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Diss Factsheets

Administrative data

Description of key information

Skin irritation: Key study. Method according to OECD 439, GLP study. Based on the study results (cell viability of 59%), the test item is not irritant to skin.

Eye irritation:

- Key study. Method according to OECD 438, GLP study. Based on the study results (overall ICE class 1xI, 1xII, 1xIII), the test item is not a severe irritant, but no prediction can be made.

- Key study. Method according to OECD 405, GLP study. The test item caused slight to severe conjunctival and moderate corneal irritant effects, fully reversible within 2 weeks. Based on the results, the test item is irritant to the eyes.

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:
From July 31st to October 9th, 2013.
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 15-30ºC, in tightly closed container.
Test system:
human skin model
Remarks:
EPISKIN-SM
Source species:
human
Cell type:
other: adult human-derived epidermal keratinocytes
Cell source:
other: human adult donors, not specified.
Source strain:
not specified
Details on animal used as source of test system:
EPISKIN-SM (Manufacturer: SkinEthic, France, Batch No.:13-EKIN-030, Expiry Date: 09 September 2013)
Justification for test system used:
The EPISKIN model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and no- skin irritating test substances (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN-SM (Manufacturer: SkinEthic, France)
- Tissue batch number(s): 13-EKIN-030
- Expiry Date: 09 September 2013
- Date of initiation of testing: 05 September 2013

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: Exposure of test material was terminated by rinsing thoroughly with PBS 1x solution. The rest of the PBS was removed from the epidermal surface with suitable pipette tip linked to a vacuum source (care was taken to avoid the damage of epidermis).
- Observable damage in the tissue due to washing: no.
- Modifications to validated SOP: no.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 3 mg/mL
- Incubation time: 3 hours (± 5 min)
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 15 minutes exposure is less than 50%.
- The test substance is considered to be non-corrosive to skin if the viability after 15 minutes exposure is greater than or equal to 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- The test item was applied in its original form, no formulation was required.
- Amount(s) applied (volume or weight with unit): 10 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 20 µL
- Concentration: PBS 1x

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 20 µL
- Concentration: Sodium Dodecyl Sulphate (SDS) 5%
Duration of treatment / exposure:
15 min (± 0.5 min)
Duration of post-treatment incubation (if applicable):
42 h (± 1h)
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 / 1
Value:
56
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 / 2
Value:
54
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 / 3
Value:
66
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
59
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: Prior to routine use of the method TOXI-COOP ZRT. demonstrated the technical proficiency, using the ten Proficiency Chemicals according to OECD Test Guideline No. 439 (data not included).

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

Table 1. Summary of results.

Substance

Optical Density (OD)

Viability (%)

Negative Control:

1 x PBS

mean

0,631

100

standard deviation (SD)

3,11

Positive Control:

SDS (5%aq.)

mean

0,228

36

standard deviation (SD)

1,33

Test Item:

IMIDAZOLE ETHANOL

mean

0,370

59

standard deviation (SD)

6,51

Following exposure with the test item, the mean cell viability was 59% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non irritant to skin. In conclusion, in thisin vitro EPISKIN model test with the test item, the results indicate that the test item is non irritant to skin.

The mean OD value of the three negative control tissues was 0.631, the positive control result showed 36 % viability, each standard deviation value (SD) of the % viability was below 18 (see table below in section 9.3). All validity criteria were within acceptable limits and therefore the study can be considered as valid.

Interpretation of results:
GHS criteria not met
Remarks:
EU criteria.
Conclusions:
The test item is not irritant to skin, with a cell viability of 59%.
Executive summary:

An in vitro skin irritation test of the test item was performed in an EPISKIN-SM reconstructed human epidermis model, according to OECD TG 439 (GLP study). Disks of EPISKIN (3 units / chemical) were treated with test item and incubated for 15 minutes at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in 5% CO2 protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically. SDS (5% aq.) and PBS treated epidermis were used as positive and negative controls, respectively. All validity criteria were met.

The mean cell viability was 59%, compared to the negative control. Therefore, the test item was not irritating to the skin.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 27th, 2013.
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 Ocular Corrosives and Severe Irritants)
Deviations:
yes
Remarks:
test item was applied dry.
Qualifier:
according to guideline
Guideline:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
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 (e.g. transport time, transport media and temperature, and other conditions): After collection, the heads were inspected for appropriate quality and wrapped with paper moistened with saline, then placed in a plastic box that can be closed (4-5 heads/box). The heads were transported to TOXI-COOP ZRT. at the earliest convenience for use approximately within 2 hours from collection. All eyes used in the assay were from the same groups of eyes collected on one specific day.
- Time interval prior to initiating testing: 2h
- indication of any existing defects or lesions in ocular tissue samples: no.
- Indication of any antibiotics used: no.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.03 g/eye.
Duration of treatment / exposure:
10 seconds.
Duration of post- treatment incubation (in vitro):
240 ± 5 minutes
Number of animals or in vitro replicates:
3 eyes for the test item and positive control, 1 eye for the negative control.
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES: 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 eye ball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, 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 appropriate number of eyes was selected and after being placed in the superfusion apparatus, the selected eyes were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the isotonic saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured using the depth measuring device on the slit lamp microscope (Haag-Streit BQ 900) with the slit-width set at 9½, equalling 0.095 nm. Any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced.

EQUILIBRATION AND BASELINE RECORDINGS: Acclimatization was conducted for approximately 45 to 60 minutes. The temperature of the circulating water was verified to be in the range of 32 ± 1.5 °C in all chambers during the acclimatization and treatment periods. 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 (-3% 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. The location of any minor findings was marked on the record sheet as a drawing, if applicable. If any eye was considered to be unsuitable following baseline assessment, it was discarded.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: 30 µL of 9 g/L NaCl, saline (Source: REANAL, Batch: PP/2012/10937) in ultra-pure water.

POSITIVE CONTROL USED: 0.03 g Imidazole (Source: Sigma-Aldrich, Batch: SLBC7446V)

APPLICATION DOSE AND EXPOSURE TIME: 0.03 g test item, exposure 10 s.

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

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: 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.
- Indicate any deviation from test procedure in the Guideline: The test item was stuck on the corneas at 30 minutes after the post-treatment rinse, so a gentle rinsing with additional 20 mL saline was performed. The cornea surfaces were not totally cleared, little volume of test item was stuck on the cornea surface at 240 min after the posttreatment rinse.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: qualitative assessment.
- Damage to epithelium based on fluorescein retention: qualitative assessment.
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: 0.095 nm. 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 nm.
- Macroscopic morphological damage to the surface: qualitative assessment.

SCORING SYSTEM:
- Mean corneal swelling (%):
Corneal swelling (CS) was calculated as follows: CS = [(corneal thickness at time t – corneal thickness at time t = 0)/ corneal thickness at time t = 0] x100
For the calculation of Maximum Swelling, small negative numbers for swelling (0 to -5%) following application are counted as zero. Large negative numbers (> -12 %) are probably due to erosion and indicate a severe effect (scored as class IV). Cases of values of -5 % to -12 % are evaluated on a case by case basis but in the absence of other findings do not indicate a severe effect.

- Mean maximum opacity score
0 No opacity
0.5 Very faint opacity
1 Scattered or diffuse areas; details of the iris are clearly visible
2 Easily discernible translucent areas; details of the iris are slightly obscured
3 Severe corneal opacity; no specific details of the iris are visible; size of the pupil is barely discernible
4 Complete corneal opacity; iris invisible

- Mean fluorescein retention score at 30 minutes post-treatment
0 No fluorescein retention
0.5 Very minor single cell staining
1 Single cell staining scattered throughout the treated area of the cornea
2 Focal or confluent dense single cell staining
3 Confluent large areas of the cornea retaining fluorescein

DECISION CRITERIA: as indicated in the TG.
Irritation parameter:
fluorescein retention score
Run / experiment:
mean
Value:
2.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class III.
Irritation parameter:
cornea opacity score
Run / experiment:
mean max.
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class I.
Irritation parameter:
percent corneal swelling
Run / experiment:
mean max. (240 min)
Value:
11
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class II.
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: The test item was stuck on the cornea surface all test item treated eyes at 30 minutes after the post-treatment rinse. It could not be removed from cornea surface by extra rinse (20 mL/eye saline solution).

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes.

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 (Appendix II).
- Range of historical values if different from the ones specified in the test guideline: yes.

Table 1. Summary of test item results.

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

10%

II

Mean maximum corneal swelling at up to 240 min

11%

II

Mean maximum corneal opacity

0.0

I

Mean fluorescein retention

2.5

III

Other Observations

The test item was stuck on the cornea surface all test item treated eyes at 30 minutes after the post-treatment rinse. It

cannot be removed from cornea surface by extra rinse (20 mL/eye saline solution).

Overall ICE Class

1xI, 1xII, 1xIII

Conclusion

Not a severe irritant

 

Table 2. Summary of positive control (imidazole) results.

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

9%

II

Mean maximum corneal swelling at up to 240 min

15%

II

Mean maximum corneal opacity

4.0

IV

Mean fluorescein 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

1xII, 2xIV

Conclusion

Corrosive, Category 1

 

 

Table 3. Summary of negative control (saline) results.

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 fluorescein retention

0.0

I

Other Observations

None.

Overall ICE Class

3xI

Conclusion

Not classified

 

Interpretation of results:
other: not severely irritating, based on EU criteria.
Conclusions:
Based on the available data (overall ICE class 1xI, 1xII, 1xIII), the test item is not a severe irritant.
Executive summary:

An in vitro (ex vivo) study was conducted in order to determine the potential severe eye damaging effects of the test item according to the OECD guideline 438 under GLP conditions. Eyeballs were isolated from chickens killed for human consumption and after the appropriate preparation were exposed to either the test item, imidazole (positive control) or saline (negative control). Three eyeballs were used for the test item and the positive control, one for the negative control. Fluorescein retention, corneal opacity and corneal swelling were evaluated, then the results of each endpoint were assigned to ICE classes according to the guideline recomendations.
According to the overall in vitro classification, no prediction can be made since the combinations of the 3 endpoints were
1xI, 1xII, 1xIII. Based on the results, the test item is not severely irritant to the eye.

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From November 12th to November 28th, 2013.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: S & K-LAP Kft. 2173 Kartal, Császár út 135, HUNGARY
- Age at study initiation: 16 weeks old
- Weight at study initiation: 3630 - 4100 g
- Housing: Animals were housed individually in metal cage.
- Diet: CRLT/ny rabbit diet produced by Szindbád Kft., 2100 Gödöllő, Szárítópuszta, Hungary, ad libitum.
- Water: tap water from watering bottles ad libitum. The drinking water is periodically analysed and is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
- Acclimation period: 40 days in first animal, 41 days in second animal and 42 days in third animal.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3 °C
- Humidity (%): 30-70 %
- Air changes (per hr): 10-15 air exchanges/hour by central air-condition system.
- Photoperiod (hrs dark / hrs light): 12 hours of light daily, from 6.00 a.m. to 6.00 p.m. / 12 h dark.
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
0.1 g of the test item was used for the study in undiluted state, as a single dose.
Duration of treatment / exposure:
24h.
Observation period (in vivo):
2 weeks.
Number of animals or in vitro replicates:
3.
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): no. The eyes of the test animals were not washed out 24 hour after test item application, because the test item had been removed from the eye of test animal by physiological mechanisms.

SCORING SYSTEM: The eye irritation scores were evaluated according to the scoring system by Draize (1979) and OECD 405 (02 October 2012).
- Cornea
a. Opacity-degree of density (Area most dense taken for reading)
No uncleration or opacity 0
Scattered or diffuse areas of opacity (other than slight dulling of normal lustre): details of iris clearly visible 1
Easily discernible translucent area: details of iris slightly obscured 2
Nacreous area: no details of iris visible: size of pupil barely discernible 3
Opaque cornea: iris not discernible through the opacity 4

b. Area of cornea involved
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. Values
Normal 0
Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia: or injection: iris reactive to light 1
Hemorrhage, gross destruction, or no reaction to light. 2

- Conjunctivae
a. Redness (Palpebral and bulbar)
Normal 0
Some blood vessels hyperaemic (injected) 1
Diffuse, crimson colour, individual vessels not easily discernible 2
Diffuse beefy red 3

b. Chemosis
Normal 0
Some 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 more than half 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, on considerable area around the eye 3
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
24 h
Score:
2
Reversibility:
fully reversible within: 14 d
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
48 h
Score:
1.33
Reversibility:
fully reversible
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
72 h
Score:
0.66
Reversibility:
fully reversible
Irritation parameter:
iris score
Basis:
mean
Time point:
24 h
Score:
0
Irritation parameter:
iris score
Basis:
mean
Time point:
48 h
Score:
0
Irritation parameter:
iris score
Basis:
mean
Time point:
72 h
Score:
0
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
24 h
Score:
1.66
Reversibility:
fully reversible within: 14 d
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
48 h
Score:
2
Reversibility:
fully reversible
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
72 h
Score:
2
Reversibility:
fully reversible
Irritation parameter:
chemosis score
Basis:
mean
Time point:
24 h
Score:
3
Reversibility:
fully reversible within: 14 d
Irritation parameter:
chemosis score
Basis:
mean
Time point:
48 h
Score:
3
Reversibility:
fully reversible
Irritation parameter:
chemosis score
Basis:
mean
Time point:
72 h
Score:
3
Reversibility:
fully reversible
Irritant / corrosive response data:
The animals’ individual mean scores (calculated as the mean scores following grading at 24, 48 and 72 hours after the treatment) were as follows:
- corneal opacity: 2.00, 1.33, 0.66
- iris: 0.00, 0.00, 0.00
- conjunctival redness: 1.66, 2.00, 2.00
- conjunctival chemosis: 3.00, 3.00, 2.00
- conjunctival discharge: 3.00, 3.00, 3.00
- 1h after the single application of test item into the eye of the rabbits, slight redness of the conjunctivae, slight to moderate chemosis of the conjunctivae, moderate to severe discharge of the conjunctivae and slight cornea opacity occurred.
- 2 weeks after treatment, all animals were free of symptoms.
Other effects:
LESIONS AND CLINICAL OBSERVATIONS:
- 1 hour after the single application of test item into the eye of the rabbits, slight redness of the conjunctivae, slight to moderate chemosis of the conjunctivae, moderate to severe discharge of the conjunctivae and slight cornea opacity occurred. The area of cornea involved was greater than half, but less than three quarters or was greater than one quarter, but less than half.
- 24 hour after the treatment the signs of eye irritation became stronger. Slight to moderate redness of the conjunctivae, moderate to severe chemosis of the conjunctivae, severe discharge of the conjunctivae and moderate cornea opacity were detected. The area of cornea involved was greater than three quarters, up to the whole area in one animal and the area of cornea involved was greater than one quarter, but less than half (score 2) in two animals.
- 1 week after the treatment one animal became free of symptoms. Slight redness of the conjunctivae, moderate to severe chemosis of the conjunctivae, severe discharge of the conjunctivae and moderate cornea opacity were observed. The area of cornea involved was greater than three quarters, up to the whole area.
- 2 weeks after treatment all animals were free of symptoms, so the study was finished.

- Other observations: No systemic toxicity was observed on the day of the treatment and during the 2-week observation period. The body weight of animals corresponded to their species and age. Signs of pain and distress as discharge was observed in all animals. These symptoms were detected between treatment day and Day 11, as well as on Day 13. During the study the control eyes of animals were symptom-free.

Table 1. Mean values of eye irritation scores for EU classification.

Animal No.

Sex

Cornea opacity

Iris

Conjunctivae

Redness

Chemosis

Discharge

00184

M

2.00

0.00

1.66

3.00

3.00

00190

M

1.33

0.00

2.00

3.00

3.00

00189

M

0.66

0.00

2.00

2.00

3.00

 

Table 2. Body weight data.

Animal No.

Beginning

of study

(g)

End

of study

(g)

Body weight

gain

(g)

00184

3630

3850

220

 

00190

3750

4098

348

 

00189

4100

4221

121

 

 

Table 3. Clinical symptoms.

Animal No.

Observations

Duration of Symptoms

Frequency

00184

Discharge

Day 0-11; Day 13

26

 

Free of symptoms

Day 12; Day 14

4

 

00190

Discharge

Day 0-6

14

 

Free of symptoms

Day 7-14

16

 

00189

Discharge

Day 0-5; Day 10

14

 

Free of symptoms

Days 6-9; Day 11-14

16

 

  

Table 4. Individual scores for ocular irritation.

Time after

treatment

Animal

No.

Conjunctivae

Opacity of cornea

Iris

Control eyes

Other signs

R

CH

D

OD

OE

R

1 h

00184

1

2

3

1

3

0

0

-

00190

1

2

2

1

2

0

0

-

00189

1

1

3

0

0

0

0

-

24 h

00184

1

3

3

2

4

0

0

-

00190

2

3

3

2

2

0

0

-

00189

2

2

3

2

2

0

0

-

48 h

00184

2

3

3

2

4

0

0

-

00190

2

3

3

2

3

0

0

-

00189

2

2

3

0

0

0

0

-

72 h

00184

2

3

3

2

4

0

0

-

00190

2

3

3

0

0

0

0

-

00189

2

2

3

0

0

0

0

-

1 week

00184

0

3

3

3

4

0

0

-

00190

0

0

0

0

0

0

0

-

00189

1

0

0

0

0

0

0

-

2 weeks

00184

0

0

0

0

0

0

0

-

00190

0

0

0

0

0

0

0

-

00189

0

0

0

0

0

0

0

-

 

 

Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Remarks:
EU criteria.
Conclusions:
The test item caused slight to severe conjunctival and moderate corneal irritant effects, fully reversible within 2 weeks. Based on the results, the test item is irritant to the eyes.
Executive summary:

An acute eye irritation study with the test item was performed in 3 male New Zealand White rabbits with normal healthy eye conditions. 0.1g of the test item were applied into the conjunctival sac of left eye of one rabbit. The untreated right eye served as control. The eyes were not washed out. As an irritant effect was observed, the confirmatory test was conducted in a sequential manner, using two further rabbits. The test item caused slight to severe conjunctival and moderate corneal irritant effects in all animals, all fully reversible within 2 weeks (at which point the study was terminated). The irritation effect of the test item was scoring according to the Draize method (Draize, 1959; OECD Guideline 405, 2012). Based on the results, the test item is irritant to the eyes.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

- Skin irritation: An in vitro skin irritation test of the test item in an EPISKIN-SM reconstructed human epidermis model, according to OECD TG 439 (GLP study) was available. Disks of EPISKIN (3 units / chemical) were treated with test item, SDS (5% aq., positive control) or PBS (negative control) and incubated for 15 minutes at room temperature. Exposure 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. All validity criteria were met. The mean cell viability was 59%, compared to the negative control. Therefore, the test item was not irritating to the skin.

- Eye irritation: An in vitro (ex vivo) study in order to determine the potential severe eye damaging effects of the test item according to the OECD guideline 438 (GLP study) was available. Eyeballs were isolated from chickens killed for human consumption and after the appropriate preparation were exposed to either the test item, imidazole (positive control) or saline (negative control). Three eyeballs were used for the test item and the positive control, one for the negative control. Fluorescein retention, corneal opacity and corneal swelling were evaluated, then the results of each endpoint were assigned to ICE classes according to the guideline recomendations. According to the overall in vitro classification, no prediction can be made since the combinations of the 3 endpoints were1xI, 1xII, 1xIII. Based on the results, the test item is not severely irritant to the eye.

An acute eye irritation study with the test item in 3 male New Zealand White rabbits with normal healthy eye conditions was available. 0.1g of the test item were applied into the conjunctival sac of left eye of one rabbit. The untreated right eye served as control. The eyes were not washed out. As an irritant effect was observed, the confirmatory test was conducted in a sequential manner, using two further rabbits. The test item caused slight to severe conjunctival and moderate corneal irritant effects in all animals, all fully reversible within 2 weeks (at which point the study was terminated). The irritation effect of the test item was scoring according to the Draize method (Draize, 1959; OECD Guideline 405, 2012). Based on the results, the test item is irritant to the eyes.

Justification for classification or non-classification

- Skin irritation: Based on available data (not irritant based on OECD 439 test), the test item is not classified for skin irritation according to CLP Regulation (EC) No. 1272/2008.


- Eye irritation: Based on the results on the OECD 405 test, the test item is classified as irritating to the eyes Category 2, H319 according to CLP Regulation (EC) No. 1272/2008.