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Description of key information

Skin and eye irritation studies were carried out following a tiered testing strategy: 
First, test item irritation/corrosion properties were assessed in the acute dermal toxicity study. No signs of irritation or corrosion were noted. Skin irritation properties were then confirmed in an in vivo study. The test item is considered slightly irritating to skin.
Eye irritation/corrosion was further assessed in an Isolated Chicken Eye Test (ICET) in vitro. The study revealed that the test item did not cause ocular corrosion or severe irritation in the enucleated chicken eyes and, moreover, the results suggest that the test item has no irritating potential. Eye irritation/corrosion potential was confirmed in an in vivo eye irritation study. The test item is considered slightly irritating to eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2500 (Acute Dermal Irritation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Toxi-Coop ZRT.
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: Adult rabbits
- Weight at study initiation: 2844 - 3085 g
- Housing: Animals were housed individually in metal cages.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3
- Humidity (%): 30 - 70
- Photoperiod: 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES:
- From: 15 Jan. 2013
- To: 19 Jan. 2013
Type of coverage:
semiocclusive
Preparation of test site:
shaved
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: The test item was used in pure state, in a single dose of 0.5 g test item
Duration of treatment / exposure:
In the first step an initial test was performed using one animal. One hour after application of the test item to the sentinel animal, the application site was examined. Severe irritation or corrosive effect was not observed in the initial test, therefore the bandage was replaced and the exposure continued for a further 3 hours (a total 4 hours exposure). 4 hours after the application the rest of the test item was removed using body temperature water. Two additional animals were treated comparably for this study.
Observation period:
Animals were examined for signs of erythema and oedema, and the responses scored at 60 minutes, 24, 48 and 72 hours after the patch removal.
Number of animals:
Three animals
Details on study design:
TEST SITE
- Area of exposure: Approximately 6 cm² area of intact skin
- Type of wrap if used: Sterile gauze pads (10 x10cm) were placed on the skin of rabbits. These gauze patches were kept in contact with the skin by a patch with a surrounding adhesive hypoallergenic plaster. The entire trunks of the animals were wrapped with plastic wrap.

REMOVAL OF TEST SUBSTANCE
- Washing: 4 hours after the application the rest of the test item was removed using body temperature water.
- Time after start of exposure: 4 hours

SCORING SYSTEM
- The dermal irritation scores were evaluated according to the scoring system by Draize (1959) shown in the Appendix III. The animals were observed for 72 hours, the duration of the study was sufficient to evaluate fully the reversibility or irreversibility of the effects observed.
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
other: 24, 48 and 72 hours after treatment
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
other: 24, 48 and 72 hours after treatment
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
erythema score
Basis:
animal #3
Time point:
other: 24, 48 and 72 hours after treatment
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
edema score
Basis:
animal #1
Time point:
other: 24, 48 and 72 hours after treatment
Score:
0
Max. score:
4
Irritation parameter:
edema score
Basis:
animal #2
Time point:
other: 24, 48 and 72 hours after treatment
Score:
0
Max. score:
4
Irritation parameter:
edema score
Basis:
animal #3
Time point:
other: 24, 48 and 72 hours after treatment
Score:
0
Max. score:
0
Irritant / corrosive response data:
1 hour after the patch removal well defined erythema (score 2) was observed in two animals (No.: 1, 2) and very slight erythema (score 1) occurred in animal No.: 3. Oedema was not observed in animals during the study.
24 hours after the patch removal well defined erythema (score 2) was recorded in animal No.: 1 and very slight erythema (score 1) was detected in two animals (No.: 2, 3).
48 hours after the patch removal very slight erythema (score 1) was observed in animal No.: 1. Two animals (No.: 2, 3) became free of symptoms.
72 hours after the treatment the study was terminated, since there were no primary irritation symptoms.
The animals’ individual mean scores (considering readings at 24, 48 and 72 hours after patch removal) were as follows:
- erythema: 1.00, 0.33, 0.33
- oedema: 0.00, 0.00, 0.00
There were no signs of corrosion. During the study the behaviour and general state of animals were normal. There were no notable body weight changes during the contact and observation period and no signs of systemic toxicity.
Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Test item treatment caused mild irritation. There were no signs of corrosion. The behavior and general state of animals was normal. No changes to body weight and no systemic toxicity were observed.
Executive summary:

An acute skin irritation study was performed in New Zealand White rabbits according to EU Method B.4, OECD Guideline 404 (Acute Dermal Irritation / Corrosion) and OPPTS 870.2500. The irritation effects of the test item were evaluated according to the Draize (1959) method.

The test item was administered in pure state, in a single dose of 0.5 g to the hairless skin of all experimental rabbits. The untreated skin of each animal served as control. After 4 hours the rest of the test item was removed with water of body temperature.

The animals were examined at 1, 24, 48 and 72 hours after the patch removal. 1 hour after the patch removal very slight to well defined erythema was observed in animals. 48 hours after the patch removal very slight erythema was found in one animal and two animals became free of symptoms. 72 hours after the treatment the study was terminated, since there were no primary irritation symptoms.

The animals’ individual mean scores (considering readings at 24, 48 and 72 hours after patch removal) were as follows:

- erythema: 1.00, 0.33, 0.33

- oedema: 0.00, 0.00, 0.00

There were no signs of corrosion. During the study the behaviour and general state of animals were normal. There were no notable body weight changes during the contact and observation period and no signs of systemic toxicity.

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

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Toxi-Coop ZRT.
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Age at study initiation: Adult rabbits
- Weight at study initiation: 3200 - 3430 g
- Housing: Animals were housed individually in metal cages
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 25 days for the first animal, 26 days for the second animal, 27 days for the third animal

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3
- Humidity (%): 30 - 70 %
- Photoperiod: 12 hours light, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES:
- From: 04 February 2013
To: 06 February 2013
Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 0.5 g
Duration of treatment / exposure:
The test item (finely ground) was placed into the conjunctival sac of left eye of each animal. The untreated right eye served as control. 0.1 g of the test item was used for the study in pure state, as a single dose. The eyes of the test animals were washed out 1 hour after test item application.
Observation period (in vivo):
Treated and control eyes were examined at 1, 24, 48 and 72 hours after the application.
Number of animals or in vitro replicates:
Three animals used sequentially.
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing: Yes
- Time after start of exposure: The eyes of the test animals were washed out with physiological saline solution (NaCl 0.9 %) 1 hour after test item application of, because the test item had not been removed from the eye of test animal by physiological mechanisms at the first observation time point.

SCORING SYSTEM
- The eye irritation scores were evaluated according to the scoring system by Draize (1979) and OECD 405 (02 October 2012)

TEST PROCEDURE
Three male animals in acceptable health condition were selected for the test. Care was taken to select only those animals with normal eye condition, any with ocular lesions were rejected.
In the first step an initial test was performed using one animal. The test item was placed into the conjunctival sac of the left eye. The eyelids were held closed gently for about one second to prevent any loss of the test item. The contralateral eye served as control.
In the initial test a slight irritant effect was observed 1 hour after treatment. The observed effect was fully reversible within 24 hours. The confirmatory test was conducted using a second animal. As in the first animal, slight irritant effects were observed 1 hour after treatment, but fully reversible within 24 hours. Following treatment of the second animal, the second confirmatory test was conducted using a third animal.
Again a slight irritant effect was observed 1 hour after the treatment. As in the first two animals effects were fully reversible within 24 hours.
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
other: 24, 48 and 72 hours
Score:
0
Max. score:
4
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
other: 24, 48 and 72 hours
Score:
0
Max. score:
4
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
other: 24, 48 and 72 hours
Score:
0
Max. score:
4
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
other: 24, 48 and 72 hours
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
animal #1
Time point:
other: 24, 48 and 72 hours
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
animal #2
Time point:
other: 24, 48 and 72 hours
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
animal #3
Time point:
other: 24, 48 and 72 hours
Score:
0
Max. score:
4
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
other: 24, 48 and 72 hours
Score:
0
Max. score:
4
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
other: 24, 48 and 72 hours
Score:
0
Max. score:
4
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
other: 24, 48 and 72 hours
Score:
0
Max. score:
4
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
other: 24, 48 and 72 hours
Score:
0
Max. score:
4
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
other: 24, 48 and 72 hours
Score:
0
Max. score:
4
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
other: 24, 48 and 72 hours
Score:
0
Max. score:
4
Irritant / corrosive response data:
Pre-existing test data
1. Determined physical-chemical properties and the chemical reactivity did not reveal potential corrosion/irritation effects to the eye.
2. A dermal toxicity test according to EU Method B.3 and OECD Guideline 402 (Acute Dermal Toxicity) did not reveal any systemic toxicity effects up to the limit dose (2000 mg/kg bw). Further, no corrosion or severe irritation was noted (see Toxi-Coop ZRT. Study no. 698.321.3773).
3. An in vivo skin irritation test according to EU Method B.4 and OECD Guideline 404 (Acute Dermal Irritation / Corrosion) did not reveal any corrosion or irritation effects (see Toxi-Coop ZRT. Study no. 698.550.4157).
4. In a preceding in vitro test according to EU Method B.48 and OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants), no ocular corrosion or severe irritation potential was observed (see Toxi-Coop ZRT. Study no. 698.549.3770).
Based on the information available further in vivo testing for eye irritation/corrosion was carried out.
In vivo eye irritation/corrosion data
Treated and control eyes were examined at 1, 24, 48 and 72 hours after the application.
One hour after the treatment hyperemic blood vessels (score 1) were observed in treated eyes of all animals.
24 hours after the treatment all animals were free of symptoms.
48 hours after the treatment all animals were free of symptoms.
72 hours after the treatment all animals were free of symptoms.
72 hours after the treatment the study was terminated, as no primary irritation symptoms were observed.
During the study the control eyes of animals were symptom-free.
Acute Eye Irritation Study of
Test Item LZ3547 in Rabbits page 19 of 51
General state and the behaviour of animals were normal throughout the study period. No systemic toxicity including clinical signs of pain and distress were observed on the day of the treatment and during the 72-hour observation period.
Other effects:
There were no notable body weight changes during the contact and observation periods.
Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In conclusion, the test item applied to the rabbits' eye mucosa caused slight conjunctival irritant effects after one hour, only. The slight redness observed was fully reversible within 24 hours. There were no signs of irritation observed 24, 48 and 72 hours.
Executive summary:

A study was carried out according to EU Method B.5, OECD Guideline 405 (Acute Eye Irritation / Corrosion) and OPPTS 870.2400. The testing of eye irritation/corrosion followed a tiered approach. Existing human and/or animal data, and/or in vitro data from validated and internationally accepted methods showing effects on eyes was assessed. All available information was evaluated to determine the need for in vivo eye testing.

Pre-existing data

Substance physical-chemical properties and chemical reactivity, a study on acute dermal toxicity according to EU Method B.3 and OECD Guideline 402 (Acute Dermal Toxicity) and in vivo dermal irritation testing according to EU Method B.4 and OECD Guideline 404 (Acute Dermal Irritation / Corrosion) revealed no systemic toxicity or local effects, including corrosion or severely irritating properties. In a preceding in vitro test using the Isolated Chicken Eye model according to EU Method B.48 and OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants), no ocular corrosion or severe irritation potential was observed either.

Based on the information available further in vivo testing for eye irritation/corrosion was carried out.

Eye irritation data

The acute eye irritation study with the test item was performed in New Zealand White rabbits. The irritation effect of the test item was evaluated according to the Draize method (Draize, 1959; OECD Guideline 405, 2012).

The test item (finely ground) was placed into the conjunctival sac of left eye of each animal. The untreated right eye served as control. 0.1 g of the test item was used for the study in pure state, as a single dose. The eyes of the test animals were washed out 1 hour after test item application.

Treated and control eyes were examined at 1, 24, 48 and 72 hours after the application.

1 hour after the single application of the test item into the eye of the rabbits, slight redness was noted.

24 hours after treatment all animals were free of symptoms.

72 hours after treatment the study was terminated, as all animals were symptom free without signs of corrosion or irritation observed.

During the study the control eyes of animals were symptom-free.

No systemic toxicity was observed. The general state and the behaviour of animals were normal throughout the study period. There were no notable body weight changes during the contact and observation periods.

In conclusion, the test item applied to the rabbits' eye mucosa caused slight conjunctival irritant effects. However, the slight redness observed was fully reversible within 24 hours.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Key study - Skin Irritation in vivo

An acute skin irritation study was performed in New Zealand White rabbits according to EU Method B.4, OECD Guideline 404 (Acute Dermal Irritation / Corrosion) and OPPTS 870.2500. The irritation effects of the test item were evaluated according to the Draize (1959) method.

The test item was administered in pure state, in a single dose of 0.5 g to the hairless skin of all experimental rabbits. The untreated skin of each animal served as control. After 4 hours the rest of the test item was removed with water of body temperature.

The animals were examined at 1, 24, 48 and 72 hours after the patch removal. 1 hour after the patch removal very slight to well defined erythema was observed in animals. 48 hours after the patch removal very slight erythema was found in one animal and two animals became free of symptoms. 72 hours after the treatment the study was terminated, since there were no primary irritation symptoms.

The animals’ individual mean scores (considering readings at 24, 48 and 72 hours after patch removal) were as follows:

- erythema: 1.00, 0.33, 0.33

- oedema: 0.00, 0.00, 0.00

There were no signs of corrosion. During the study the behaviour and general state of animals were normal. There were no notable body weight changes during the contact and observation period and no signs of systemic toxicity.

Key study - Eye irritation in vivo

A study was carried out according to EU Method B.5, OECD Guideline 405 (Acute Eye Irritation / Corrosion) and OPPTS 870.2400. The testing of eye irritation/corrosion followed a tiered approach. Existing human and/or animal data, and/or in vitro data from validated and internationally accepted methods showing effects on eyes was assessed. All available information was evaluated to determine the need for in vivo eye testing.

Pre-existing data

Substance physical-chemical properties and chemical reactivity, a study on acute dermal toxicity according to EU Method B.3 and OECD Guideline 402 (Acute Dermal Toxicity) and in vivo dermal irritation testing according to EU Method B.4 and OECD Guideline 404 (Acute Dermal Irritation / Corrosion) revealed no systemic toxicity or local effects, including corrosion or severely irritating properties. In a preceding in vitro test using the Isolated Chicken Eye model according to EU Method B.48 and OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants), no ocular corrosion or severe irritation potential was observed either.

Based on the information available further in vivo testing for eye irritation/corrosion was carried out.

Eye irritation data

The acute eye irritation study with the test item was performed in New Zealand White rabbits. The irritation effect of the test item was evaluated according to the Draize method (Draize, 1959; OECD Guideline 405, 2012).

The test item (finely ground) was placed into the conjunctival sac of left eye of each animal. The untreated right eye served as control. 0.1 g of the test item was used for the study in pure state, as a single dose. The eyes of the test animals were washed out 1 hour after test item application.

Treated and control eyes were examined at 1, 24, 48 and 72 hours after the application.

1 hour after the single application of the test item into the eye of the rabbits, slight redness was noted.

24 hours after treatment all animals were free of symptoms.

72 hours after treatment the study was terminated, as all animals were symptom free without signs of corrosion or irritation observed.

During the study the control eyes of animals were symptom-free.

No systemic toxicity was observed. The general state and the behaviour of animals were normal throughout the study period. There were no notable body weight changes during the contact and observation periods.

In conclusion, the test item applied to the rabbits' eye mucosa caused slight conjunctival irritant effects. However, the slight redness observed was fully reversible within 24 hours.

Supporting study - Eye irritation in vitro

A study was carried out according to EU Method B.48 and OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants). The purpose of this Isolated Chicken Eye Test (ICET) was to evaluate the potential ocular corrosivity or severe irritancy of the test item by its ability to induce toxicity in enucleated chicken eyes. Each eye used in this study was collected in the slaughterhouse, eliminating the need for laboratory animals. The test compound was applied in a single dose onto the cornea of isolated chicken eyes to assess the potential for causing ocular corrosion or irritation. Signs of corrosion or irritation include corneal swelling, opacity, reduced fluorescein retention and morphological changes. All parameters were evaluated pre-treatment and at approximately 30, 75, 120, 180, and 240 minutes after the post-treatment rinse. The endpoints evaluated were corneal swelling, opacity, fluorescein retention, and morphological effects. All endpoints, with the exception of fluorescein retention (which was determined only at pre-treatment and 30 minutes after test substance exposure), were determined at each of the above time points.

The test item and Imidazole (positive control) were ground before use in the study. Both substances were applied in a volume of 0.03 g/eye by powdering the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance or positive control. Three test item treated eyes and three positive control eyes were used in this study. One negative control eye was treated with 30 μL saline solution. After exposure periods of 10 seconds following application, the cornea surfaces were rinsed thoroughly with ca. 20 mL saline solution at ambient temperature. The procedure was repeated for each eye.

In this study the test item did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. Moreover, the results suggest that the test item has no irritating potential. Positive and negative controls showed expected results. The experiment was considered to be valid.


Effects on skin irritation/corrosion: slightly irritating

Effects on eye irritation: slightly irritating

Justification for classification or non-classification

Based on the data available the substance is not classified and labeled according to Regulation 1272/2008/EEC (CLP) and Directive 67/548/EEC (DSD).