Copper glycinate sulfate

Acute Toxicity: oral

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Bis(glycinato)copper

EC Number:

236-783-2

EC Name:

Bis(glycinato)copper

Cas Number:

13479-54-4

Molecular formula:

C4H8CuN2O4

IUPAC Name:

copper;2-aminoacetate

Test material form:

solid: granular

Details on test material:

Substance Name: Copper bisglycinate
Chemical Name(s): Bis(glycinato)copper(II) monohydrate, copper aminoacetate
CAS number n. a. (anhydrous compound: 13479‐54‐4)
Chemical formula C4 H10 Cu N2 O5
Molecular weight: 229,68 g/mol
Storage: stored in a cool and dry place in original packaging
Physical state (20°C): solid
Odour: odourless
Colour: grey -blueish

Test animals

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

Administration / exposure

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).

Results and discussion

Effect levels

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.