Copper glycinate sulfate

Endpoint summary

Description of key information

Copper monoglycinate sulfate dihydrate is classified as Category 4 for acute oral toxicity according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS).

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

acute toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across hypothesis is based on transformation of the target and source substances to common compounds (scenario 1 of the RAAF). The target substance copper monoglycinate sulfate and the source substances copper sulfate and copper bisglycinate consist of the Cu2+ cation and the respective anion. The amino acid glycine is constituent of both the target substance copper monoglycinate sulfate and the source substance copper bisglycinate.
It is generally accepted that the Cu2+ cation (as measure for dissolved copper species) is the determining factor for toxicity and ecotoxicity, but not sulfate or glycine.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target substance copper monoglycinate sulfate is a chelate-complex which consists of the divalent copper ion as centre-ion and glycine as ligand. The remaining sulfate group stabilizes the center ion within the complex.
Copper monoglycinate sulfate and the source substance copper sulfate are ionic and consist of the Cu2+ cation and the respective anions. It is generally accepted that the copper cation is the determining factor for toxicity and ecotoxicity. Therefore, this read-across approach is based on the assumption that the metal cation of both the target and the source substance, copper, is the relevant component for assessment of toxicity and ecotoxicity.
The anion of the target substance is the essential amino acid glycine and the sulfate anion. In the source substance, it is the sulfate anion. These anions are not considered as (eco)toxicologically relevant at the given concentrations.
Please refer to the justification for read-across analogue approach in Chapter 13.2 for more detailed information.
3. ANALOGUE APPROACH JUSTIFICATION
Please refer to the justification for read-across analogue approach in Chapter 13.2 for more detailed information.
4. DATA MATRIX
Please refer to the justification for read-across analogue approach in Chapter 13.2 for more detailed information.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Sex:

male/female

Dose descriptor:

LD50

Effect level:

2 000 mg/kg bw

Based on:

test mat.

Remarks:

copper bisglycinate

Remarks on result:

other: registrant's study

Sex:

male/female

Dose descriptor:

LD50

Effect level:

2 558 mg/kg bw

Based on:

other: copper monoglycinate sulfate dihydrate

Remarks on result:

other: registrant's study

Sex:

male

Dose descriptor:

LD50

Effect level:

359.6 mg/kg bw

Based on:

element

Remarks:

Cu2+

Remarks on result:

other: Tang et al., 2018

Sex:

male

Dose descriptor:

LD50

Effect level:

1 532 mg/kg bw

Based on:

other: copper monoglycinate sulfate dihydrate

Remarks on result:

other: Tang et al., 2018

Sex:

male

Dose descriptor:

LD50

Effect level:

640 mg/kg bw

Based on:

element

Remarks:

Cu2+

Remarks on result:

other: Lee et al., 2016

Sex:

male

Dose descriptor:

LD50

Effect level:

2 726 mg/kg bw

Based on:

other: copper monoglycinate sulfate dihydrate

Remarks on result:

other: Lee et al., 2016

Sex:

female

Dose descriptor:

LD50

Effect level:

571 mg/kg bw

Based on:

element

Remarks:

Cu2+

Remarks on result:

other: Lee et al., 2016

Sex:

female

Dose descriptor:

LD50

Effect level:

2 432 mg/kg bw

Based on:

other: copper monoglycinate sulfate dihydrate

Remarks:

copper monoglycinate sulfate dihydrate

Remarks on result:

other: Lee et al., 2016

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

The test item is classified as Category 4 for acute oral toxicity according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS).

Executive summary:

In an acute oral toxicity study (OECD guideline 425), groups of male and female Sprague Dawley rats (8-12 weeks) were given a single oral dose of copper bisglycinate in water at doses of 175, 550, or 2000 mg/kg bw and observed for 14 days. No gross abnormalities in the internal organs were identified at the scheduled necropsy in experimental animals treated with a single intragastric dose of copper bisglycinate at 175 mg/kg, 550 mg/kg and 2000 mg/kg. The LD50 value for copper bisglycinate was 2000 mg/kg following single-dose intragastric administration to male and female rats.

In the study by Lee et al. (2016), the acute oral toxicity of copper ions (Cu²⁺) was examined in a study according to guideline OECD 420 and 423. Nine week-old female and male Sprague-Dawley rats were dosed with 156 mg/kg, 312 mg/kg, 625 mg/kg and 1250 mg/kg of copper ions and the concurrent vehicle via gavage. The dose levels were selected according to a preliminary study. The LD50 of Cu²⁺ was 571 mg/kg bw in female rats and 640 mg/kg bw in male rats, respectively.

In the study by Tang et al. (2018), the acute oral toxicity of copper ions was examined in a study according to guideline OECD 420. The LD50 of Cu²⁺ in male rats was reported to be 359.6 mg/kg bw.

According to the EU Voluntary Risk Assessment (2008), the most reliable LD50 values for copper sulphate pentahydrate in rats (rated Klimisch 1) were 482 mg/kg bw (male and female combined) and 666 mg/kg bw (females). However, the corresponding publications are not available, and it is unclear whether effect levels were determined based on the total fraction or on copper ions. For this reason, the effect levels reported in the EU Voluntary Risk Assessment have not been considered here for estimating the acute oral toxicity of the target substance.

Acute oral toxicity effects levels of source substances:

Data source	Source substance	LD50 females	LD50 males	Equivalent to copper monoglycinate sulfate dihydrate LD50:
Lee et al., 2016	Cu^2+	571 mg/kg bw	640 mg/kg bw	2432 mg/kg bw (females) 2726 mg/kg bw (males)
Tang et al., 2018	Cu^2+	-	359.6 mg/kg bw	1532 mg/kg bw (males)
Registrant’s study, 2019	Copper bisglycinate	2000 mg/kg bw	2000 mg/kg bw	2558 mg/kg bw (females and males)

These results substantiate the assumption that the presence of glycine in the chemical structure does not increase but lower the acute oral toxicity of copper and that Cu²⁺ is the toxicologically relevant component for assessing acute oral toxicity. It is generally accepted that the copper ion, but not glycine, is the compounds’ moiety which determines acute toxicity in vertebrates. A read-across approach referring to copper ions does thus represent a worst-case approach. Therefore, and for reasons of animal welfare, it is not considered necessary to conduct an acute oral toxicity study with the target substance copper monoglycinate sulfate.

Based on the calculation with the source substances copper bisglycinate and Cu²⁺ according to Lee et al. (2016), the target substance does not require classification according to Regulation (EC) No 1272/2008 (CLP), but classification as Category 5 for acute oral toxicity according to the Globally Harmonized System for Classification and Labelling of Chemicals (GHS). In contrast, the calculation based on the data provided by Tang et al. (2018) results in an LD50 of 1532 mg copper monoglycinate sulfate dihydrate/kg bw in male rats which leads to a classification as Category 4 for acute oral toxicity.

For precautionary reasons, the lowest LD50 was chosen und therefore, copper monoglycinate sulfate dihydrate is classified as Category 4 for acute oral toxicity according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS).

Reference 2

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-08-07 to 2019-09-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 425 (Acute Oral Toxicity: Up-and-Down Procedure)

Version / remarks:

Federal Law No. 61–FZ “On the Circulation of Medicinal Products” of 12 April 2010 (as amended);Federal Law No. 323–FZ “On the Basics of Health Protection of the Citizens in the Russian Federation” of 21 November 2011 (as amended)

GLP compliance:

yes

Test type:

up-and-down procedure

Limit test:

no

Species:

rat

Strain:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: NPO House of Pharmacy (inhouse bred)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8-12 weeks
- Fasting period before study: 16h
- Housing: individually in standard transparent plastic cages. Wood pellets were used as bedding.
- Diet (e.g. ad libitum): Feed for laboratory animals PK-120-1 prepared in accordance with GOST R50258-92 "Compound Feeds for Laboratory Animals. Specifications” was given ad libitum
- Water (e.g. ad libitum): purified water normalized in respect of organoleptic properties, pH, solids, reducing substances, carbon dioxide, nitrates and nitrites, ammonia, chlorides, sulphates, calcium and heavy metals in accordance with SanPiN 2.1.4.1074-01 "Drinking Water. Hygienic Requirements for the Quality of Water from Centralized Drinking Water Supply Systems. Quality Control". Water in standard drinking bowls with steel nose caps was given ad libitum.
- Acclimation period: 5 days
- Method of randomisation in assigning animals to test and control groups: Randomization was not expected in this study, since dosing occurred in stages and individually. The main criterion for including an animal in the experiment was its body weight.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26 °C
- Humidity (%): 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From:
No. 2.0-31.05/19 of 31 May 2019
No. 2.0-30.06/19 of 01 July 2019

Route of administration:

oral: gavage

Vehicle:

other: 1% starch solution

Details on oral exposure:

VEHICLE
- Concentration in vehicle: Before administration, suspensions of the test article was prepared with concentrations of ≈ 29.2 mg/mL for 175 mg/kg, ≈ 91.7 mg/mL for 550 mg/kg and 333.3 mg/mL for 2000 mg/kg.
- Amount of vehicle (if gavage): 1.5 mL per 250 g rat
- Lot/batch no. (if required): М-4.38/19

MAXIMUM DOSE VOLUME APPLIED: 333.3 mg/mL

CLASS METHOD
- Rationale for the selection of the starting dose:since the test article is presumably a low toxicity substance, 175 mg/kg was selected as the starting dose for a single intragastric administration to one male and one female rat. The following doses were tested in accordance with the described approach: 175 mg/kg, 550 mg/kg and 2000 mg/kg.
The test stopping criteria, according to OECD protocol No. 425, are as follows:
1. No deaths of 3 animals of the same sex, sequentially included in the experiment at the maximum dose of 2000 mg/kg
2. Recording 5 changes in directions of the "response/no response" parameter in 6 animals of the same sex sequentially included in the experiment, i. e. no death of 3 animals of the same sex that received the same dose, and deaths of 3 animals of the same sex that received the next dose in the selected sequence
3. Recording a change in the direction of the “response/no response” parameter in at least 4 animals after the first recorded change in the parameter direction
The test stopping criterion in this study was criterion No. 2: recording 5 changes in directions of the "response/no response" parameter in 6 males and females sequentially included in the experiment.

Doses:

175, 550, 2000 mg/kg bw

No. of animals per sex per dose:

175 mg/kg bw: 1male and 1 female animal; 550 mg/kg bw: 3 male and 4 female animals; 2000 mg/kg bw 5 male and 6 female animals

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: On day 1, 2, 7 and 15
- Necropsy of survivors performed: yes
- Clinical signs: daily
- Other examinations performed: other:Clinical examination on day 2, 7, 14 and local tolerance evaluation on day 15

Statistics:

Calculation of LD50 with confidence intervals was performed using the AOT 425 StatPgm program (Westat, USA).

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

act. ingr.

Mortality:

Deaths of the animals were recorded within 48 hours after administration (see table under 'Any other information on results incl. tables'). Delayed death was observed in female No. 21 treated with the test article at a dose of 2000 mg/kg on Day 6 of the experiment (not taken into account in Table under 'Any other information on results incl. tables'). By means of the specialized software AOT 425 StatPgm (Westat, USA) used in accordance with OECD Test No. 425, LD50 for the test article following intragastric administration to male and female rats was found to be 2000 mg/kg, with the 95% confidence interval being 793.2–2000.0 mg/kg (for males) and 734.0–2160.0 mg/kg (for females).

Clinical signs:

other: Throughout the study, the condition of all survived experimental animals was satisfactory, except for one female who received the test article at 550 mg/kg. In males and females that received the test article at 2000 mg/kg, depressed behaviour, dyspnoea,

Gross pathology:

No abnormalities were found at the injection site.

Other findings:

The muscle tone in all rats was moderate. The fur of the animals was smooth, shiny, without foci of alopecia.
The skin had no signs of irritation or inflammation. The skin turgor and integrity were within normal limits, palpable masses were absent.
Visible mucous membranes were pale pink, shiny, with intact integrity.
None of the animals had exophthalmos, swelling, or hyperaemia of the mucous membrane of the eyes. No lacrimation was observed.
The nasoscope was moderately moist, no abnormal discharge was observed. The local temperature of the ear skin was not elevated; no suppuration, inflammation, fouling during the entire observation period was observed in any animal. The teeth of all animals were intact. No salivation was observed.
No impairments of coordination of movement were observed. Respiration was normal in all experimental animals.
Defecation and urination were not impaired. No unusual behaviour was observed.
Thus, intoxication symptoms were recorded in all animals that received the test article at 2000 mg/kg. The animals that received the test article at doses of 175 mg/kg and 550 mg/kg exhibited no symptoms of intoxication.
Histological examination: The test article at 175 mg/kg (in 1 female) and at 2000 mg/kg (in 1 male) following single-dose intragastric administration had a moderate local irritant effect, which was manifested as catarrhal gastritis. The formation of ulcers was also noted in two males that received the test object at 2000 mg/kg.

Mortality of experimental animals following administration of the test article (number of dead animals/number of animals treated with the similar dose)

Products	Animal sex	Dose, mg/kg
		175	550	2000
Test article	Males	0/1	0/3	3/5
	Females	0/1	0/4	4/6

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

The LD50 value of the test substance is 2000 mg/kg following single-dose intragastric administration to male and female rats. According to Regulation (EU) No. 1272/2008 (CLP), the test item can be classified as toxicity category 4.

Executive summary:

In an acute oral toxicity study (OECD guideline 425), groups of male and female Sprague Dawley rats (8-12 weeks) were given a single oral dose of bis(glycinato)copper in water at doses of 175, 550, or 2000 mg/kg bw and observed for 14 days.

Over the entire experiment, deaths of animals treated with the test article at 2000 mg/kg were recorded: 3 males and 5 females died. The immediate cause of animal death was acute heart failure.

Clinical signs of intoxication were manifested as depressed behaviour, dyspnoea, diarrhoea, decreased muscle tone and response to stimuli in animals treated with the test article at 2000 mg/kg. The condition of animals following single-dose intragastric administration of the test article, bis(glycinato)copper, at 175 mg/kg and 500 mg/kg was satisfactory.

Single-dose intragastric administration of bis(glycinato)copper at 175 mg/kg, 550 mg/kg and 2000 mg/kg did not affect the body weights of the experimental animals. All animals experienced physiological weight gain.

No gross abnormalities in the internal organs were identified at the scheduled necropsy in experimental animals treated with a single intragastric dose of bis(glycinato)copper at 175 mg/kg, 550 mg/kg and 2000 mg/kg.

The test article, bis(glycinato)copper, at 175 mg/kg and 2000 mg/kg following single-dose intragastric administration had a moderate local irritant effect. The data obtained allow concluding that the NOAEL for bis(glycinato)copper is < 175 mg/kg following single-dose intragastric administration to rats.

Conclusion:

The LD50 value for bis(glycinato)copper is 2000 mg/kg following single-dose intragastric administration to male and female rats.

Reference 3

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

not reported

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method)

Version / remarks:

2001

Deviations:

yes

Remarks:

Male rats

Qualifier:

according to guideline

Guideline:

OECD Guideline 425 (Acute Oral Toxicity: Up-and-Down Procedure)

Version / remarks:

LD50 values of the test samples were calculated using the AOT425 program.

GLP compliance:

not specified

Remarks:

The publication does not contain information about GLP compliance.

Test type:

fixed dose procedure

Limit test:

no

Specific details on test material used for the study:

Copper ions (CuCl2 ·2H2O, lot: F1620012), manufactured by Aladdin Industrial Corporation (Shanghai, China).

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Chengdu Dossy Biological Technology Co., Ltd. (Chengdu, China)
- Rationale for use of males: not provided
- Age at study initiation: 9 weeks
- Weight at study initiation: 100 -120 g one week before start of study
- Housing: 3 animals per cage in specific pathogen-free conditions
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 1 week
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 – 24 °C
- Humidity (%): 30 – 70 %
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

methylcellulose

Remarks:

1% HPMC, Hydroxypropyl Methylcellulose (Shanghai Ryon Biological Technology Co. Ltd. Shanghai, China)

Details on oral exposure:

VEHICLE
- Amount of vehicle (if gavage): The administration volume (10 mL/kg body weight) was calculated based on the most recently recorded body weight of each animal.
- Justification for choice of vehicle: 1% HPMC was chosen as vehicle for the oral gavage, because apart from the test substance, Cu NPs have been tested which required 1% HPMC as a vehicle.

Doses:

200 mg/kg bw

No. of animals per sex per dose:

6

Control animals:

yes

Details on study design:

- Frequency of observations and weighing: Each animal was observed twice daily throughout the test period for any clinical signs of toxicity or mortality.
- Necropsy of survivors performed: yes
- Clinical signs including body weight: yes
- Other examinations performed: organ weights, histopathology, hematology, serum biochemistry

Statistics:

All statistical results were expressed as the mean ± standard deviation and all experimental values were compared with their corresponding control values. Differences between mean values were analyzed by SPSS 19.0 with one-way ANOVA, and differences with a P-value ≤0.05 were considered statistically significant.

Preliminary study:

Cu ions administered at a dose of 200 mg/kg caused symptoms of hematuria, paleness, piloerection, diarrhea and death. Therefore, the 200 mg/kg dose was selected for the main study.

Key result

Sex:

male

Dose descriptor:

LD50

Effect level:

359.6 mg/kg bw

Based on:

test mat.

Mortality:

Cu ions administered at a dose of 200 mg/kg caused acute death after 2 hours.

Clinical signs:

diarrhoea

other: Piloerection, hematuria, paleness

Body weight:

lower than 10% body weight loss

Gross pathology:

no findings reported

Other findings:

- Organ weights: decrease in lung and testes weight.
- Histopathology: kidney: renal tubular epithelial cells were slightly swollen.
- hematology: increased monocyte count, decrease in mean corpuscular hemoglobin concentration, increase in red blood cell volume distribution width.
- serum biochemistry: Total protein, globulin and glucose levels were decreased. Albumin/globulin, urea and creatine kinase levels were increased.

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

The LD50 of the test item was 359.6 mg /kg bw in male rats.

Executive summary:

In the study by Tang et al., 2018, the acute oral toxicity of copper ions (Cu²⁺) was examined in a study according to guideline OECD 420. Male rats were administered a dose of 200 mg/kg bw in 1 % HPMC per gavage once per day for 7 days. The dose level was chosen because it caused symptoms of hematuria, paleness, piloerection, diarrhea and death in a preliminary study. Organ weights were decreased in lung and testes. Histopathology revealed that renal tubular epithelial cells were slightly swollen. Hematology proved an increased monocyte count, decrease in mean corpuscular hemoglobin concentration and an increase in red blood cell volume distribution width. In the serum biochemistry analysis, total protein, globulin and glucose levels were decreased while albumin/globulin, urea and creatine kinase levels were increased.

The LD50 of Cu²⁺ in male rats was reported to be 359.6 mg/kg bw.

Reference 4

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

not reported

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method)

Version / remarks:

2001

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Version / remarks:

2001

GLP compliance:

not specified

Remarks:

The publication does not contain information about GLP compliance.

Test type:

fixed dose procedure

Limit test:

no

Specific details on test material used for the study:

Copper ions were released from copper(II)chloride (CAS no 10125-13-0; CuCl2, > 99.9% purity, molecular weight: 134.45 g/mol) purchased from Sigma-Aldrich Co. (St Louis, MO, USA).

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Samtako Co. (Osan, Republic of Korea).
- Females nulliparous and non-pregnant: not specified
- Age at study initiation: 9 weeks
- Weight at study initiation: males ca. 330 g, females ca. 230 g
- Housing: Two rats were housed in each stainless wire mesh cage.
- Diet: commercial rodent chow (Samyang Feed, Wonju, Korea) ad libitum
- Water: tap water sterilized by ultraviolet irradiation ad libitum
- Acclimation period: 1 week before experimental start
- Microbiological status when known: rats were obtained from a specific pathogen-free colony
- Method of randomisation in assigning animals to test and control groups: randomisation method is not described.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23°C ± 3°C
- Humidity (%): 50% ± 10%
- Air changes (per hr): 13–18
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

methylcellulose

Remarks:

1% HPMC, Hydroxypropyl Methylcellulose (Sigma-Aldrich Co.)

Details on oral exposure:

VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg bw
- Justification for choice of vehicle: 1% HPMC was chosen as vehicle for the oral gavage, because apart from the test substance, Cu NPs have been tested which required 1% HPMC as a vehicle.

Doses:

concurrent vehicle, 156 mg/kg, 312 mg/kg, 625 mg/kg and 1250 mg/kg of Cu ions

No. of animals per sex per dose:

5 of each sex

Control animals:

yes

Details on study design:

- Frequency of observations and weighing: Each animal was observed twice daily throughout test period for any clinical signs of toxicity or mortality. The body weight of each rat was measured on days 0, 1, 3, 7, and 14.
- Necropsy of survivors performed: yes
- Clinical signs including body weight: yes
- Other examinations performed: organ weights, histopathology, serum biochemistry

Statistics:

The numerical data were presented as mean ± SD. All statistical comparisons were analyzed by one-way analysis of variance followed by Dunnett’s multiple comparison test. A P-value of ,0.05 was considered as statistically significant. Statistical analyses were performed using the GraphPad InStat Version 3.0 (GraphPad Software, Inc., La Jolla, CA, USA).

Preliminary study:

The dose levels were selected based on the results of a preliminary study.

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

571 mg/kg bw

Based on:

test mat.

Key result

Sex:

male

Dose descriptor:

LD50

Effect level:

640 mg/kg bw

Based on:

test mat.

Mortality:

Dead females: 1 in the 312 mg/kg group, 1 in the 625 mg/kg group, 3 in the 1250 mg/kg group at the day after treatment.
Dead males: 1 in the 625 mg/kg group, 4 in the 1250 mg/kg group at the day after treatment.

Clinical signs:

diarrhoea

other: soft feces, piloerection, hematuria, depression, paleness, prone position, nasal discharge

Body weight:

lower than 10% body weight loss

Other findings:

- Organ weights: the relative weights of kidneys in male rats at 625 mg/kg increased significantly when compared with that of the control group. In female rats, the absolute and relative weights of the spleen in the group treated with at 625 mg/kg decreased significantly, whereas the relative weights of thymus and kidneys increased significantly when compared with those of the control group.
- Histopathology: mild inflammatory cell infiltration and dilated sinusoids in the liver, mild-to-moderate degree of inflammatory cell infiltration, hyaline cast/cell debris in tubules, dilated tubules, atrophy of glomeruli in the kidneys.
- Potential target organs: liver, kidney, spleen
- Serum biochemistry: CPK and LDH levels increased significantly, while TG levels decreased significantly in male rats in the 312 mg/kg group when compared with those of the control group. The serum levels of AST, alkaline phosphatase, CPK, and LDH in male rats and CPK and LDH levels in female rats increased significantly, whereas the TG levels in male rats decreased significantly in the 625 mg/kg group when compared with those of the control group.

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

The LD50 of the test material was 571 mg/kg bw in female and 640 mg/kg bw in male rats.

Executive summary:

In the study by Lee et al., 2016, the acute oral toxicity of copper ions (Cu²⁺) was examined in a study according to guideline OECD 420 and 423. Nine week-old female and male Sprague-Dawley rats were dosed with 156 mg/kg, 312 mg/kg, 625 mg/kg and 1250 mg/kg of copper ions and the concurrent vehicle, 1% hydroxypropylmethylcellulose, via gavage. The dose levels were selected according to a preliminary study.

The relative weights of kidneys in male rats at 625 mg/kg increased significantly when compared with that of the control group. In female rats, the absolute and relative weights of the spleen in the group treated with at 625 mg/kg decreased significantly, whereas the relative weights of thymus and kidneys increased significantly when compared with those of the control group.

Mild inflammatory cell infiltration and dilated sinusoids in the liver, mild-to-moderate degree of inflammatory cell infiltration, hyaline cast/cell debris in tubules, dilated tubules, atrophy of glomeruli in the kidneys.

CPK and LDH levels increased significantly, while TG levels decreased significantly in male rats in the 312 mg/kg group when compared with those of the control group. The serum levels of AST, alkaline phosphatase, CPK, and LDH in male rats and CPK and LDH levels in female rats increased significantly, whereas the TG levels in male rats decreased significantly in the 625 mg/kg group when compared with those of the control group.

The LD50 of Cu²⁺ was 571 mg/kg bw in female rats and 640 mg/kg bw in male rats, respectively.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

ca. 1 532 mg/kg bw

Quality of whole database:

Studies rated Klimisch 1 oder 2 have been considered for the read-across approach.

Acute toxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Acute toxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Acute toxicity data are available for copper in form of the copper ion Cu²⁺, which is released from copper sulfate upon solving, as well as copper bisglycinate and glycine. As source and target substances only differ in the number of glycine molecules in their chemical structure, it is assumed that the difference in composition does not contribute to the acute oral toxicity of the substance because glycine has been proven to be non-toxic via the oral route in rats (Shibui et al. 2013, Yakuri 1977).

The studies which have been chosen here for determining the acute oral toxicity of copper ions are no older than 2016, conducted according to OECD guidelines and considered the most reliable recent data. Apart from that, an acute oral toxicity study guideline study with the source substance copper bisglycinate is available. Furthermore, the comparably large database for copper and copper compounds has already been evaluated in an EU Voluntary Risk Assessment (2008).

In an acute oral toxicity study (OECD guideline 425), groups of male and female Sprague Dawley rats (8-12 weeks) were given a single oral dose of copper bisglycinate in water at doses of 175, 550, or 2000 mg/kg bw and observed for 14 days. No gross abnormalities in the internal organs were identified at the scheduled necropsy in experimental animals treated with a single intragastric dose of copper bisglycinate at 175 mg/kg, 550 mg/kg and 2000 mg/kg. The LD50 value for copper bisglycinate was 2000 mg/kg following single-dose intragastric administration to male and female rats.

In the study by Lee et al. (2016), the acute oral toxicity of copper ions (Cu²⁺) was examined in a study according to guideline OECD 420 and 423. Nine week-old female and male Sprague-Dawley rats were dosed with 156 mg/kg, 312 mg/kg, 625 mg/kg and 1250 mg/kg of copper ions and the concurrent vehicle via gavage. The dose levels were selected according to a preliminary study. The LD50 of Cu²⁺ was 571 mg/kg bw in female rats and 640 mg/kg bw in male rats, respectively.

In the study by Tang et al. (2018), the acute oral toxicity of copper ions was examined in a study according to guideline OECD 420. The LD50 of Cu²⁺ in male rats was reported to be 359.6 mg/kg bw.

According to the EU Voluntary Risk Assessment (2008), the most reliable LD50 values for copper sulphate pentahydrate in rats (rated Klimisch 1) were 482 mg/kg bw (male and female combined) and 666 mg/kg bw (females). However, the corresponding publications are not available, and it is unclear whether effect levels were determined based on the total fraction or on copper ions. For this reason, the effect levels reported in the EU Voluntary Risk Assessment have not been considered here for estimating the acute oral toxicity of the target substance.

Table 1: Acute oral toxicity effects levels of source substances.

Data source	Source substance	LD50 females	LD50 males	Equivalent to copper monoglycinate sulfate dihydrate LD50:
Lee et al., 2016	Cu^2+	571 mg/kg bw	640 mg/kg bw	2432 mg/kg bw (females) 2726 mg/kg bw (males)
Tang et al., 2018	Cu^2+	-	359.6 mg/kg bw	1532 mg/kg bw (males)
Registrant’s study, 2019	Copper bisglycinate	2000 mg/kg bw	2000 mg/kg bw	2558 mg/kg bw (females and males)

These results substantiate the assumption that the presence of glycine in the chemical structure does not increase but lower the acute oral toxicity of copper and that Cu²⁺ is the toxicologically relevant component for assessing acute oral toxicity. It is generally accepted that the copper ion, but not glycine, is the compounds’ moiety which determines acute toxicity in vertebrates. A read-across approach referring to copper ions does thus represent a worst-case approach. Therefore, and for reasons of animal welfare, it is not considered necessary to conduct an acute oral toxicity study with the target substance copper monoglycinate sulfate.

Based on the calculation with the source substances copper bisglycinate and Cu²⁺ according to Lee et al. (2016), the target substance does not require classification according to Regulation (EC) No 1272/2008 (CLP), but classification as Category 5 for acute oral toxicity according to the Globally Harmonized System for Classification and Labelling of Chemicals (GHS). In contrast, the calculation based on the data provided by Tang et al. (2018) results in an LD50 of 1532 mg copper monoglycinate sulfate dihydrate/kg bw in male rats which leads to a classification as Category 4 for acute oral toxicity.

For precautionary reasons, the lowest LD50 was chosen und therefore, copper monoglycinate sulfate dihydrate is classified as Category 4 for acute oral toxicity according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS).