 1, 24, 48 and 72 hours after removal of the dressing. The scores of each animal at the following reading times (24, 48, 72 hours) were used in calculating the respective mean values for each type of lesion.

The primary irritation score was calculated by totaling the individual cumulative scores at 24, 48 and 72 hours and then dividing by the number of data points. The primary irritation score was 0.00 (max. 8.0).

Very slight erythema was observed in one male animal (no. 91) one hour after treatment. Local signs (mean values from 24 to 72 hours) consisted of grade 0.00 erythema and grade 0.00 oedema.

Light blue staining produced by the test item of the treated skin was observed in all animals after removal of the dressing and during the whole study period. No corrosive effects were noted on the treated skin of any animal at any measuring interval.

Based upon the referred classification criteria (EEC Commission Directive 93/21 /EEC of April 27, 1993), BLUE MGi 1037 is considered to be "not irritating" to rabbit skin.

Eye:

For the in vitro evaluation of the eye irritating properties of chemicals, one single in vitro assay may not be sufficient for a conclusive classification according to CLP criteria. Following the Guidance Document on an Integradted Approach on the Testing and Assessment (IATA) for Serious Eye damage and Eye Irritation, multiple ex vivo/in vitro assays were performed. The final assessment is based on the results of the assays in an weight of evidence approach.

The eye irritation/serious eye damaging potential of the test item was assessed in an ex vivo assay (BCOP) according to OECD guideline 437.The induced opacity and increased permeability was investigated in isolated bovine corneas after exposure to the test item as a 20% (w/v) solution in a 0.9% sodium chloride solution. As negative control 0.9% sodium chloride solution and as positive control 20% (w/v) Imidazole was used. Three corneas were used per group (negative control, positive control or test substance group). After a first opacity measurement of the untreated bovine corneas, 750 µL of the dissolved test item, positive or negative control were applied on the corneas and incubated for 240 minutes. After the incubation phase the test item, the positive, and the negative control were rinsed from the corneas and the opacity was measured again. After the opacity measurements, the permeability of the corneas was determined by application of a fluorescein solution for 90 minutes. The amount of fluorescein solution that crossed the cornea was measured spectrophotometrically. The opacity and permeability assessments were combined to determine an In Vitro Irritancy Score (IVIS). After treatment with the negative control the calculated IVIS was 1.31. Treatment with the positive control revealed an IVIS of 122.9. The study fulfilled the acceptance criteria and was considered as valid. The mean IVIS obtained after treatment with the test substance was 5.52 and, thus, above 3 but below 55, i.e. according to OECD 437 the test substance is not Category 1, but no further classification can be made.

In an in vitro eye irritation study according to OECD guideline 438, using the Isolated Chicken Eye (ICE) model, no ocular corrosion or severe irritation potential was observed after exposure to the test item. The overall ICE score was 1xI, 2xII. Based on the evaluation criteria, no prediction regarding te eye irritating properties of the test item could be made.

An in vitro study eye irritation study according to OECD guideline 492 was performed to assess the eye irritation potential of the test item by means of the Human Cornea Model Test.

The test item proved to be an MTT reducer in the MTT pre-test. Also, its intrinsic colour was intensive and it proved to dye water and isopropanol in the colour interference pre-test. Therefore, additional tests with freeze-killed and viable tissues had to be performed. The viability values resulted in these additional tests were used to correct the values gained in the normal tests.

Each 50 mg of the test item, were applied to each of duplicate tissue for 6 hours. Each 50 µL of the negative control (deionised water) and of the positive control (methyl acetate) were also applied to duplicate tissues each.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of OD > 0.8 and < 2.5, thus showing the quality of the tissues. Treatment with the positive control induced a decrease below 50 % viability compared with the negative control value in the relative absorbance, thus ensuring the validity of the test system. The difference of viability between the two relating tissues was < 20 % in the same run (for test item tissues, positive and negative control tissues). Irritating effects were observed following incubation with the test item. Compared with the value of the negative control, the relative mean absorption value corresponding to the viability of the tissues decreased below 60 % (determined value for the test item 3.3 %). In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item possesses an eye irritating potential.

Conclusion

Based on the in vitro results a classification of the test item as Category 2 (eye irritating) deemed necessary for the substance Blue MGi 1037-Na. However, in the OECD TG 405 study performed with the structurally similar read-across substance Blue MGi 1037 the eyes of animals showed slight blue colouration, which was not reversible until day 21. Based on this persistent colouration the read-across substance is classified for eye damage Cat.1. Therefore, the substance Blue MGi 1037-Na is also classified for serious eye damage Cat.1 since persistent coloration of eyes is expected also for the substance Blue MGi 1037-Na.

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 1272/2008 (CLP). As a result the test substance is considered not to be classified for skin irritation under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) 2019/521. In contrast, the test substance is considered to be classified for eye damage (UN GHS Category 1) under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) 2019/521.