Registration Dossier

The skin irritation potential of Copper glucoheptonate (CAS 1821694 -24 -9) was tested in vivo in rabbits. It was applied undiluted, semiocclusive for 4 h. It was reported to be not irritating (Patel, 2016A, acc. OECD 404).

The eye irritation potential of Copper glucoheptonate (CAS 1821694 -24 -9) was tested in vivo in rabbits. It was applied as 1 % dilution in distilled water. It was reported to be not irritating (Patel, 2016B, acc. OECD 405).

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 15, 2016 - March 10, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well documented GLP Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

yes

Remarks:

Exception to guideline 404, for not testing weight of evidence analysis by the test facility.

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Sainath Agencies, Hyderabad, India
- Age at study initiation: 4 to 5 months
- Weight at study initiation: Minimum: 2.406, Maximum: 2.560
- Housing: individually in stainless steel wire meshed cages were used.
- Diet (e.g. ad libitum): Teklad certified Global High Fiber Rabbit pellet Feed manufactured by Harlan, USA ad libitum
- Water (e.g. ad libitum): UV sterilised drinking water filtered through Kent Reverse Osmosis water filtration system ad libitum
- Acclimation period: 6 to 8 days

Animal Identification
Each rabbit was uniquely numbered on the ear using a tattoo machine. Appropriate labels were attached to the cages indicating the study number, test item code, sex, dose, type of study, cage number and animal number.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 22 °C
- Humidity (%): 63 to 65%
- Air changes (per hr): minimum 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 h artificial light and 12 h darkness, light hours being 06:00 - 18:00 h

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 500 mg
- Concentration (if solution): 1 %

Duration of treatment / exposure:

4 hours

Observation period:

The skin reactions of each rabbit were observed at 1, 24, 48 and 72 h post patch removal.

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: 6 cm²
- Type of wrap if used: gauze patch that was secured at the margins by non-irritating tape for a period of 4 h

REMOVAL OF TEST SUBSTANCE
- Washing (if done): At the end of the 4 h exposure period, the residual test item was removed with cotton soaked in distilled water.
- Time after start of exposure: 4 hours

SCORING SYSTEM:
Irritation was scored according to OECD 404.

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: 24

Score:

0

Reversibility:

other: not applicable

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: 48

Score:

0

Reversibility:

other: not applicable

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: 72

Score:

0

Reversibility:

other: not applicable

Irritation parameter:

edema score

Basis:

mean

Time point:

other: 24

Score:

0

Reversibility:

other: not applicable

Irritation parameter:

edema score

Basis:

mean

Time point:

other: 48

Score:

0

Reversibility:

other: not applicable

Irritation parameter:

edema score

Basis:

mean

Time point:

other: 72

Score:

0

Reversibility:

other: not applicable

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

other: 24, 48 and 72 h

Score:

0

Irritant / corrosive response data:

There were no signs of systemic adverse effect in any treated rabbits.
Erythema was evident at 1 h in all rabbits which resolved by 24 h post patch removal in all rabbits. The mean dermal irritation scores at 24, 48 and 72 h post-patch removal for individual animals were 0.00, 0.00, 0.00 for erythema and 0.00, 0.00, 0.00 for oedema, respectively.

Mean Dermal Irritation Score

The mean dermal irritation scores at 24, 48 and 72 h post-patch removal for animal Nº 1, 2 and 3 were 0.00, 0.00, 0.00 for erythema and 0.00, 0.00, 0.00 for oedema, respectively (TABLE 2).

Narrative Description of Skin Reactions

Following the 4 h exposure period (day 0), the skin of each rabbit was observed at 1, 24, 48 and 72 h post patch removal.

At 1 h post patch removal, the treated skin site revealed very slight erythema (barely perceptible) in all three rabbits (TABLE 1).

At 24 h post patch TIA, the treated skin site recovered completely and appeared normal in all three rabbits throughout the experimental period (TABLE 1).

The control skin sites of all three rabbits were normal with no erythema and no oedema observed throughout the experimental period (TABLE 1).

Clinical Observations other than Dermal Irritation

No clinical signs were observed in any rabbit throughout the experimental period (TABLE 2).

Interpretation of Results

The mean scores of erythema (0.00) and oedema (0.00) observed at the 24, 48 and 72 h post patch removal observation time-points indicated that the DABQUEL COMPLEX CuP is a non-irritant under the described experimental conditions.

Acute Dermal Irritation Study of DABQUEL COMPLEX CuP in Rabbits

TABLE1: Dermal Irritation Scores

Control Site: Untreated Sex: Male

Rabbit N°	Site of Application	Observations after Patch Removal
		Erythema				Oedema
		Hour				Hour
		1	24	48	72	1	24	48	72
1	Right	0	0	0	0	0	0	0	0
2	Right	0	0	0	0	0	0	0	0
3	Left	0	0	0	0	0	0	0	0

Treated Site: 500 mg DABQUEL COMPLEX CuP (pulverised and moistened with 0.5 mL distilled water)

Rabbit N°	Site of Application	Observations after Patch Removal
		Erythema				Oedema
		Hour	Hour
		1	24	48	72	1	24	48	72
1	Left	1	0	0	0	0	0	0	0
2	Left	1	0	0	0	0	0	0	0
3	Right	1	0	0	0	0	0	0	0

Note: Refer section 2.12 for Irritation Scores

Acute Dermal Irritation Study of DABQUEL COMPLEX CuP in Rabbits

Table 2: Clinical Observations and Body Weight (kg) of Individual Rabbit

Sex: Male

Rabbit N°	Clinical Observations made on Day				Body Weights (kg)
Rabbit N°	0	1	2	3	Before Treatment	At Termination
1	1	1	1	1	2.426	2.515
2	1	1	1	1	2.406	2.531
3	1	1	1	1	2.560	2.651

Key: 0 = Day of dermal application

Clinical Sign: 1 = Normal

Interpretation of results:

not irritating

Conclusions:

Based on the results of this study, and the Globally Harmonized System of Classification and Labelling of Chemicals (GHS 2015), DABQUEL COMPLEX CuP is not classified as a skin irritant

Executive summary:

In an acute dermal irritation study, three adult male New Zealand White rabbits were dermally exposed to 500 mg DABQUEL COMPLEX CuP (pulverised and moistened with 0.5 mL distilled water), for 4 h (day 0), applied to approximately 6 cm² area of skin. Initially one rabbit was tested with a single patch applied evenly to the intact skin for a period of 4 h. Based on the observations at 24 h post patch removal, two additional rabbits were tested simultaneously to confirm the irritation response. The control skin site of each rabbit was untreated. The treated and the control sites were covered with a gauze patch that was secured at the margins by non-irritating tape for a period of 4 h. At the end of the 4 h exposure period, the residual test item was removed with cotton soaked in distilled water. The skin reactions of each rabbit were observed at 1, 24, 48 and 72 h post patch removal. Irritation was scored according to OECD 404.

There were no signs of systemic adverse effect in any treated rabbits.

Erythema was evident at 1 h in all rabbits which resolved by 24 h post patch removal in all rabbits. The mean dermal irritation scores at 24, 48 and 72 h post-patch removal for individual animals were 0.00, 0.00, 0.00 for erythema and 0.00, 0.00, 0.00 for oedema, respectively.

Based on the results of this study, an indication of the classification for DABQUEL COMPLEX CuP is as follows:

Globally Harmonized System of Classification and Labeling of Chemicals (GHS 2015): Not classified as a skin irritant

Endpoint conclusion:

no adverse effect observed (not irritating)

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 15, 2016 - March 10, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well documented GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

yes

Remarks:

Exception to OECD guideline 405, for not testing weight of evidence analysis by the test facility.

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Sainath Agencies, Hyderabad, India
- Age at study initiation: 4 to 5 months
- Weight at study initiation: Minimum: 2.358, Maximum: 2.392
- Housing: individually in stainless steel wire meshed cages
- Diet (e.g. ad libitum): Teklad certified Global High Fiber Rabbit pellet Feed manufactured by Harlan, USA ad libitum
- Water (e.g. ad libitum): UV sterilised drinking water filtered through Kent Reverse Osmosis water filtration system ad libitum
- Acclimation period: 8 - 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 22 °C
- Humidity (%): 64 to 65%
- Air changes (per hr): Minimum 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 h artificial light and 12 h darkness, light hours being 06:00 - 18:00 h except light was kept ON at the time of the subcutaneous injection during night hours. Photoperiod was maintained through automatic timer.

Animal Identification
Each rabbit was serially numbered on the ear using a tattoo machine on day 1 of acclimatisation. Appropriate labels were attached to the cages indicating the study number, test item code, sex, dose, type of study, cage number and animal number.

Vehicle:

other: distilled water

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL
- Concentration (if solution): 1 % solution in distilled water

VEHICLE
- Amount(s) applied (volume or weight with unit): distilled water, test item solved in it
- Concentration (if solution): 1 %

Duration of treatment / exposure:

8 hours

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

3

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): with 0.9 % normal saline
- Time after start of exposure: 24 after treatment

SCORING SYSTEM: according to OECD 405

TOOL USED TO ASSESS SCORE: fluorescein

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

other: 24, 48 and 72 hours

Score:

0

Reversibility:

other: not applicable

Irritant / corrosive response data:

Conjunctival redness was evident at 1, 24 and 48 h in rabbit N° 1, which was resolved by 72 h and at 1 and 24 h in rabbit N° 2 and 3, which was resolved by 48 h post TIA. Conjunctival chemosis was evident at 1 and 24 h in rabbit N° 1, which was resolved by 48 h post TIA and at 1 h post TIA in rabbit N° 2 and 3, which was resolved by 24 h post TIA. The animal mean eye irritation scores of the 24, 48 and 72 h post-TIA observation were 0.00, 0.00, 0.00 for corneal opacity, 0.00, 0.00, 0.00 for iris effects, 0.67, 0.33, 0.33 for conjunctival redness and 0.33, 0.00, 0.00 for conjunctival chemosis for rabbit N° 1, 2 and 3, respectively (TABLE 1). Examination with fluorescein dye and cobalt blue filter at 24 h post TIA revealed no corneal epithelium damage in all rabbits.

Mean Eye Irritation Scores

The animal mean eye irritation scores observed for corneal opacity (0.00), iritis (0.00), conjunctival redness (0.33 to 0.67) and conjunctival chemosis (0.00 to 0.33) following grading at 24, 48 and 72 h post TIA.

Narrative Description of Eye Irritation

Treated Eye

At 1 h post-TIA, the treated eye of all three rabbits revealed conjunctival redness [some blood vessels definitely hyperaemic (injected); score of 1] and conjunctival chemosis [some swelling above normal (includes nictitating membranes); score of 1].

At 24 h post-TIA, the treated eye revealed conjunctival redness [some blood vessels definitely hyperaemic (injected) in all three rabbits; score of 1] and conjunctival chemosis [some swelling above normal (includes nictitating membranes) in rabbit N° 1, score of 1].

At 48 h post-TIA, the treated eye revealed conjunctival redness (some blood vessels definitely hyperaemic (injected) in rabbit N°1; score of 1) while rabbit N°2 and 3 recovered completely and appeared normal throughout the experimental period.

At 72 h post-TIA, the treated eye of rabbit N° 1 recovered completely and appeared normal.

Corneal opacity and iritis were not observed in any of the rabbits throughout the experimental period.

Examination with fluorescein dye and cobalt blue filter [corneal epithelium damage showing as green fluorescein staining] revealed no (area) corneal epithelium damage in all three rabbits at 24 h post TIA.

Control Eye

No abnormalities were detected in the control eye of each rabbit during the course of this study (Table 3). No disruption of corneal epithelium was observed during the examination with fluorescein dye and cobalt blue filter.

Clinical Observations other than Eye Irritation

Other than eye irritation, no signs of systemic toxicity including clinical observation and body weight were observed in the rabbits throughout the experimental period.

Interpretation of Results

Conjunctival redness was evident at 1, 24 and 48 h in rabbit N° 1 which was resolved by 72 h post-TIA and at 1 and 24 h in rabbit N° 2 and 3 which resolved by 48 h post TIA. Conjunctival chemosis was evident at 1 and 24 h in rabbit N° 1 which resolved by 24 h post-TIA. The animal mean eye irritation scores of the 24, 48 and 72 h post-TIA observation were 0.00, 0.00, 0.00 for corneal opacity, 0.00, 0.00, 0.00 for iris effects, 0.67, 0.33, 0.33 for conjunctival redness and 0.33, 0.00, 0.00 for conjunctival chemosis for rabbit N° 1, 2 and 3, respectively (TABLE 1). Examination with fluorescein dye and cobalt blue filter at 24 h post TIA revealed no corneal epithelium damage in all rabbits.

Conclusion

Based on the results of this study, an indication of the classification for DABQUEL COMPLEX CuP is as follows:

Globally Harmonized System of Classification and Labelling of Chemicals (GHS 2015): Not classified as an eye irritant

Acute Eye Irritation Study of DABQUEL COMPLEX CuP in Rabbits

TABLE1: Mean Eye Irritation Scores

Sex: Female

Rabbit N°	Mean Score at 24, 48 and 72 Hours
	Opacity: Degree of Density	Iris Lesion	Conjunctivae
	Opacity: Degree of Density	Iris Lesion	Redness	Chemosis
1	0.00	0.00	0.67	0.33
2	0.00	0.00	0.33	0.00
3	0.00	0.00	0.33	0.00

Acute Eye Irritation Study of DABQUEL COMPLEX CuP in Rabbits

Table 2: Clinical Observations (Non Ocular) and Body Weight (kg) of Individual Rabbit

A. Clinical Observation (Non Ocular)

Sex: Female

Rabbit N°	Observations made on Day
Rabbit N°	0	1	2	3
1	1	1	1	1
2	1	1	1	1
3	1	1	1	1

Key: 0 = Day of treatment

1 = Normal

B. Body Weight Record

Rabbit N°	Body Weight (kg)
Rabbit N°	Initial (Day 0)	Termination (72 h)
1	2.358	2.460
2	2.392	2.493
3	2.387	2.513

Acute Eye Irritation Study of DABQUEL COMPLEX CuP in Rabbits

Table 3 :Individual Scores of Eye Reactions Post Application

Control Eye

Sex: Female

Rabbit N°	1				2				3
Site of Application	Left				Left				Left
Reaction	Hour				Hour				Hour
Reaction	1	24	48	72	1	24	48	72	1	24	48	72
Opacity: Degree of Density	0	0	0	0	0	0	0	0	0	0	0	0
Iris	0	0	0	0	0	0	0	0	0	0	0	0
Conjunctivae (Redness)	0	0	0	0	0	0	0	0	0	0	0	0
Conjunctivae (Chemosis)	0	0	0	0	0	0	0	0	0	0	0	0

Treated Eye

Rabbit N°	1				2				3
Site of Application	Right				Right				Right
Reaction	Hour				Hour				Hour
Reaction	1	24	48	72	1	24	48	72	1	24	48	72
Opacity: Degree of Density	0	0	0	0	0	0	0	0	0	0	0	0
Iris	0	0	0	0	0	0	0	0	0	0	0	0
Conjunctivae (Redness)	1	1	1	0	1	1	1	0	1	1	0	0
Conjunctivae (Chemosis)	1	1	0	0	1	0	0	0	1	0	0	0

Acute Eye Irritation Study of DABQUEL COMPLEX CuP in Rabbits

Table 4: Individual Corneal Observations 24 h Post Application using Fluorescein Dye Staining

Control Eye

Sex: Female

Rabbit N°	Control Eye	Fluorescein Staining Response	Details of Corneal Damage Observed in Control Eye
1	Left	Negative	No corneal epithelium damage was observed.
2	Left	Negative	No corneal epithelium damage was observed.
3	Left	Negative	No corneal epithelium damage was observed.

Treated Eye

Rabbit N°	Treated Eye	Fluorescein Staining Response	Details of Corneal Damage Observed in Treated Eye
1	Right	Negative	No corneal epithelium damage was observed.
2	Right	Negative	No corneal epithelium damage was observed.
3	Right	Negative	No corneal epithelium damage was observed.

Acute Eye Irritation Study of DABQUEL COMPLEX CuP in Rabbits

Table 5: Details of Injections and Applications

Rabbit N°	On Days	Administration of Systemic Analgesics (Subcutaneous Injection)		Administration of Topical Anaesthetic (1 to 2 drops) 0.5% Proparacaine Hydrochloride	Time of Test Item Application (0.1 mL)
Rabbit N°	On Days	Buprenorphine Hydrochloride (0.01 mg/kg body weight)	Meloxicam (0.5 mg/kg body weight)		Time of Test Item Application (0.1 mL)
1	0	11:45 am & 8:49 pm	8:49 pm	12:40 pm	12:45 pm
	1	8:36 am and 8:53 pm	8:53 pm	-	-
	2	8:36 am and 8:51 pm	8:51 pm	-	-
	3	8:51 am	-	-	-
2 and 3	0	11:42 am to 11:43 am & 8:52 pm to 8:53 pm	8:52 pm to 8:53 pm	12:37 pm to 12:38 pm	12:42 pm to 12:43 pm
	1	8:53 am to 8:54 am and 8:40 pm to 8:41 pm	8:40 pm to 8:41 pm	-	-
	2	8:51 am to 8:52 pm	-	-	-

Key : - = Not applicable

Note: Day 0 = Day of treatment

Interpretation of results:

not irritating

Conclusions:

Examination with fluorescein dye and cobalt blue filter at 24 h post TIA revealed no corneal epithelium damage in all rabbits. Based on the results of this study, an indication of the classification for DABQUEL COMPLEX CuP is as follows:
Globally Harmonized System of Classification and Labelling of Chemicals (GHS 2015): Not classified as an eye irritant.

Executive summary:

In an acute eye irritation study, 3 adult male New Zealand White rabbits were given a single ocular application of 0.1 mL DABQUEL COMPLEX CuP in the right eye of the rabbit while the contralateral eye remained untreated and served as the control. Initially one rabbit was tested. Based on the results obtained at 24 h post Test Item Application (TIA), the irritation response was confirmed by testing two additional rabbits simultaneously. Observations were made at 1, 24, 48 and 72 h post TIA. General health status was also checked.

Conjunctival effects were evident at 1 h, 24 h and 48 h which resolved by 72 h post TIA. The animal mean eye irritation scores of the 24, 48 and 72 h post TIA observation were 0.00, 0.00, 0.00 for corneal opacity, 0.00, 0.00, 0.00 for iris effects, 0.67, 0.33, 0.33 for conjunctival redness and 0.33, 0.00, 0.00 for conjunctival chemosis for rabbit N° 1, 2 and 3, respectively.

Examination with fluorescein dye and cobalt blue filter at 24 h post TIA revealed no corneal epithelium damage in all rabbits.

The control eye did not show any abnormal reaction during the study. Moreover, there were no signs of systemic toxicity in any animal observed.

Based on the results of this study, an indication of the classification for DABQUEL COMPLEX CuP is as follows:

Globally Harmonized System of Classification and Labelling of Chemicals (GHS 2015): Not classified as an eye irritant.

Endpoint conclusion:

no adverse effect observed (not irritating)

Endpoint conclusion:

no adverse effect observed (not irritating)

Skin Irritation

Key study

The skin irritation potential of Copper glucoheptonate (CAS 1821694-24-9) was tested in rabbits (Patel, 2016A, acc. OECD 404). For this acute dermal irritation study, three adult male New Zealand White rabbits were dermally exposed to 500 mg copper glucoheptonate (pulverised and moistened with 0.5 mL distilled water, Copper content 12.5 %), for 4 h (day 0), applied to approximately 6 cm² area of skin. Initially one rabbit was tested with a single patch applied evenly to the intact skin for a period of 4 h. Based on the observations at 24 h post patch removal, two additional rabbits were tested simultaneously to confirm the irritation response. The control skin site of each rabbit was untreated. The treated and the control sites were covered with a gauze patch that was secured at the margins by non-irritating tape for a period of 4 h. At the end of the 4 h exposure period, the residual test item was removed with cotton soaked in distilled water. The skin reactions of each rabbit were observed at 1, 24, 48 and 72 h post patch removal. Irritation was scored according to OECD 404. As a result, there were no signs of systemic adverse effect in any treated rabbits. Erythema was evident at 1 h in all rabbits which resolved by 24 h post patch removal in all rabbits. The mean dermal irritation scores at 24, 48 and 72 h post-patch removal for individual animals were 0.00, 0.00, 0.00 for erythema and 0.00, 0.00, 0.00 for oedema, respectively.

Read-across approach

Copper glucoheptonate is a chelate compound consisting of two molecules of sugar like glucoheptonic acid and two copper ions. Data on structurally similar gluconic acid, its derivatives and organic and inorganic copper compounds have been taken into account to assess the irritating potential of copper glucoheptonate.

Gluconic acid and its derivatives

Primary dermal irritation tests with 0.5 mL of a 50% solution of gluconic acid (pH 1.8) in 12 albino rabbits demonstrated, that – as all the effects have cleared up after a 72 hours observation period – the test substance is not a dermal irritant (TNO, 1984, cited in SIDS, 2004).

The 2014 Cosmetic Ingredient Review Expert Panel acknowledged that the group of monosaccharides, disaccharides, and their related Ingredients, including calcium gluconate and gluconic acid, are safe for humans at concentrations as used in cosmetics. Based on the clinical experience of the Panel, there is little concern that these ingredients are irritants or sensitizers (CIR, 2014).

Organic and inorganic copper compounds

There are several unpublished animal study results provided for the preparation of a voluntary Risk Assessment Report (EU VRAR, 2007). Since Copper glucoheptonate is a water soluble copper compound, the results of the irritation studies with soluble and slightly soluble inorganic copper compounds i.e. copper sulphate pentahydrate and copper oxychloride have been taken into account. In guideline studies on dermal irritation, no signs of skin irritation or weak irritation of the skin were observed in animals treated with these compounds. The dermal irritation scores were under the cut-off values triggering C&L as skin irritant. The available data for copper sulphate pentahydrate and copper oxychloride do not meet the EU classification criteria requiring classification as irritant. There are no data available indicating that copper compounds are skin irritants in humans (EU VRAR, 2007). Similarly, a copper containing organic substance Tripeptide-Copper Complex Glycyl-L-Histidyl-L-Lysine-Cu2+ (CAS 89030-95-5) was also evaluated as safe for the use in cosmetics as a skin condition agent (CIR, 2014b). Moreover, copper showed wound healing effect in in vivo and in in vitro studies (Simeon et al., 1999; McCormack et al., 2001; please refer to robust study summary in the section "Skin sensitisation").

EFSA (Scientific Opinion, 2015) has evaluated the safety and efficacy of copper compounds (E4) (cupric acetate, monohydrate; basic cupric carbonate, monohydrate; cupric chloride, dihydrate; cupric oxide; cupric sulphate, pentahydrate; cupric chelate of amino acids, hydrate; cupric chelate of glycine, hydrate) as feed additives for all animal species. Copper salts can act as skin and eye irritants: skin eczema, conjunctivitis and even ulceration and turbidity of the cornea may occur in exposed workers (ACGIH, 1991, cited in EFSA, 2015). In the absence of specific studies and considering the toxicological profiles of copper compounds, the FEEDAP Panel retains that the compounds under evaluation should be considered as irritants to the skin and eye (EFSA, 2015).

There are conflicting results from animal studies and case reports mentioned in SCOEL (2013) and WHO (2002) reports on copper and its inorganic compounds. The skin irritation potential seems to depend on the anion (sulphate, chloride, carbonate etc.). The following statements have been found in the reports: "Dermal contact with copper salts may cause irritation to the skin, itching and erythema" in animals and humans (no further details) (WHO 2002); "Dermal application of metallic copper caused follicular reactions in guinea pigs (Greim, 2004, cited in SCOEL., 2013)". Copper monochloride (CAS 7758 -89 -6) was irritating in an acute dermal toxicity study in rats (SIDS, 2005). Necroses were observed after dermal exposure of mice to copper chloride in DMSO at concentrations ≥2.5 % (Basketter et al., 1999). On the other hand, "Copper sulphate probably is mildly or non-irritant to intact skin" is reported in WHO report (2002).

Eye irritation

Key study

The eye irritation potential of copper glucoheptonate (CAS 1821694-24-9) was tested in vivo in rabbits (Patel, 2016B, acc. OECD 405). For this acute eye irritation study, 3 adult male New Zealand White rabbits were given a single ocular application of 0.1 mL copper glucoheptonate (pulverised and moistened with distilled water, Copper content 12.5 %) in the right eye, while the contralateral eye remained untreated and served as the control. Initially one rabbit was tested. Based on the results obtained at 24 h post Test Item Application (TIA), the irritation response was confirmed by testing two additional rabbits simultaneously. Observations were made at 1, 24, 48 and 72 h post TIA. General health status was also checked.

As a result, conjunctival effects were evident at 1 h, 24 h and 48 h which resolved by 72 h post TIA. The animal mean eye irritation scores of the 24, 48 and 72 h post TIA observation were 0.00, 0.00, 0.00 for corneal opacity, 0.00, 0.00, 0.00 for iris effects, 0.67, 0.33, 0.33 for conjunctival redness and 0.33, 0.00, 0.00 for conjunctival chemosis for rabbit N° 1, 2 and 3, respectively. Examination with fluorescein dye and cobalt blue filter at 24 h post TIA revealed no corneal epithelium damage in all rabbits. The control eye did not show any abnormal reaction during the study. Moreover, there were no signs of systemic toxicity in any animal observed. Based on the results of this study, an indication of the classification for copper glucoheptonate is as follows: Globally Harmonized System of Classification and Labelling of Chemicals (GHS 2015): Not classified as an eye irritant.

Read-across

Gluconic acid and its derivatives

The eye irritation potential of the structurally similar analogues gluconic acid and its derivatives was evaluated by the 2014 Cosmetic Ingredient Review Expert Panel (CIR, 2014). The results of the following studies originate also from the OECD SIDS report (2004) on gluconate category.

In an in vitro study, the ocular irritation potential of a 50% aq. solution of gluconic acid was evaluated in vitro in enucleated rabbit eyes (TNO, 1984; cited in OECD SIDS, 2004). The test material was applied to four eyes and observed over a period of 4 h following application. Slight corneal swelling and slight permeability of the superficial epithelial cells were not considered to be of any toxicological significance.

In an in vivo study, a 50% aq. solution of gluconic acid was not irritating to rabbit eyes. A 50% solution of gluconic acid (pH 1.8; 0.1 ml) was instilled into the conjunctival sac of one eye in nine New Zealand white rabbits; the contralateral eye served as an untreated control. The eyes of three animals were rinsed after 2 sec, and of another three animals after 4 sec; the eyes of the remaining three animals were not rinsed. The eyes were examined for irritation 1, 24, 48, and 72 h and 7 days after instillation. Slight redness and conjunctival swelling were observed initially; however, no signs of irritation were observed after 72 h.

The Expert Panel concluded that monosaccharides, disaccharides, and their related Ingredients, including calcium gluconate and gluconic acid, are safe for humans at concentrations as used in cosmetics (CIR, 2014). The substances in this category are not irritating to eyes. The same conclusion is done in the OECD SIDS report for gluconates and the derivatives: "These compounds are neither irritant to the eye or the skin nor show sensitizing properties" (OECD SIDS, 2004).

Organic and inorganic copper compounds

Similarly to skin irritation potential of inorganic copper compounds, the grade of eye irritation depends on anion and the oxidation state of copper. A number of unpublished animal studies have been made available which have investigated eye irritant properties of several copper compounds and copper powder (EU VRAR, 2007). It is reported, that Copper (I) oxide caused eye irritation in rabbits in a number of tests, mostly of mild to moderate severity and is proposed to be classified as eye irritant. Copper sulphate pentahydrate caused fairly severe and persistent eye irritation in rabbits. In contrast, Copper (II) oxide, copper oxychloride and copper powder all caused mild and reversible eye irritation in rabbits; the data do not meet the criteria requiring these substances to be classified as irritant.

Other literature sources report that contact of copper salts with the eye may lead to conjunctivitis, ulceration, turbidity of the cornea and adhesion of the eyelids to the eye (SCOEL, 2013; WHO, 2002). Eye irritation has been reported by workers exposed to copper dust (ATSDR, 2004; Askergren and Mellgren, 1975).

EFSA (Scientific Opinion, 2015) has evaluated the safety and efficacy of copper compounds (E4) (cupric acetate, monohydrate; basic cupric carbonate, monohydrate; cupric chloride, dihydrate; cupric oxide; cupric sulphate, pentahydrate; cupric chelate of amino acids, hydrate; cupric chelate of glycine, hydrate) as feed additives for all animal species. Copper salts can act as skin and eye irritants: skin eczema, conjunctivitis and even ulceration and turbidity of the cornea may occur in exposed workers (ACGIH, 1991, cited in EFSA, 2015). In the absence of specific studies and considering the toxicological profiles of copper compounds, the FEEDAP Panel retains that the compounds under evaluation should be considered as irritants to the skin and eye (EFSA, 2015).

Respiratory irritation

Cases of irritation of the respiratory tract after inhalation of copper containing compounds are reported in occupationally exposed workers, in unintentionally exposed persons and in animal inhalation studies (SCOEL, 2013; ATSDR, 2004; WHO, 2002).

In Scientific Opinion of EFSA (2015) on the safety and efficacy of copper compounds (E4) (cupric acetate, monohydrate; basic cupric carbonate, monohydrate; cupric chloride, dihydrate; cupric oxide; cupric sulphate, pentahydrate; cupric chelate of amino acids, hydrate; cupric chelate of glycine, hydrate) as feed additives for all animal species, it was concluded that these substances should be considered as hazardous by inhalation. Since the dusting potential of these additives - except the cupric chloride, dihydrate - is high (209 to 11800 mg/m³), the estimates of copper exposure resulting from the amount of copper in dust would lead to an exceedance of the OEL for copper (0.01 mg/m³ respirable fraction) by several orders of magnitude (EFSA, 2015).

Based on the results of the available in vivo key study (Patel, 2016a,b), Copper glucoheptonate (CAS 1821694 -24 -9) does not meet criteria for classification and labelling as a skin or eye irritant according to European Regulation (EC) No. 1272/2008.

Based on the available medical and scientific literature data, most of the organic and inorganic copper compounds were irritaing to skin and eyes and caused respiratory irritation. Structurally similar gluconates and their derivatives are assessed to be not irritating to skin and eyes. Since copper glucoheptonate is a water soluble compound, attention was given to the test results of soluble copper compounds. Insoluble copper compounds do not possess irritating properties or are weakly irritating. In contrast, well soluble copper compounds are irritating to skin and severe irritating to eyes. However, although Copper glucoheptonate is a soluble compound it was not irritating to eyes of rabbits in the in vivo study.

Since Copper glucoheptonate has a respirable particle fraction (9.86 %) under 100 µm (please refer to granulometry study; Dabeer, 2014), theoretically, a respiratory irritating hazard by inhalation cannot be ruled out. However, in the absence of dusting potential of copper glucoheptonate, and in the absence of cases of respiratory irritation to this product in humans, no conclusion can be made on the relevance of this fraction for respiratory irritation. Therefore, Copper glucoheptonate does not need to be classified and labelled as respiratory irritant taking into account the provisions laid down in Council Directive 67/548/EEC and CLP Regulation (EC) No 1272/2008.