Reaction mass of 2-ethyl-4-methyl-1H-imidazole-1-propiononitrile and 2-ethyl-5-methyl-1H-imidazole-1-propiononitrile

Endpoint summary

Description of key information

OECD 405 (Hong, 2018): Eye Irrit. 2 (H319)

OECD 437 (Volker, 2018): Not Corrosive

OECD 492 (Grahl, 2018): Inconclusive

 

The in vitro eye irritation/corrosion studies conducted were not conclusive for classification therefore the classification of the test substance required the use of an additional in vivo study. The results from the in vivo OECD TG 405 were suffiient to classify the test item to be mildly irritating to the eye with a Category 2 classification according to CLP Regulation (EC) No 1272/2008.

Key value for chemical safety assessment

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:

key study

Study period:

12 -14 Dec 2017

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:

adopted 28 Jul 2015

Deviations:

yes

Remarks:

- the test substance showed reduction of MTT and colouring as compared to the vehicle control. - the non-specific reduction of MTT was not determined - no killed tissue controls were performed

Qualifier:

equivalent or similar 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:

adopted 9 Oct 2017

Deviations:

yes

Remarks:

- the test substance showed reduction of MTT and colouring as compared to the vehicle control. - the non-specific reduction of MTT was not determined - no killed tissue controls were performed

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Deutschland

Species:

human

Details on test animals or tissues and environmental conditions:

- in vitro test method: Eye Irritation Test (EIT) with reconstructed human cornea-like epithelium (RhCE) tissue, EpiOcular™ (MatTek)
- Lot number: 27017
- Keratinocyte strain: 4F1188
- no biological contaminants (virus, bacteria, yeast or fungi) detected
- Acceptance criteria for tissue viability met: 1.604 ± 0.08 (Acceptance criteria: OD (540-570 nm) [1.1 - 3.0])
- Acceptance criteria for Barrier function met: 21.7 min (Acceptance criteria: ET-50 [12.2 - 37.5 min])

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 µL undiluted

NEGATIVE CONTROL
- Amount(s) applied: 50 µL
- Lot/batch no.: Sigma, RNBF7110

POSITIVE CONTROL
- Amount(s) applied: 50 µL
- Lot/batch no.: Merck, S6943111

Duration of treatment / exposure:

30 ± 2 min

Duration of post- treatment incubation (in vitro):

12 ± 2 min (post-soak plate)
120 ± 15 min (post-treatment plate)

Number of animals or in vitro replicates:

duplicates for each treatment and control group; from each tissue, 2 absorbance measurements after MTT incubation were performed

Details on study design:

- RhCE tissue construct used, including batch number: Epiocular™, Test Kit name: OCL-200-EIT (MatTek), Lot number: 27017

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: exposure: 30 ± 2 min at 37 ± 1°C, post exposure: 12 ± 2 min at room temperature (post-soak plate), 120 ± 15 min (post-treatment plate) at 37 ± 1°C

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: The ability of the test substance to directly reduce MTT and to change the color of the MTT medium was assessed in a pre-experiment.

Mixture of the test substance with MTT medium showed non-specific reduction of MTT compared to the solvent. The mixture turned blue/purple. Since the mean relative tissue viability of the test substance treated tissues (TM) was below the 60% threshold value no killed tissue controls were performed, since the test substance has to be classified as irritant in any case.
Mixture of test substance with distilled water or isopropanol showed colouring as compared to the solvent. Since the mean relative tissue viability of the test substance treated tissues (TM) was below the 60% threshold value no coloured tissue controls were performed, since the test substance has to be classified as irritant in any case.

- Number of tissue replicates used per test chemical and controls: 2

- Wavelength: 570 nm ± 30 nm

- Spectrophotometer: plate spectrophotometer

- Description of evaluation criteria used: The test substance is considered to be not irritating to the eye if the tissue viability is > 60%. The test substance is considered to be irritating to eye if the tissue viability is ≤ 60% (no distinction between category 1 or 2 possible).

The test meets acceptance criteria if:
- mean absolute OD570 of the negative control is > 0.8 and < 2.5,
- mean relative tissue viability of the positive control is < 50%,
- mean relative tissue viability difference between replicate tissues is < 20%.

Irritation parameter:

other: % tissue viability mean value of 2 tissues

Value:

6.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Mixture of the test substance with MTT medium showed non-specific reduction of MTT compared to the solvent. The mixture turned blue/purple. Since the mean relative tissue viability of the test substance treated tissues (TM) was below the 60% threshold value no killed tissue controls were performed, since the test substance has to be classified as irritant in any case.

- Mixture of test substance with distilled water or isopropanol showed colouring as compared to the solvent. Since the mean relative tissue viability of the test substance treated tissues (TM) was below the 60% threshold value no coloured tissue controls were performed, since the test substance has to be classified as irritant in any case.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the OD of the negative control was 1.720, 1.808, 1.959 and 1.952, and thus within the acceptibility range (OD > 0.8 and < 2.5).
- Acceptance criteria met for positive control: Yes, the mean positive control reduced the cell viability at 2.0% and fulfilled the acceptance criteria (< 50%).
- Acceptance criteria for variabilties: Differences of two tissue cell viabilities in the negative control substance, the positive control substance and the test substance groups were 10.3%, 0.3% and 0.4%, respectively, and thus < 20%.

Table 2: Summary of Results

Name	Negative Control		Positive Control		Test Substance
Tissue	1	2	1	2	1	2
OD570 values (blank-corrected)	1.720225	1.959125	0.039525	0.035025	0.109525	0.118925
	1.808325	1.952425	0.040225	0.032025	0.109225	0.115125
mean of the duplicates	1.764	1.956	0.040	0.034	0.109	0.117
mean OD	1.860^*		0.037		0.113
mean SD OD	0.12		0.00		0.00
tissue viability [%]	94.9	105.1	2.1	1.8	5.9	6.3
relative tissue viability difference [%]^***	10.3		0.3		0.4
mean tissue viability [%]	100.0		2.0^**		6.1

*         Corrected mean OD570 of the negative control corresponds to 100% absolute tissue viability

**        mean relative tissue viability of the positive control is < 50%

***       relative tissue viability difference of replicate tissues is < 20%.

SD = standard deviation

OD = optical density

Interpretation of results:

other: irritating potential according to OECD 492

Conclusions:

Under the conditions of the RhCE test method the test substance showed irritant effects, but no prediction on the potential for serious eye damage can be made and additional testing should be conducted for classification and labeling purposes.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 Jan 2018

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 i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted 9 Oct 2017

Deviations:

yes

Remarks:

The in vitro irritation score obtained with the positive control did not fell within the two standard deviations of the current historical mean as suggested by OECD 437.

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: A. Moksel AG, Buchloe, Germany
- Storage, temperature and transport conditions of ocular tissue: The freshly isolated eyes were collected from the slaughterhouse and transported in Hank's Buffered Salt Solution (HBSS) containing Penicillin/Streptomycin to the laboratories.
- Time interval prior to initiating testing: The corneae were isolated on the same day after delivery of the eyes and directly used in the BCOP test.
- Indication of any existing defects or lesions in ocular tissue samples: All eyes were visually inspected for defects and any defective cornea was discarded.

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.75 mL
- Concentration: used as provided by the sponsor

NEGATIVE CONTROL
- Amount applied: 0.75 mL
- Concentration: physiological saline 0.9% NaCl (w/v)

POSITIVE CONTROL
- Amount applied: 0.75 mL
- Concentration: 100 % (v/v)

Duration of treatment / exposure:

10 min

Number of animals or in vitro replicates:

triplicates for each treatment and control group

Details on study design:

SELECTION AND PREPARATION OF CORNEAS:
The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS. Each cornea was mounted in a specially designed cornea holder.

QUALITY CHECK OF THE ISOLATED CORNEAS:
An initial measurement was performed on each of the corneas using the opacitometer. Three corneas with illuminance readings approximately equivalent to the median illuminance of all corneas were selected as negative-control corneas. The illuminance of each cornea was read and recorded. Only corneas that had an initial illuminance reading I > I 0/1.1651 lux were used for the assay.

TREATMENT METHOD:
The cornea holder consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. The corneas were mounted in corneal holders (Duratec GmbH) with the endothelial side against the O-ring of the posterior chamber. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with Roswell Park Memorial Institute medium (RPMI) (without phenol red) containing 1% fetal bovine serum FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber was always filled first. The corneas were incubated at 32 ± 1 °C for one hour. After the equilibration period, the medium was removed from both chambers and replaced with fresh complete RPMI. The medium was removed from the anterior chamber and replaced with the test substance or control. 750 µL of the test substance or the control substance was introduced into the anterior chamber. After 10 minutes incubation at 32 ± 1 °C either the test substance or the control substance was removed and the epithelium washed at least three times with minimum essential medium (MEM) (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red). The anterior chamber was refilled with complete RPMI and an illuminance measurement was performed after 2 hours incubation at 32 ± 1 °C. Also, each cornea was observed visually and pertinent observations were recorded. After the illuminance measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI. 1 mL of a 4 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 ± 1 °C. Then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer (Jenway 6405 UV/VIS).

NUMBER OF REPLICATES: 3 corneae per test group

REMOVAL OF TEST SUBSTANCE: The test substance was rinsed off from the application side with minimum essential medium (MEM).

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer (BASF-OP3.0, Duratec GmbH).
- Corneal permeability: After incubation with fluorescein dye at 32 ± 1 °C for 90 min, passage of sodium fluorescein dye was measured using spectrophotometer (Jenway 6405 UV/VIS) at 490 nm (OD490).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS), IVIS = mean opacity value + (15 x mean permeability OD490 value)

DECISION CRITERIA:
Test substance with an IVIS > 55 was regarded as potentially causing serious eye damage and labelled Category 1 according to CLP/EPS/GHS.
Test substance with an IVIS ≤ 3 was regarded as non-irritant and labelled in no category.
Test substance with an IVIS > 3; ≤ 55 no prediction can be made.

Irritation parameter:

in vitro irritation score

Remarks:

mean value of 3 corneas

Value:

31.16

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

The negative control responses resulted in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

Positive controls validity:

valid

Remarks:

positive control did not fall within the two standard deviations of the current historical mean. Since the positive control is “more positive” as expected this assay might still considered to be functional and valid.

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control resulted in opacity and permeability values that were less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.
- Acceptance criteria were not met for positive control: The in vitro irritation score obtained with the positive control did not fall within the two standard deviations of the current historical mean as suggested by OECD 437. However, the obtained in vitro irritation score of the positive control was higher than the range defined by the two standard deviations of the current historical mean. Since the positive control is “more positive” as expected this assay might still considered to be functional and valid.

Table 2: In vitro Irritation Score

Cornea Number	Test substance	Corrected Opacity	Corrected OD490 Value	IVIS
1	Negative Control	0.19	0.008
2		- 0.12	0.005
3		0.00	0.018
Mean Value (MV)		0.02	0.010	0.18
4	Positive Control	24.25	6.010
5		24.28	5.075
6		25.37	5.600
MV		24.63	5.561	108.05
7	Test Substance	4.29	0. 461
8		10.57	2.130
9		11.87	1.860
MV		8.91	1.583	31.16

 

Interpretation of results:

other: non-corrosive according to OECD 437

Conclusions:

Application of the test substance to bovine corneae resulted in a calculated mean IVIS of 31.16.
The IVIS cut-off value for identifying test substances as inducing serious eye damage (UN GHS Category 1, IVIS > 55) and test substances not requiring classification for eye irritation or serious eye damage (UN GHS No Category, IVIS ≤ 3 ) are both not met.
The test substance is not corrosive to the eyes. No prediction can be made regarding the classification of the test substance according to the evaluation criteria. Further testing is required for calssification and labelling purposes.

Reference 3

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 December 2017 to 09 February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

"Regulation on Test Methods for Chemical Substances"
Notification No. 2017-4, National Institute of Environmental Research, Republic of Korea (Mar. 14, 2017)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (measurement value 18.7 - 20.0˚C , permissible rangeL 15 - 25˚C), sealed
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: NA
- Reactivity of the test substance with the solvent/vehicle /test medium (if applicable): NA

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: NA - applied neat
- Preliminary purification step (if any): NA
- Final dilution of a dissolved solid, stock liquid or gel: NA
- Final preparation of a solid: NA

Species:

rabbit

Strain:

New Zealand White

Remarks:

(Yac:NZW (KBL)), SPF

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source:Producer - Yonam College, Yonam Laboratory Animal, Supplier - Orientbio InC., Republic of Korea
- Sex: Male
- Number of animals: 3
- Age at study initiation: 11 weeks
- Weight at study initiation: 2.03 - 2.11 kg
- Housing:Stainless wire mesh cages (450Wx600Dx360H (mm))
- Diet: ad libitum - Purina experimental diet for rabbit 38302AF
- Water: ad libitum - public tap water filtered and irradiated
- Acclimation period: Upon receipt, all animals were subjected to the clinical examinations with body weights recorded. All animals were observed for clinical signs once daily for 8 days in the initial test and for 10 days in the confirmatory test. On the last day of the quarantine-acclimation period, the measurement of body weights and examination of health conditions were conducted.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Measured value - 19.0 - 21.2. Permissible range: 18.0 - 24.0
- Humidity (%): Measurement value : 40.0 -57.4. Permissible range: 30.0-70.0
- Air changes (per hr): 10-15 clean, fresh, filtered air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour light/dark cycle

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1mL test substance instilled
- Concentration (if solution): 100%

VEHICLE
- Amount(s) applied (volume or weight with unit): NA
- Concentration (if solution): NA
- Lot/batch no. (if required): NA
- Purity: NA

Duration of treatment / exposure:

24 hours

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

three male rabbits

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): NA
- Time after start of exposure:

SCORING SYSTEM:

Observation

Cornea
A. Opacity- degree of density (area which is most dense taking for reading)
- No opacity - 0
-Scattered or diffuse areas, details of iris clearly visible - 1
- Easily discernible translucent areas, details of iris slightly obscured - 2
- Opalescent areas, no details of iris visible, size of pupil barely discernible - 3
Opaque, iris visible - 4

B. Area of cornea involved
- Normal - 0
- One quarter (or less) but not zero - 1
- Greater than one quarter but less than half - 2
- Greater than half, but less than three quarters - 3
- Greater than three quarters, up to whole area - 4

Iris
A. Iris
- Normal - 0
- Folds above normal, congestion, swelling, circumcorneal injection (any or all of these or combination of any thereof) iris still reacting to light (sluggish reaction is positive) - 1
- No reaction to light, haemorrhage, gross destruction (any of these) - 2

Conjunctive
A. Redness (refers to palpebral and bulbar conjunctive excluding cornea and iris)
- Vessels normal - 0
- Vessels definitely injected above normal - 1
- More diffuse, deeper crimson red, individual bessels not easily discernible - 2
- Diffuse beefy red - 3

B. Chemosis
-No swelling - 0
- Any swelling above normal (includes nictitating membrane) -1
- Obvious swelling with partial eversion of lids - 2
- Swelling with lids about half closed - 3
- Swelling with lids about half closed to completely closed - 4

C. Discharge
- No discharge - 0
- Any amount different from normal (does not include small amounts observed in inner canthus of normal animals) - 1
- Discharge with moistening of the lids and hairs just adjacent to lids - 2
- Discharge with moistening of the lids and hairs and considerable area around the eye - 3


TOOL USED TO ASSESS SCORE: hand-slit lamp

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

72 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: 7 days

Remarks on result:

probability of mild irritation

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

48 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: 7 days

Remarks on result:

probability of mild irritation

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: 7 days

Remarks on result:

probability of mild irritation

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

72 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: 7 days

Remarks on result:

probability of mild irritation

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

48 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: 7 days

Remarks on result:

probability of mild irritation

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: 7 days

Remarks on result:

probability of mild irritation

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

72 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: 7 days

Remarks on result:

probability of mild irritation

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

48 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: 7 days

Remarks on result:

probability of mild irritation

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: 7 days

Remarks on result:

probability of mild irritation

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

72 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: 7 days

Remarks on result:

probability of mild irritation

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

48 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: 7 days

Remarks on result:

probability of mild irritation

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: 7 days

Remarks on result:

probability of mild irritation

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

72 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: 7 days

Remarks on result:

probability of mild irritation

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

48 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: 7 days

Remarks on result:

probability of mild irritation

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: 7 days

Remarks on result:

probability of mild irritation

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

72 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: 7 days

Remarks on result:

probability of mild irritation

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

48 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: 7 days

Remarks on result:

probability of mild irritation

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: 7 days

Remarks on result:

probability of mild irritation

Irritation parameter:

iris score

Basis:

animal #3

Time point:

72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 96hr

Remarks on result:

probability of mild irritation

Irritation parameter:

iris score

Basis:

animal #3

Time point:

48 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 96h

Remarks on result:

probability of mild irritation

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 96hr

Remarks on result:

probability of mild irritation

Irritation parameter:

iris score

Basis:

animal #2

Time point:

72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 96h

Remarks on result:

probability of mild irritation

Irritation parameter:

iris score

Basis:

animal #2

Time point:

48 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 96h

Remarks on result:

probability of mild irritation

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 96h

Remarks on result:

probability of mild irritation

Irritation parameter:

iris score

Basis:

animal #1

Time point:

72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 96h

Remarks on result:

probability of mild irritation

Irritation parameter:

iris score

Basis:

animal #1

Time point:

48 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 96h

Remarks on result:

probability of mild irritation

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 96h

Remarks on result:

probability of mild irritation

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

72 h

Score:

0

Max. score:

0

Reversibility:

other: No effect seen

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

48 h

Score:

0

Max. score:

0

Reversibility:

other: No effect seen

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24 h

Score:

0

Max. score:

0

Reversibility:

other: No effect seen

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

72 h

Score:

0

Max. score:

0

Reversibility:

other: No effect seen

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

48 h

Score:

0

Max. score:

0

Reversibility:

other: No effect seen

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24 h

Score:

0

Max. score:

0

Reversibility:

other: No effect seen

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

72 h

Score:

0

Max. score:

0

Reversibility:

other: No effect seen

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

48 h

Score:

0

Max. score:

0

Reversibility:

other: No effect seen

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24 h

Score:

0

Max. score:

0

Reversibility:

other: no effect seen

Remarks on result:

no indication of irritation

Table 1: Individual eye irritation scores

Test group (Conc.)	Intervals (hours)		Animal ID	Cornea		Score I^1)	Iris	Score II^2)	Conjunctivae			Score III^3)	ITS	MTS
				Opacity	Area				Redness	Chemosis	Discharge
GI (100%)	1	Initial	1101	0	0	0	1	5	2	3	3	16	21	21.0
		Confirmatory	1102	0	0	0	1	5	2	3	3	16	21
			1103	0	0	0	1	5	2	3	3	16	21
	24	Initial	1101	0	0	0	1	5	2	2	2	12	17	17.0
		Confirmatory	1102	0	0	0	1	5	2	2	2	12	17
			1103	0	0	0	1	5	2	2	2	12	17
	48	Initial	1101	0	0	0	1	5	2	2	0	8	13	13.0
		Confirmatory	1102	0	0	0	1	5	2	2	0	8	13
			1103	0	0	0	1	5	2	2	0	8	13
	72	Initial	1101	0	0	0	1	5	2	2	0	8	13	13.0
		Confirmatory	1102	0	0	0	1	5	2	2	0	8	13
			1103	0	0	0	1	5	2	2	0	8	13
	96	Initial	1101	0	0	0	0	0	2	1	0	6	6	6.0
		Confirmatory	1102	0	0	0	0	0	2	1	0	6	6
			1103	0	0	0	0	0	2	1	0	6	6

Table 1: Continued 

Test group (conc)	Intervals (days)		Animal ID	Cornea		Score I^1)	Iris	Score II^2)	Conjunctivae			Score III^3)	ITS	MTS
				Opacity	Area				Redness	Chemosis	Discharge
GI (100%)	5	Initial	1101	0	0	0	0	0	1	1	0	4	4	4.0
		Confirmatory	1102	0	0	0	0	0	1	1	0	4	4
			1103	0	0	0	0	0	1	1	0	4	4
	6	Initial	1101	0	0	0	0	0	0	0	0	0	0	0
		Confirmatory	1102	0	0	0	0	0	0	0	0	0	0
			1103	0	0	0	0	0	0	0	0	0	0

 

hour: Observation after application

1): Opacity x Area x 5

2): Iris x 5

3): (REdness +Chemosis + dishcharge) x 2

a): No fluorescein staining mark

ITS: Individual total score = Score 1+ Score II + Score III

MTS: Mean total score

Conc. Concentration

 

Table 2: Summary of Eye Irritation Scores  

Test group (conc)		No. of animals	Mean Total Score (MTS) within 96 hours					MMTS	Classification of Eye Irritation
			1 hour	24 hours	48 hours	72 hours	96 hours	21.0	Moderately Irritating
GI (100%)	Initial	1	21.0	17.0	13.0	13.0	6.0
	Confirmatory	2

Table 2: Continued

Test group (conc)		No. of animals	Mean total Score (MTS)
			Day 5	Day 6
GI (100%)	Initial	1	4.0	0.0
	Confirmatory	2

 

Table 3: Summary of Clinical Signs

Test group		Animal ID	Day
			1	2	3	4	5	6
GI	Initial	1101	-	-	-	-	-	-
	Confirmatory	1102	-	-	-	-	-	-
		1103	-	-	-	-	-	-

- : No Observable Abnormality

Table 4: Clinical Signs of Pain and Distress

Test group		Animal ID	Day	0			1			2			3			4			5				6
			Times	Immediately after test substance application	1^st a)	2^nd b)	1^st a)	2^nd b)	3^rd b)	1^st a)	2^nd b)	3^rd b)	1^st a)	2^nd b)	3^rd b)	1^st a)	2^nd b)	3^rd b)	1^st a)	2^nd b)	3^rd b)		1^st a)	2^nd b)		3^rd b)
GI	Initial	1101		-	ET	ET	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-		-	-	-
	Confirmatory	1102		-	ET	ET	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-		-	-	-
		1103		-	ET	ET	-	-	-	-	-	-	-	-	-	-	-	-	-	-		-	-	-	-

a) Several Observation Times

b) Prior to Systemic Analgesics

ET: Excessive tearing

-: No Observed Abnormality

 

Table 5: Mean body weights (kg)

Test group		Animal ID	Day		Gain
			0	6
GI	Initial	1101	2.03	2.17	0.14
	Confirmatory	1102	2.11	2.28	0.17
		1103	2.06	2.18	0.12
		Mean	2.07	2.21	0.14
		S.D.	0.04	0.06	0.03
		No. of animals	3	3	3

 

Interpretation of results:

Category 2B (mildly irritating to eyes) based on GHS criteria

Remarks:

Category 2 according to CLP

Conclusions:

Under the conditions of this study, the test item is mildly irritating to the eye and a Category 2 Eye Irritant according to CLP Regulation (EC) No 1272/2008.

Executive summary:

The study was performed to OECD TG 405 under GLP to assess the irritancy potential of the test material to the eye following a single application in the New Zealand White rabbit.

A volume of 0.1ml of the test material was placed into the conjunctival sac of one eye of two animals. The other remained untreated and was used for control purposes. The test was conducted in a stepwise manner conducted singularly. Assessment of ocular irritation was made at approx. 1 hour then at 24, 48 and 72 hours following application, additional observations was made at day 5 and 6.

The application of test material to the eye of three rabbits produced no corneal effects. Iritis was noted in all animals 1 hour after treatment and persisted to 72 hours post treatment. Moderate chemosis was noted in all animals 1-hour post treatment and persisted to observation at day 5. Conjunctival redness was also noted 1-hour post treatment and persisted to day 5 observation. Moderate discharge was noted in all animals 1-hour post treatment and was observed until 24 hour observation. Treated eyes for all animals appeared normal at the day 6 observation. Under the conditions of this study, the test item was considered to be moderately irritating to the eye, and was therefore classified as a Category 2 Eye irritant according to CLP Regulation (EC) No. 1272/2008.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Additional information

Initial assessment for eye irritation/corrosion was conducted under OECD TG 492: Reconstructed Human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage (Grahl, 2018). Under the conditions of the RhCE test method the test substance showed irritant effects. However, no prediction on the potential for serious eye damage could be made, therefore additional testing was required in order to conclude on the classification of the substance.

In accordance with the accepted testing strategy under REACH, an additional in vitro endpoint study was performed. Secondary assessment was undertaken according to OECD TG 437: Bovine Corneal Opacity and Permeability Test method  (BCOP) for identifying i) chemicals inducing serious eye damage and ii) chemicals not requiring classification for eye irritation or serious eye damage (Volker, 2018). Application of the test substance to bovine corneae resulted in a calculated mean IVIS of 31.16. Neither the IVIS cut-off value for identifying test substances as inducing serious eye damage (UN GHS Category 1, IVIS > 55), nor test substances not requiring classification for eye irritation or serious eye damage (UN GHS No Category, IVIS ≤ 3) were met. It was therefore concluded that the test substance is not corrosive to the eye. The results were not adequate to enable classification and therefore no prediction could be made according to the CLP evaluation criteria. 

As the results of both in vitro studies did not provide adequate information to classify the substance, it was necessary to complete an in vivo eye irritation/corrosion study (Hong 2018). The study was performed to OECD TG 405 (Acute eye irritation/corrosion) under GLP conditions to assess the irritancy potential of the test material to the eye following a single application to New Zealand White rabbits. 0.1ml of the test material was placed into the conjunctival sac of one eye of two animals, the other remained untreated, used for control purposes. Assessment of ocular irritation was made at approx. 1 hour and 24, 48,72 hours following application, with additional observations was made on day 5 and 6. The application of test material to the eye of three rabbits produced no corneal effects. Iritis was noted in all animals 1 hour after treatment and persisted to 72 hours post treatment. Moderate chemosis was noted in all animals 1-hour post treatment and persisted to day 5 observation. Conjunctival redness was also noted 1-hour post treatment which persisted to day 5 observation. Moderate discharge was noted in all animals 1-hour post treatment and was observed until 24 hours. Treated eyes for all animals appeared normal at the day 6 observation.

Using a weight of evidence based on adequate information from both EpiOcular and BCOP in vitro studies and acute eye irritation/corrosion in vivo tests, the test item is determined to be mildly irritating to the eye, and therefore classified as a Category 2 Eye Irritant according to CLP Regulation (EC) No 1272/2008.

Justification for classification or non-classification

Results from the key study OECD TG 405 (Hong, 2018) were sufficient for Category 2 Eye Irritant classification according to CLP Regulation (EC) No 1272/2008.