Copper(2+), bis[N-[amino(imino-.kappa.N)methyl]urea-.kappa.O]-, nitrate (1:2)

Endpoint summary

Description of key information

- Skin irritation: moderately irritating in human skin model, OECD guideline 439, EU B.46 method, GLP. Mean relative viability was < 40% relative to the negative control in the MTT assay. 
- Eye irritation: Causes serious eye damage (severe corneal opacity) in rabbits, OECD guideline 405, EU B.5 method, GLP.
Means scores were: marked to severe chemosis (score 3.0), severe redness of the conjunctiva (score 3.0), iris lesions (score 1.0) and corneal opacity (score 2.7). No clinical signs indicative of systemic toxicity were observed in any animals.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 february 2012 to 26 june 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: study performed in accordance to OECD guideline 439

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

yes

Remarks:

during the preliminary test for the detection of colouring potential of the test item, an additional colour evaluation after 1 hour of incubation was performed. It was considered not to have compromised the validity or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Species:

other: human skin model

Strain:

other: EpiskinTM reconstituted human epidermis

Details on test animals or test system and environmental conditions:

TEST CELLS
- Source: SkinEthic Laboratories, Nice, FRANCE
- batch No. 12-EKIN-014
- Passage: second
- origin: adult donor
- quality control: histology scoring 21.7+/-0.3 (>or=19.5) and IC50 determination (SDS concetration, MTT test) 2.1 mg/ml (> or equal 1.5 mg/ml)
- expiration date: april 9, 2012

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 37°C
- Humidity (%): saturated
- CO2: 5%

EXPERIMENT DATES: From: march 27, 2012 To: april 6, 2012

Type of coverage:

not specified

Preparation of test site:

other: no preparation was needed because of in vitro test

Vehicle:

unchanged (no vehicle)

Remarks:

original form

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied to each tissue: 10 mg +/- 2 mg

Duration of treatment / exposure:

test item: 15 (+/-1) minutes
recovery time after treatment: 42 (+/- 1) hours

Observation period:

no observation period. MTT viability assay was assessed after the end of the recovery period

Number of animals:

each condition was assessed in triplicate tissue

Details on study design:

NEGATIVE CONTROL
- Phosphate-Buffered saline 1X
- Lot/batch no. (if required): prepared by CiToxLAB, 020412A
-dose: 10 µl applied to negative control tissue

POSITIVE CONTROL
- sodium dodecyl sulphate (SDS) as a 5% (w/v) aqueous solution
- Lot/batch no. (if required): Bio-Rad, 111011A
-dose: 10 µl applied to positive control tissue

REMOVAL OF TEST SUBSTANCE
- at the end of incubation period (15 minutes)
- Washing (if done): test item was removed from the well of treatment plate and rinced with D-PBS.

Irritation / corrosion parameter:

% tissue viability

Value:

16

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 15 min exposure + 42h recovery. Reversibility: other: not relevant: in vitro test. (migrated information)

Other effects / acceptance of results:

irritant
no other effect

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Remarks:

Migrated information

Conclusions:

CuGUN is considered to be irritant to skin as the mean relative viability was <50% relative to the negative control. According to these results, the classification is irritant (R38), category 2 (H315)

Executive summary:

The objective of this study was to evaluate the skin irritation potential of the test item,Copper Bis Guanylurea Nitrate (CuGUN),using the Episkin^TMreconstituted human epidermis model. The study design was based upon international guidelines (OECD Guideline No. 439 and Commission Regulation (EC) No. 640/2012, B.46) and the study was conducted in compliance with CIT’s standard operating procedures and the principles of Good Laboratory Practice.

 

Methods

Preliminary tests were performed to detect the ability of the test item to directly reduce MMT as well as its colouring potential.

Following the preliminary tests, the skin irritation potential of the test item was tested in the main test. The test item and both the negative and positive controls were applied topically on triplicate tissues and incubated at room temperature for 14 to 15 minutes. At the end of the treatment period, each tissue was rinsed with D-PBS and incubated for 41 hours and 55 minutes at, 5% CO₂in a humidified incubator. The cell viability was then assessed by means of the colourimetric MTT reduction assay. Relative viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (reference viability).

 

Results

Preliminary test

In the preliminary test, theMTT solution containing the test item was found blue when compared with the negative control. However according to the NSMTT% results of the CIT study No. 38756 TIC for the same test item, it was considered that the test item did not interact with MTT. The blue colouration of the MTT solution containing the test item was probably due to the intrinsic colour of the test item, consequently no additional controls were performed on water-killed tissues in parallel to the main test. The test item was found to have a colouring potential in the preliminary test as the water solution containing the test item was coloured. As a result, additional controls were used in parallel to the main test (two viable tissues were used: one was treated with the test item and the other with the negative control; these two tissues were not incubated with MTT)for the evaluation of the non specific OD.

Main test

The acceptance criteria for this test were:

.            the mean cOD of the three negative controls (PBS) was≥0.6 and the standard deviation value of the % viability was≤18%,

.            relative mean viability of the positive control (SDS) was ≤ 40% of the relative mean viability of the negative control and the standard deviation value of the % viability was ≤ 18%. All acceptance criteria for the negative and positive controls were fulfilled. The study was therefore considered to be valid. Following a 15 minutes exposure and a 42-hour recovery period, the true relative mean viability of the tissues treated with the test item was 16%.

 

Conclusion

 Under the experimental conditions of this study, the test item,Copper Bis Guanylurea Nitrate (CuGUN), is considered to be irritant to skin as the mean relative viability was<50%relative to the negative control (see the classification table below). According to these results, the classification of the test item is the following:

.            irritant R38 (EU classification); Category 2 (H315) (Regulation EC No. 1272/2008 (CLP Regulation)).

 

Criteria forin vitroclassification	Interpretation	Directive 67/548/EEC	Regulation EC No. 1272/2008 (CLP Regulation)
Mean relative viability is ≤ 50%	Irritant	Irritant (R38)	Category 2 (H315)
Mean relative viability is > 50%	Non-irritant	Not classified	No category

 

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 february 2012 to 26 june 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: study performed in accordance to OECD guideline 431

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

INVITTOX protocol No.118, Episkin TM skin corrosivity test published by ECVAM

Deviations:

yes

Remarks:

for the 3 minutes time exposure, the test item was applied for an exposure of 3 min 07 sec to 3 min 21 sec instead of 3 min +/- 5 sec. It is considered by the study director not to have compromised the viability or integrity of the study

GLP compliance:

yes (incl. QA statement)

Species:

other: human skin model

Strain:

other: EpiskinTM reconstituted human epidermis

Details on test animals or test system and environmental conditions:

TEST CELLS
- Source: SkinEthic Laboratories, Nice, FRANCE
- batch No. 12-EKIN-011
- Passage: second
- origin: adult donor
- quality control: histology scoring 21.6+/-0.2 (>or=19.5) and IC50 determination (SDS concetration, MTT test) 1.9 mg/ml (> or equal 1.5 mg/ml)
- expiration date: march 19, 2012

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 37°C
- Humidity (%): saturated
- CO2: 5%

EXPERIMENT DATES: From: february 21, 2012 To: march 15, 2012

Type of coverage:

not specified

Preparation of test site:

other: no preparation was needed because of in vitro test

Vehicle:

unchanged (no vehicle)

Remarks:

original form

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20 mg +/- 2 mg

Duration of treatment / exposure:

3 minutes, 1hour and 4 hours (test item)
4 hours (negative and positive controls)

Observation period:

no observation period. MTT viability assay was assessed immediately after the end of exposure and washing

Number of animals:

each condition was assessed in duplicate tissue

Details on study design:

NEGATIVE CONTROL
- NaCl 0.9%
- Lot/batch no. (if required): CDM Lavoisier-OF1961.

POSITIVE CONTROL
- glacial acetic acid
- Lot/batch no. (if required): Sigma-aldrich-SZBB1820V

REMOVAL OF TEST SUBSTANCE
- at the end of incubation period (3 min, 1 or 4 hours)
- Washing (if done): test item was removed from the well of treatment plate and rinced with D-PBS. Rinsing was achieved by gently filling and emptying each tissue insert 12 times with 2 mL D-PBS to gently remove any removal dosage form.

Irritation / corrosion parameter:

% tissue viability

Remarks:

3 min

Run / experiment:

Mean of duplicate tissue

Value:

68

Vehicle controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Remarks:

1 hour

Run / experiment:

Mean of duplicate tissue

Value:

58

Vehicle controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Remarks:

4 hours

Run / experiment:

Mean of duplicate tissue

Value:

40

Vehicle controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

no corrosive
no other effects

Interpretation of results:

not classified

Remarks:

Migrated information irritant in vitro test will be assessed Criteria used for interpretation of results: other: regulation 440/0028 (B.40bis) and INVITTOX protocol No.118, Episkin TM skin corrosivity test published by ECVAM

Conclusions:

non corrosive to the skin

Executive summary:

The objective of this study was to evaluate the corrosive potential of the test itemCopper Bis Guanylurea Nitrate (CuGUN), using the Episkin^TMreconstituted skin model.The study design was based on the following guidelines:OECD Guideline No. 431, 13th April 2004 andCommission Regulation (EC) No. 440/2008, B.40bis, 30 May 2008.

This study was conducted in compliance with CIT’s standard operating procedures and the principles of Good Laboratory Practices.

 

Method

 Preliminary tests were performed to detect the ability of the test item to directly reduce MMT.

Following the preliminary tests, the skin corrosive potential of the test item was tested in the main test on viable and water-killed tissues. The test item and both the negative and positive controls were applied topically on duplicate tissues and incubated at room temperatureas follows: positive and negative controls for 4 hours; test item for 3 minutes 21 seconds on viable tissues and 3 minutes 07 seconds on dead tissues, 1 hour on both viable and dead tissues and 4 hours for both viable and dead tissues.

At the end of the treatment period, each tissue was rinsed with D-PBS. The cell viability was then assessed by means of the colourimetric MTT reduction assay and a relative viability (%) calculated for each tissue relative to control tissues.

Results

 Preliminary test

In the preliminary test, thetest item was found to have direct MTT reducing properties. As a result, additional controls were performed on water-killed tissues in parallel to the main test (performed on viable tissues). The MTT‑reducing test item was applied to two water-killed tissues for each exposure time and two water‑killed tissues remained untreated.Based on the OD obtained with these control tissues, the NSMTT% was found negative, consequently these OD were not take into account in the final calculation of viability of the viable test item treated tissues.

 

Main test

The acceptance criteria for this test were:

-  the mean cOD of the negative control (0.9% NaCl solution) was between 0.115 and 0.400,

-  relative mean viability of the positive control (99.8% glacial acetic acid solution) was ≤ 20% of the relative mean viability of the negative control.

The acceptance criteria were fulfilled and the study was therefore considered to be valid. The blue discolouration of the viable test item-treated tissues following the 3-minute, 1-hour and 4-hour exposure periods was representative of viable tissue. The true relative mean viabilities of the test item-treated tissues were:

-  3-minute exposure: 68%,

- 1-hour exposure: 58%,

-   4-hour exposure: 40%.

Conclusion

 Under the experimental conditions of this study, the test item,Copper Bis Guanylurea Nitrate (CuGUN)(batch No. 40), tested in its original form, is considered to be non corrosive to the skin as the mean viabilities were > 35% relative to the negative control for the three exposure periods. According to these results, the classification of the test item is  non corrosive (Directive 67/548/EEC, UN 2009 and Regulation 1272/2008/EEC).

Reference 3

Endpoint:

skin irritation: in vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study need not be conducted because the available information indicates that the criteria are met for classification as corrosive to the skin or irritating to eyes

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

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:

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: CEGAV, Argenvilliers, France
- Age at study initiation: 2 to 4 months
- Weight at study initiation: mean body weight of 3358 g (range: 2710 g to 4005 g)
- Housing: The animals were individually housed in noryl cages (Tecniplast, 65.3 cm x 65.3 cm x 45 cm). The cages were suspended in batteries over trays with absorbant paper and each cage contained a platform and enrichment (dumbbell).
- Diet (e.g. ad libitum): All animals had free access to pelleted breeding diet "type 110C", batch No. 12102, (SAFE, Augy, France)
- Water (e.g. ad libitum): All animals had free access to tap water (filtered using a 0.22 µm filter) contained in bottles.
- Acclimation period: the animals were acclimated to the study conditions for a period of at least 5 days before treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 ± 3°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: From: 2012-12-11 To: 2012-12-20

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.1 g/animal
The full amount of 0.1 g was not administered to animal A30381 because its left conjunctival sac was not large enough to receive all the test item

Duration of treatment / exposure:

1 hour

Observation period (in vivo):

evaluation of ocular reaction: 1 hour, 24, 48 and 72 hours.
Since animal A30381 showed persistent ocular reactions at the 72-hour reading, the evaluation of ocular reactions was extended up to day 10.

Number of animals or in vitro replicates:

2

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Just after the 1-hour scoring, as the test item was not evacuated from the eye by the animal’s own physiological mechanisms; the eye was rinsed to remove any residual solid test item. Both eyes were rinsed with sterile isotonic saline solution (0.9% NaCl), under a volume and velocity of flow which not caused injury to the eyes.
- Time after start of exposure: Just after the 1-hour scoring

SCORING SYSTEM:
Chemosis (lids and/or nictitating membranes)
0: no swelling
1: any swelling above normal
2: obvious swelling with partial eversion of lids
3: swelling with lids about half-closed
4: swelling with lids more than half-closed

Redness (refers to the most severe reading for the palpebral and bulbar conjunctivae, excluding the cornea and iris)
0: blood vessels normal
1: some blood vessels hyperemic (injected)
2: diffuse, crimson color, individual vessels not easily discernible
3: diffuse, beefy red

Iris lesions
0: normal
1: markedly deepened rugae, congestion, swelling, moderate circum-corneal hyperemia or injection; any or a combination of any there findings, but iris still reacting to light (a sluggish reaction being considered positive)
2: no reaction to light, haemorrhage, gross destruction (any or all of these)

corneal lesions: Opacity (degree of intensity: area most dense taken for reading)
0: no ulceration or opacity
1: scattered or diffuse areas of opacity (other than slight dulling or normal lustre), details of iris clearly visible
2: easily discernible translucent area, details of iris slightly obscured
3: nacreous areas, no details of iris visible, size of pupil barely discernible
4: opaque cornea, iris not discernible through the opacity

corneal lesions: Area of opacity
1: one quarter (or less) but not zero
2: greater than one quarter, but less than a half
3: greater than one half, but less than three quarters
4: greater than three quarters, up to whole area.

TOOL USED TO ASSESS SCORE: hand-slit lamp / biomicroscope / fluorescein
From the 24-hour scoring, the presence of corneal opacification was examined under a UV lamp after instillation of one or two drops of 0.5% sodium fluorescein solution (a clear fluorescence is visible in the areas of opacification). As findings were noted at 24 hours, a fluoresceine examination was conducted at each subsequent scoring time-point until a negative response was obtained.

Irritation parameter:

chemosis score

Basis:

animal #1

Remarks:

rabbit A30381

Time point:

24/48/72 h

Score:

3

Max. score:

3

Reversibility:

not specified

Remarks:

animal A30381 was prematurely killed on day 10 of its observation period before the end of the ocular reactions reversibility

Irritation parameter:

conjunctivae score

Basis:

animal #1

Remarks:

rabbit A30381

Time point:

24/48/72 h

Score:

3

Max. score:

3

Reversibility:

not specified

Remarks:

animal A30381 was prematurely killed on day 10 of its observation period before the end of the ocular reactions reversibility

Irritation parameter:

iris score

Basis:

animal #1

Remarks:

rabbit A30381

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

not specified

Remarks:

animal A30381 was prematurely killed on day 10 of its observation period before the end of the ocular reactions reversibility

Irritation parameter:

cornea opacity score

Basis:

animal #1

Remarks:

rabbit A30381

Time point:

24/48/72 h

Score:

>= 2 - <= 3

Max. score:

3

Reversibility:

not specified

Remarks:

animal A30381 was prematurely killed on day 10 of its observation period before the end of the ocular reactions reversibility

Remarks on result:

other: mean 2.7

Irritation parameter:

cornea opacity score

Basis:

animal #2

Remarks:

rabbit A30382

Time point:

24/48 h

Score:

4

Max. score:

4

Reversibility:

not reversible

Remarks:

Animal A30382 was sacrificed on day 3 of the observation period for ethical reasons (serious eye damage)

Irritation parameter:

iris score

Basis:

animal #2

Remarks:

rabbit A30382

Time point:

24/48 h

Score:

1

Max. score:

1

Reversibility:

not specified

Remarks:

Animal A30382 was sacrificed on day 3 of the observation period for ethical reasons (serious eye damage)

Irritation parameter:

conjunctivae score

Basis:

animal #2

Remarks:

rabbit A30382

Time point:

24/48 h

Score:

3

Max. score:

3

Reversibility:

not specified

Remarks:

Animal A30382 was sacrificed on day 3 of the observation period for ethical reasons (serious eye damage)

Irritation parameter:

chemosis score

Basis:

animal #2

Remarks:

rabbit A30382

Time point:

24/48 h

Score:

>= 3 - <= 4

Max. score:

4

Reversibility:

not specified

Remarks:

Animal A30382 was sacrificed on day 3 of the observation period for ethical reasons (serious eye damage)

Irritant / corrosive response data:

In the left treated eye of animal A30381, marked chemosis and severe redness of the conjunctiva were observed from day 1 to day 5. Then, moderate chemosis and redness persisted up to day 7. Slight conjunctiva redness was still observed until day 9.
Iris lesions were noted from day 1 until premature sacrifice on day 10.
Marked corneal opacity was recorded on days 2 and 3; moderate then slight corneal opacity persisted up to premature sacrifice on day 10.
In addition, neovascularisation was noted from day 2 to day 9.
In the left treated eye of animal A30382, marked to severe chemosis, severe redness of the conjunctiva and iris lesions were observed from day 1 until sacrifice on day 3.
A severe opacity was observed on almost the whole area of the cornea on days 2 and 3.
In addition, neovascularisation and lacrimation were noted from day 1 or 2, respectively.
As severe corneal opacity was observed for 2 consecutive days on almost the whole area of this animal, no mean scores were calculated and the test item was considered as inducing serious eye damage

Other effects:

No clinical signs indicative of systemic toxicity were observed in any animals.
The body weight of the animals was unaffected by the test item treatment.
Animal A30382, showing a severe corneal opacity intensity persisting for 48 hours, was humanely sacrificed (see § Pathology) on the third day of the observation period and discarded without macroscopic post-mortem examination.
Therefore decision was also taken to sacrifice animal A30381 on the same day (i.e. on day 10 of its observation period). Due to severity of lesions noted in animal A30382, it was not appropriate to observe the reversibility of ocular reactions for animal A30381.

No ocular reactions were observed in the right untreated control eye.

As the test item is a blue powder, bluish discoloration was noted in the left treated eye of both animals on day 1. Neovascularisation was noted for rabbit A30381 from day 2 to day 9 and for rabbit A30382 from day 1. Lacrimation were noted for rabbit A30382 from day 2. The body weight of the animals was unaffected by the test item treatment. No clinical signs indicative of systemic toxicity were observed in any animals

Interpretation of results:

Category 1 (irreversible effects on the eye)

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the experimental conditions of this study, the test item, Copper Guanylurea Nitrate (CuGUN) (batch No. 40) was severely irritant when administered by ocular route to rabbits.
According to the criteria of CLP Regulation, the test item should be classified category 1 and assigned the signal word "danger" and the hazard statement "H318: causes serious eye damage".

Executive summary:

The objective of this study was to evaluate the potential irritant properties of the test item for the eye, Copper Guanylurea Nitrate (CuGUN), following a single administration to rabbits.

 

Methods

The test item, Copper Guanylurea Nitrate (CuGUN)(batch No. 40),was first administered to a single male New Zealand White rabbit (animal A30381).

As mean value from grading at 24, 48 and 72 hours after instillation was ≥ 2 for conjunctival edema (chemosis) and for conjunctival redness, ≥ 1 for iris lesions and for corneal opacity, the test item was administered in the left eye of a second animal (animal A30382).

 After administration to animal A30382, asserious eye damage was observed, no other animal was treated.

 The test item was administered inthe conjunctival sac of the left eye. The right eye remained untreated and served as control.

A quantity of 0.1 g/animal was used.

Just after the 1-hour scoring, both eyes were rinsed with a sterile isotonic saline solution (0.9% NaCl).

 Each animal was observed once a day for mortality and clinical signs.Ocular reactions were observed approximately 1 hour, 24, 48 and 72 hours after the administration and then daily until the sacrifice of animals, when applicable. The mean values of the scores for chemosis, redness of the conjunctiva, iris lesions and corneal opacity were calculated for animal A30381 only. Body weight was recorded on the day of treatment and at sacrifice.

At sacrifice, the animals were discarded without macroscopicpost‑mortemexamination.

 

Results

Animal A30382 was sacrificed on day 3 of the observation period for ethical reasons (serious eye damage).

Therefore, animal A30381 was prematurely killed on day 10 of its observation period before the end of the ocular reactions reversibility.

 No clinical signs indicative of systemic toxicity were observed in any animals.

 The body weight of the animals was unaffected by the test item treatment.

 In the left treated eye of animal A30381, marked chemosis and severe redness of the conjunctiva were observed from day 1 to day 5. Then, moderate chemosis and redness persisted up to day 7. Slight conjunctiva redness was still observed until day 9.

Iris lesions were noted from day 1 until premature sacrifice on day 10.

Marked corneal opacity was recorded on days 2 and 3; moderate then slight corneal opacity persisted up to premature sacrifice on day 10.

In addition, neovascularisation was noted from day 2 to day 9.

Mean scores calculated for animal A30381 over 24, 48 and 72 hours were as follows:

.         chemosis : 3.0; showing eye irritation,

.         redness of the conjunctiva: 3.0; showing eye irritation,

.         iris lesions: 1.0; showing eye irritation,

.         corneal opacity: 2.7; showing eye irritation.

In the left treated eye of animal A30382, marked to severe chemosis, severe redness of the conjunctiva and iris lesions were observed from day 1 until sacrifice on day 3.

A severe opacity was observed on almost the whole area of the cornea on days 2 and 3.

In addition, neovascularisation and lacrimation were noted from day 1 or 2, respectively.

As severe corneal opacity was observed for 2 consecutive days on almost the whole area of this animal, no mean scores were calculated and the test item was considered as inducing serious eye damage.

 

Conclusion

 Under the experimental conditions of this study, the test item, Copper Guanylurea Nitrate (CuGUN)(batch No. 40)was severely irritant when administered by ocular route to rabbits.

 According to the criteria of CLP Regulation,the test item should be classified category 1 and assigned the signal word"danger"and the hazard statement"H318: causes serious eye damage".

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6 march 2012 to 16 august 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: study performed in accordance to OECD guideline 437

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

no historical data for comparison to study results were available at the time of the study realization. Clear and unequivocal results were obtained with the 20% imidazole solution. It was considered to not have compromised the validity of the study

GLP compliance:

yes (incl. QA statement)

Species:

other: bovine cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Bovine eyes were obtained from freshly slaughtered cattle (EVA, Saint-Pierrre-sur-Dives, FRANCE)
Eyes were transported to CiToxLAB at ambient temperature, immerged in buffered Hanks medium containing an antibiotic solution
Corneas were mounted in corneal holders. Both chambers of the corneal holder were filled with complemented MEM culture media (cMEM) and pre-incubated for 1 hour at 32°C

Vehicle:

physiological saline

Remarks:

0.9% NaCl

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
-Concentration (if solution): 20% (w/v) in vehicle
-Dosage form application: a volume of 750 µL +/- 8 µL was applied on each cornea

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 µL
- Lot/batch no. (if required): Lavoisier-1A043

POSITIVE CONTROL
-20% imidazole solution in 0.9% NaCl (w/v)
-batch No.: Sigma-Aldrich-050M01771V (imidazole)
-amount applied: 750 µl on each cornea

Duration of treatment / exposure:

4 hours (+/-5 minutes)

Observation period (in vivo):

immediately after treatment: opacity measurement
after 90 minutes incubation with fluorescein: permeability measurement

Number of animals or in vitro replicates:

no animals
3 corneas per condition

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): the test item was removed from the front opening of the anterior chamber and the epithelium was rinsed with cMEM.
- Time after start of exposure: 4 hours

SCORING SYSTEM: in vitro irritancy score (IVIS) determined from the opacity and permeability measurement.
IVIS=corrected opacity + (15xcorrectedOD490nm)

TOOL USED TO ASSESS SCORE:
-opacity: an opacitometer was used to measure light transmission though the center of each cornea. opacity was measured before (OPT0) and after (OPT2) treatment. corrected opacity=OPT2-OPT0
-permeability: the medium of the anterior chamber was removed and filled with a 5 mg/mL fluoresceine solution. The holders were then incubated vertically at 32°C (+/-1°C) for 90 minutes (+/-5) minutes. At the end of the incubation, the optical density of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea. correctedOD490nm = OD490nm(test item) - OD490nm (vehicle)

Irritation parameter:

in vitro irritation score

Run / experiment:

3 corneas per condition

Value:

ca. 2.9

Vehicle controls validity:

valid

Remarks:

0.9% NaCl

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

20% imidazole solution in 0.9% NaCl (w/v)

Remarks on result:

no indication of irritation

Remarks:

IVIS < 55.1

Other effects / acceptance of results:

no corrosive or no severely irritant to the eye
Macroscopic examinations:
No notable opaque spots or irregularities were observed on vehicle corneas following the treatment.
Fluoresceine fixation was observed on the corneas treated with the test item following treatment.

Interpretation of results:

not classified

Remarks:

Migrated information IVIS<55.1 Criteria used for interpretation of results: other: Regulation EC 1152/2010 (B.47)

Conclusions:

According to OECD guideline 437, further testing should be conducted for classification and labeling purposes

Executive summary:

The objective of this study was to evaluate the potential corrosive or severe irritant properties of the test item on bovine eyes.

The study was performed according to the Guideline OECDincompliance with CIT’s standard operating proceduresand with the principles of Good Laboratory Practices.

 

Method

Corneas obtained from freshly slaughtered calves were mounted in corneal holders. Both chambers of the corneal holder were filled with complemented MEM culture media (cMEM) and pre-incubated for 1 hour at 32°C. Three corneas were used for each treated series (test item, positive control and vehicle control). Before the treatment, a first opacity measurement was performed using an opacitometer. The test item was tested at the concentrations of 20% (w/v) in the vehicle (0.9% NaCl), in a single experiment using a treatment time of 4 hours. At the completion of the treatment period, the test item was removed from the front opening of the anterior chamber and the epithelium was rinsed. A second opacity measurement was then performed. After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluoresceine solution. The holders were then incubated vertically for 90 minutes at. At the end of the incubation, the optical density of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea. Then the cornea was observed for opaque spots and other irregularities.

Results

No notable opaque spots or irregularities were observed on vehicle corneas following the treatment.

Fluoresceine fixation was observed on the corneas treated with the test item following treatment.

TheIn VitroIrritancy Score (IVIS) was: 2.9.

As the test item induced an IVIS < 55.1, it was not identified as ocular corrosive or severe irritant. 

Conclusion

Under the experimental conditions of this study, the test item, Copper Guanylurea Nitrate (CuGUN), is considered to be non corrosive to the eye. According to the OECD Guideline 437, further testing should be conducted for classification and labeling purposes.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Based on the available information, CuGUN was observed to be moderately irritating for skin in human skin model and corrosive for eyes in rabbits. No human information is available for this endpoint but there is no reason to believe that these results would not be applicable to humans.

No data are available for respiratory irritation. However, this potential hazard is taken into account for risk assessment even if CuGUN is not classified as respiratory irritant.

Justification for selection of skin irritation / corrosion endpoint:
The study was performed in accordance to OECD 439 and EC B.46 guideline, and to GLP. One deviation was observed during the preliminary test but it was considered not to have compromised the validity or integrity of the study.

Justification for selection of eye irritation endpoint:
The study was performed in accordance to OECD 405 and EC B.5 guideline, and to GLP. No deviation was observed.

Effects on skin irritation/corrosion: moderately irritating

Effects on eye irritation: corrosive

Justification for classification or non-classification

According to 1272/2008 regulation (CLP), the substance is classified as:

- Skin irritant, category 2, based on the mean relative viability to the negative control in the MTT assay that was < 50 %.

- Serious eyes damage, category 1, based on the irreversible corneal opacity.