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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
September-December 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted study under GLP
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
except for effects on kidneys 5 animals/sex/group were used; males were exposed for at least 12 weeks
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Chemical name: Ethylenediaminetetraacetic acid, manganese disodium complex
Purity: 92.3%
Batch no: CFC 9380
Expiry date: 31 August 2012

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutshland, Sulzfeld, Germany
- Age at study initiation: 10-11 weeks
- Weight at study initiation: mean weight males 171-175 g; mean weight females
- Fasting period before study: not applicable
- Housing: 4 per sex in macrolon cages, with wood shavings as bedding material, and paper strips as environmental enrichment
- Use of restrainers for preventing ingestion (if dermal): not applicable
- Diet (e.g. ad libitum): ad lib
- Water (e.g. ad libitum): ad lib
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2 degrees C
- Humidity (%): at least 45% and not exceeding 65%. During several periods, humidity was outside the limits reaching a minimum of 39.9% and a maximum of 93.7% during a short period
- Air changes (per hr): ca. 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 16 September To: 25 December 2009

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Preparation of the test formulations was performed one day before the first day of the dosing period and at weekly interval thereafter until the completion of the dosing phase of the study. The concentration of the test item in tap water was prepared by stirring on a magnetic stirrer. Subsequently, under continuous stirring, 8 aliquots (7 days plus 1 extra) were taken according to the volume required for each dosing. Aliqouts were stored in a refrigerator. On each subsequent day, one aliquot for each group was removed from the refrigerator and allowed to equilibrate to ambient temperature. The test item solutions were continuously stirred on a magnetic stirrer during the entire daily administration period, in order to maintain the homogeneity of the test item in the vehicle.

DIET PREPARATION (applicable to the additional group that got a surplus of zinc)
The animals of this group received a diet with a surplus level of Zn added. Hereto, an appropriate amount of zinc carbonate was mixed with the RM3 diet in a mechanical blender (Lödige, Paderborn, Germany). Two batches of this Zn-containing diet were prepared that were stored at room temperature (15 September and 25 November 2009).

VEHICLE: tap water
- Concentration in vehicle: 0, 15, 50 and 150 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations of managanese measured by ICP-AES in the gavage liquids prepared on 15 September, 17 November and 8 December 2009, respectively were ‘close to intended’ for all gavage liquids at all dose levels, except for the mid-dose level liquids prepared on 15 September and 17 November 2009 (+13.6% and +11.6%, respectively).

Zinc in the diets was also measured by ICP-AES and considered to be homogeneously distributed in the diet of group 5 which was prepared on 15 September 2009. Partly due to the higher than anticipated zinc concentration in the basal diet (77.9 mg/kg instead of 52 mg/kg) the content of zinc in the diet of group 5 was higher than intended (560 mg/kg diet instead of 500 mg/kg diet).
Duration of treatment / exposure:
10 weeks pre-mating, 1 week mating, 3 weeks gestation, and 4 days lactation
Frequency of treatment:
single daily application by gavage
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 150, 500 and 1500 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on studies done with EDTA
- Rationale for animal assignment (if not random): computer randomization proportionately to BW
Positive control:
An additional group was included to examine differences in chelating effects of the high dose in the presence of an extra amount of dietary zinc (ca. 500 ppm instead of ca. 50 ppm), if any.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: observations outside the home cage were made once weekly

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: in week 8 of the pre-mating period
- Dose groups that were examined: all
- Battery of functions tested: FOB (including sensory activity and grip strength) and spontaneous motor activity

BODY WEIGHT: Yes
- Time schedule for examinations: weekly (males and females) and on day 1 and 4 of lactation (females)

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined: weekly (at same time as measurement of bw)

WATER CONSUMPTION: Yes
- Time schedule for examinations: two times 2 days in 2 weeks towards the end of the pre-mating period (because it appeared that animals of the high dose groups were drinking more).

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: one week prior to mating
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: Yes (water freely available)
- How many animals: 5 sex/group
- Parameters checked: haemoglobin
packed cell volume
red blood cell count
reticulocytes
total white blood cell count
differential white blood cell counts (neutrophils, lymphocytes, eosinophils, basophils, monocytes)
prothrombin time
thrombocyte count
mean corpuscular volume (MCV; calculated)
mean corpuscular haemoglobin (MCH; calculated)
mean corpuscular haemoglobin concentration (MCHC; calculated).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: one week prior to mating
- Animals fasted: Yes (water freely available)
- How many animals: 5 sex/group
- Parameters checked: alkaline phosphatase activity (ALP), bilirubin (total), aspartate aminotransferase activity (ASAT), cholesterol (total), alanine aminotransferase activity (ALAT), triglycerides, gamma glutamyl transferase activity (GGT), phospholipids, total protein, calcium (Ca), albumin, sodium (Na), ratio albumin to globulin (calculated), potassium (K), urea, chloride (Cl), creatinine, inorganic phosphate (PO4), glucose (fasting)

URINALYSIS: Yes
- Time schedule for collection of urine: on days 63-65 of the study (based on the increased water intake)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No (free access to water and food)
- Parameters checked: volume, osmolarity, creatinine and sodium

Sacrifice and pathology:
SACRIFICE
- Male animals: All surviving animals as soon as possible after mating (at least 12 weeks of treatment)
- Maternal animals: All surviving animals at or shortly after day 4 of lactation (almost 14 weeks of treatment)

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera

ORGAN WEIGHTS:
- testes, epididymides (12 rats/group)
- kidneys (12 rats/sex/group)
- adrenals, brain, heart, liver, spleen, thymus (5 rats/sex/group)

HISTOPATHOLOGY:
- ovaries, uterus (12 rats/group; control and high dose groups (with and without additional zinc))
- testes, epididymides, seminal vesicles, prostate, coagulating glands (12 rats/group; control and high dose groups (with and without additional zinc))
- adrenals, axillary lymph nodes, brain, caecum, colon, femur, Peyer's patches, heart, liver, lungs, mesenteric lymph nodes, peripheral nerve, rectum, small intestines, spinal cord, spleen, stomach, thymus, thyroid, trachea/bronchi, urinary bladder (5 rats/sex/group; control and high dose groups (with and without additional zinc))
- kidneys (all animals of all groups)
Other examinations:
See at reproduction and developmental toxicity
Statistics:
- Clinical findings were evaluated by Fisher's exact probability test.
- Body weight, body weight gain, organ weights and food consumption data were subjected to one way analysis of variance (ANOVA).
- Mortality data and data of the pathology of parent females were evaluated by the Fisher’s exact probability test.
- Functional observational battery: one-way analysis of variance followed by Dunnett’s multiple comparison tests (continuous data), Kruskal-Wallis non-parametric analysis of variance followed by multiple comparison tests (rank order data) or Pearson chi-square analysis (categorical data).
- Motor activity data-total distance moved: one-way analysis of variance followed by Dunnett’s multiple comparison tests; habituation of activity: repeated measures analysis of variance on time blocks (each session consists of 5 time blocks of 6 minutes each).

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):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
no effects observed
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:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY: no effects

BODY WEIGHT AND FOOD CONSUMPTION: decreased body weight in females of the high concentration groups (with and without extra zinc); most probably due to increased fetal mortality

TEST SUBSTANCE INTAKE: no effects (gavage)

WATER CONSUMPTION: increased intake in animals of the high concentration groups (with and without extra zinc); most probably due to increased fetal mortality

OPHTHALMOSCOPIC EXAMINATION: not measured

HAEMATOLOGY: no treatment-related effects

CLINICAL CHEMISTRY: no treatment-related effects

URINALYSIS: increased urinary sodium concentration in animals of the high concentration groups (with and without extra zinc)

NEUROBEHAVIOUR: no treatment-related effects

ORGAN WEIGHTS: increased kidney weight and decreased spleen weight in animals of the high concentration groups (with and without extra zinc)

GROSS PATHOLOGY: no effects

HISTOPATHOLOGY (NON-NEOPLASTIC): very slight diffuse subcortical tubular dilatation in the kidneys of the high concentration groups (with and without extra zinc)

HISTOPATHOLOGY (NEOPLASTIC): no changes

HISTORICAL CONTROL DATA: not needed

Effect levels

Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: water consumption, urinary sodium concentration, kidneys weight and histopathology

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table – Changes in male animals

 

0 mg/kg bw

150 mg/kg bw

500 mg/kg bw

1500 mg/kg bw

1500 mg/kg bw + extra Zn

Urinary Na (mmol/L) ± SD

(n=5)

155.0 ± 46.0

161.2 ± 35.8

217.8 ± 40.1

326.2 ± 34.5**

319.8 ± 39.4**

Urinary sodium / creatinine ratio (mol/mol) ± SD (n=5)

14.94 ± 1.25

17.20 ± 2.15

17.56 ± 4.06

25.64 ± 3,76**

26.70 ± 3.16**

Relative kidney weight (g/kg) ± SE

(n=12)

5.88 ± 0.06

6.11 ± 0.07

6.19 ± 0.09

6.98 ± 0.11*

6.83 ± 0.10*

Very slight diffuse subcortical tubular dilatations

(n=12)

1

2

0

6

6

*p<0.05; **p<0.01

 

Table – Changes in female animals

 

0 mg/kg bw

150 mg/kg bw

500 mg/kg bw

1500 mg/kg bw

1500 mg/kg bw + extra Zn

Urinary Na (mmol/L) ± SD

(n=5)

111.2 ± 9.5

120.4 ± 21.1

144.2 ± 14.1

197.2 ± 32.2**

217.2 ± 43.2**

Urinary sodium / creatinine ratio (mol/mol) ± SD (n=5)

19.50 ± 2.52

20.56 ± 2.33

23.62 ± 2.04

32.34 ± 0.96**

27.24 ± 4.47**

Relative kidney weight (g/kg) ± SE

(n)

5.88 ± 0.06

(11)

6.29 ± 0.12

(10)

6.30 ± 0.15

(11)

6.93 ± 0.28**

(3)

6.74 ± 0.21***

(7)

Very slight diffuse subcortical tubular dilatations

(n=12)

0

0

1

8**

5*

*p<0.05; **p<0.01; ***p<0.001

Applicant's summary and conclusion

Conclusions:
Based on the changes in water consumption, urinary sodium concentration, kidney weight and histopathological effects of kidneys as observed in the animals treated with the highest concentration of the test item, the No Observed Adverse Effect Level (NOAEL) is 500 mg/kg body weight/day.
Executive summary:

The objective of this study was to provide data on the possible effects of the test item EDTA-MnNa2on reproductive performance of rats and the development of pups consequent to daily oral administration of various concentrations of the test item by gavage to male and female rats during a premating period of 10 weeks and during mating (1 week), gestation and lactation until postnatal day 4 (PN day 4). A 10-week pre-mating period was used to cover a full sperm cycle. Additionally, an extra group was included in the study. The animals of this group were treated with the highest concentration of the test item by gavage and received a surplus dietary level of Zn. This group with additional dietary zinc was added to the study to compensate for possible (repro-) toxic effects, if any, due to the zinc-chelating properties of EDTA.

Data with regard to fertility/reproduction/developmental toxicity are presented under 'toxicity to reproduction', and 'developmental toxicity'.

The test item EDTA-MnNa2was considered to be homogeneously distributed in the gavage liquids at all dose levels. The concentrations of managanese measured in the gavage were ‘close to intended’ for all gavage liquids at all dose levels, except for the mid-dose level liquids of which the concentrations were higher than intended on 2 occasions (+13.6% and +11.6%, respectively).

Zinc was considered to be homogeneously distributed in the diet of group 5, but, partly due to the higher than anticipated zinc concentration in the basal diet (77.9 mg/kg instead of 52 mg/kg) the content of zinc in the diet of group 5 was higher than intended (560 mg/kg diet instead of 500 mg/kg diet).

Daily clinical observations during the premating, mating, gestation and lactation period did not reveal any treatment-related changes in the animals’appearance, general condition or behaviour.

Detailed clinical observations, functional observational battery observations and motor activity assessment did not indicate treatment related effects on neurobehaviour.

No treatment-related effects on body weights and body weight changes of male and female animals were observed except for females in the high dose groups that showed a decreased mean body weight during the last week of the gestation period which was most probably related to an increased fetal mortality.

No statistically significant adverse effects were observed on food consumption of male and females animals during the entire study.

Water consumption was measured during 2 consecutive days of two weeks during the premating period. During all these 4 days, water consumption of particularly male and also of female animals treated with the highest concentration of the test item was increased. Most probably, this effect was due to the high sodium exposure of these animals via the test item.

No treatment-related adverse effects were observed on haematology and clinical chemistry parameters between the control and treatment groups.

The volume of urine was increased in the male animals of the mid- and highest dose groups and in the female animals of the high dose group which resulted in an increased concentration of creatinine. The absolute amount of creatinine excreted was not affected. The sodium concentration and the sodium/creatinine ratio was statistically significantly increased in both male and female animals of the two groups treated with the highest concentration of the test item (irrespectively of dietary zinc supplementation).

Both the absolute and relative weights of the kidneys of the male and females of the two groups treated wih the highest concentration of the test item (irrespectively of dietary zinc supplementation) were statistically significantly increased. Furthermore, the absolute and relative weights of the spleen of the female animals of the highest dose group with supplementary zinc was statistically significantly decreased.

At necropsy no treatment related gross changes were observed in male and female animals.

In the two groups treated wih the highest concentration of the test item (irrespectively of dietary zinc supplementation) an increase in the incidence of rats showing very slight diffuse subcortical tubular dilatation was observed in the kidneys, reaching the level of statistically significance in the female animals only.

Based on the results of of this repeated study (specifically water consumption, urinary sodium concentration, weight of and histopathological effects in kidneys as observed in the animals treated with the highest concentration of the test item), the No Observed Adverse Effect Level (NOAEL) is 500 mg/kg body weight/day.