Zinc EDDHA

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1995-04-06 to 1995-06-28

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Acetic acid, oxo-, sodium salt, reaction products with ethylenediamine and phenol, iron sodium salts

EC Number:

283-044-5

EC Name:

Acetic acid, oxo-, sodium salt, reaction products with ethylenediamine and phenol, iron sodium salts

Cas Number:

84539-55-9

Molecular formula:

non specified (UVCB substance)

IUPAC Name:

Acetic acid, oxo-, sodium salt, reaction products with ethylenediamine and phenol, iron sodium salts

Test material form:

other: microgranules

Details on test material:

- Test article: CGA65047 SG 100 (A-5787 A)
- Additional specification: Sesquestrene 138 Fe 100 SG
- Physical state: granules
- Analytical purity: 100 % (UVCB), 8.18% (total iron)
- Storage condition of test material: room temperature

Test animals

Species:

rat

Strain:

other: Sprague-Dawley derived; Tif:RAIf (SPF); hybrids of RII/1 x RII/2 (Sprague-Dawley derived)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Animal Production, CIBA-GEIGY Limited, 4332 Stein, Switzerland
- Age at start of acclimation period: approximately 5 weeks old
- Weight range at acclimation period: 124.8-155.9 g (males), 126.8-148.0 g (females)
- Housing: in groups of 5 animals, in macrolon cages type 4 with wire mesh tops and standardised granulated soft wood bedding
- Diet: pelleted, certified standard diet (Nafag No. 8900 FOR GLP), provided ad libitum (exception: food was withheld overnight prior to blood removal performed at the end of the treatment period)
- Water: tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 55 ± 10 %
- Air changes: 16-20 air changes/hour
- Photoperiod: 12 hours dark / 12 hours light

IN-LIFE DATES: From: 22-May-1995 To: 28-Jun-1995

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % CMC and 0.1 % Tween 80 in distilled water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Suspensions of the test article in the vehicle at the appropriate concentrations were freshly prepared every day immediately prior to the dosing of the animals and administered within about 2 hours.

VEHICLE
- Justification for use and choice of vehicle: standard procedure
- Amount of vehicle: 10 mL/kg bw of suspension were applied

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to the beginning of the study, suspensions containing the test article at concentrations of 0.1, 1, 10 and 100 mg/mL were analyzed for determination of content, homogeneity and stability. Control analyses were performed once per experimental week. Samples were analysed by RCC Umweltchemie AG, 4452 Itingen, Switzerland (RCC Project Number 393322).
The results of these analyses showed that the contents of CGA 65047 SG 100, (A-5787 A) in the vehicle were in agreement with the nominal concentrations and that the samples were homogeneous and stable for 4 hours at room temperature.

Duration of treatment / exposure:

28 days

Frequency of treatment:

once per day, 7 times per week

Doses / concentrations

Remarks:

Doses / Concentrations:
50, 200 and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

5 animals

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
50 mg/kg bw/day: this dose level was expected to cause no observable effects.
200 mg/kg bw/day: this dose level was expected to cause slight effects, if any.
1000 mg/kg bw/day: this dose level was considered to cause observable effects but no or few fatalities to permit a meaningful evaluation of the study.
- Rationale for animal assignment (if not random): randomised (by means of computer-generated random numbers).

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
- Time schedule: twice daily for mortality and once daily for general observations.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: at least weekly

BODY WEIGHT:
- Time schedule: once weekly

FOOD CONSUMPTION:
- Food consumption for each animal was determined weekly and mean daily diet consumption calculated as g food/kg bw/day.

WATER CONSUMPTION
- Time schedule for examinations: once weekly

HAEMATOLOGY:
- Time schedule for collection of blood: once, at the end of the treatment period
- Anaesthetic used for blood collection: ether
- Animals fasted: food was withheld overnight prior to blood sampling
- How many animals: all animals
- Parameters examined: erythrocyte count, haematocrit, mean corpuscular volume, red cell volume distribution width, haemoglobin concentration, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, haemoglobin concentration distribution width, prothrombin time and leukocyte, neutrophil, eosinophil, basophil, lymphocyte, monocyte, large unstained cells, reticulocyte and thrombocyte counts

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: once, at the end of the treatment period
- Animals fasted: food was withheld overnight prior to blood sampling
- How many animals: all animals
- Parameters examined: glucose, urea, creatinine, total bilirubin, total protein, albumin, globulin, cholesterol, sodium, potassium, calcium, chloride and inorganic phosphorus concentration, and A/G ratio and aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase activity

URINALYSIS:
- Time schedule for collection of urine: once, at the end of the treatment period
- Metabolism cages were used for collection of urine
- Animals fasted: food was withheld overnight prior to blood sampling
- Parameters examined: urine volume, relative density, pH-value, urine colour and protein, glucose, ketone, bilirubin, blood and urobilinogen content

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY:
The following organs or tissues were examined microscopically:
spleen, lung, heart, liver, kidneys, testis, epididymides, ovary, adrenal gland, mesenteric lymph node, thymus and any organ with gross lesions

Other examinations:

ORGAN WEIGHT
The weights of the following organs were determined at necropsy:
brain, heart, liver, kidneys, adrenals, thymus, ovaries or testes, and spleen

Statistics:

Each treated group was compared to the control group by Wilcoxon's two-sample test (non-parametric) and tested for increasing or decreasing trends from control up to the respective dose group by Jonckheere's test for ordered alternatives (parametric).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no test item related clinical findings. No deaths occurred.

BODY WEIGHT AND WEIGHT GAIN
Mean body weight was reduced and there was a mean body weight gain depression at 200 and 1000 mg/kg bw/day.
Compared to the control group, the body weight development of animals in groups 3 and 4 (200 and 1000 mg/kg) was impaired. At week 4, the mean weights of group 4 (1000 mg/kg bw) were 25% (males) and 22% (females), and the mean body weight gains (week -1 to week 4) 44% (males) and 54% (females) below that of the control group, respectively. The mean body weights of group 3 (200 mg/kg bw) were 6% (males) and 11% (females), and the mean body weight gains 11% (males) and 27% (females) below that of the control group, respectively. Body weight development in group 2 (50 mg/kg bw) was not affected by treatment.

FOOD CONSUMPTION
Food consumption was decreased at 200 and 1000 mg/kg bw/day:
"Overall food consumption (week 1 to 4) was reduced by 29% in males and 26% in females of group 4 (1000 mg/kg bw), and by 10% in males and 8% in females of group 3 (200 mg/kg bw)".

WATER CONSUMPTION
The mean water consumption was markedly increased at 1000 mg/kg bw/day.

HAEMATOLOGY
Anaemia was observed at 200 and 1000 mg/kg bw/day: "It was accompanied by a slight hyperchromasia of red blood cells. The release of reticulocytes from erythropoietic organs was not increased, suggesting a lack of an adaptive response. Furthermore, males of group 4 (1000 mg/kg bw) had slightly lower values of white blood cells, predominantly of lymphocytes and basophils. In addition, males of groups 3 and 4 (200 mg/kg bw and 1000 mg/kg bw) had minimally higher platelet counts. Slightly lower values of prothrombin time, as recorded for males of group 2 (50 mg/kg), and for animals of groups 3 and 4 (200 and 1000 mg/kg bw) are considered without toxicological relevance".

CLINICAL CHEMISTRY
Minimal or slight changes in several clinical chemistry parameters were noted at 1000 mg/kg bw/day. Minimally increased creatinine concentrations were observed in males at 200 mg/kg bw/day:
"Plasma creatinine concentrations were minimally increased in males of group 3 (200 mg/kg) and slightly increased in males and females of group 4 (1000 mg/kg). In group 4 (1000 mg/kg bw), plasma cholesterol levels were slightly increased in males and females and plasma potassium concentrations were decreased in females. Furthermore, in the high dose group (1000 mg/kg) plasma bilirubin levels were increased in males, plasma albumin concentrations were minimally elevated and plasma globulins were minimally decreased in females, resulting in an increased A/G ratio".

URINALYSIS
Excretion of a larger amount of reddish discoloured urine and mild bilirubinuria were observed in males at 1000 mg/kg bw/day. There was no evidence that treatment with the test article had affected other urine parameters investigated.

ORGAN WEIGHTS
The mean body weight relative weights of the heart, kidneys, adrenals and spleen were changed at 200 and/or 1000 mg/kg bw/day as compared to the concurrent control group:
"At necropsy the mean carcass weight was decreased in group 3 (200 mg/kg; males -8%, females -10%) and in group 4 (1000 mg/kg; males -27%, females -23%), compared to that of the control group.
Heart to body weight ratio was significantly increased in males of group 3 (+12%) and group 4 (+30%), and in females of group 4 (+18%). Kidney to body weight ratio was increased in males of group 3 (+12%) and group 4 (+51%), and in females of group 4 (+31%). Adrenal to body weight ratio was increased in males of group 2 (+11%), group 3 (+13%) and group 4 (+28%). Spleen to body weight ratio was increased in females of group 4 (+24%).
Changes in absolute organ weights of liver, thymus, adrenals, testis and spleen, which attained statistical significances in group 4, are considered consequent to the body weight decrease in this group, and are therefore of no toxicological relevance. Other differences which attained a level of statistical significance were dose-independent and, therefore, not considered of experimental relevance".

GROSS PATHOLOGY
There were no test item related macroscopical findings.

HISTOPATHOLOGY
Microscopical examination revealed cytoplasmic vacuolisation of cortical tubules of the kidneys at 1000 mg/kg bw/day:
"On morphological grounds this lesion was compatible with osmotic nephrosis (C. Gopinath et al, 1987) which is known to be induced in rats by hypertonic sugar solutions including dextran and some chelating agents (P. Greaves, 1990)".

Effect levels

Dose descriptor:

NOAEL

Basis for effect level:

other: This study was a range finding study for a 90-day repeated dose oral toxicity study (Novartis Crop Protection AG, 1998). The latter study was used for NOEL derivation.

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

CHEMICAL ANALYSIS OF DOSE FORMULATIONS

The calculated mean contents of the test item in the dose formulations used for the low, mid and high dose groups were 105, 112 and 107 % of the nominal concentrations, respectively.

Applicant's summary and conclusion

Conclusions:

No NoAEL is identified. This study was used as scientific basis for dose level selection for a subsequent 90 -day repeated dose oral toxicity study in the rat.

Executive summary:

In a dose range-finding subacute oral toxicity study (CIBA-GEIGY Limited, 1996), FeNaEDDHA in 0.5 % CMC and 0.1 % Tween 80 in distilled water was administered to 5 Sprague-Dawley derived rats/sex/dose level by oral gavage (10 mL/kg bw) at dose levels of 50, 200 or 1000 mg/kg bw/day for a period of 28 days. Male and female animals of the concurrent control group were treated with the vehicle only. Treatment with the test item resulted in impaired body weight development of rats treated at 200 and 1000 mg/kg bw/day and correspondent lower food intake. An anaemia without erythropoietic response was noted at 200 and 1000 mg/kg bw/day. At the same dose levels, the kidney was revealed as target organ by microscopical examination, by blood chemistry data and by organ weight evaluation. In addition, body weight relative organ weight changes were noted in the heart, adrenals and spleen. However, the relevance of these findings was considered as equivocal. This study was used as scientific basis for dose level selection for a subsequent 90 -day repeated dose oral toxicity study in the rat.

This subacute oral toxicity study in the rat is acceptable and satisfies the requirement for test guideline OECD 407.