Registration Dossier

Administrative data

Description of key information

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
no data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Well conducted non-GLP study. The study was performed according to methods similar to OECD407/408. Only male rats were used. 10 male animals were sacrificided at the beginning of the experiment in order to establish baseline levels of the analytical parameters to be measured. No other data were collected for these animals. No further control group was included.
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Reason / purpose:
read-across source
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Principles of method if other than guideline:
- Only male rats were used.
- 31 or 61 days of exposure
- 10 male animals were sacrificed at the beginning of the experiment in order to establish baseline levels of the analytical parameters to be measured. No other data were collected for these animals. No further control group was included.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany (SPF colony)
- Age at study initiation: 5-6 weeks
- Weight at study initiation: no data
- Fasting period before study: not applicable
- Housing: individually under conventional laboratory conditions in one room, in suspended stainless-steel cages fitted with wire-mesh floor and front.
- Diet (e.g. ad libitum): Prior to the start of the experiment, a batch of modified AIN-93G (Reeves et al., 1993) diet was prepared freshly at
TNO and stored at or below 18C until use. Two batches of a commercially available low-iron AIN-93M diet ( < 5 mg iron/kg diet; lot nos 8315-6 and 8345-4) were obtained from Dyets Inc. (Bethlehem, PA, USA).
- Water (e.g. ad libitum): no data
- Acclimation period: ± 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data

IN-LIFE DATES: From: To: no data
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): no data
- Mixing appropriate amounts with (Type of food): FeEDTA or FeSO4 were incorporated in the low-iron AIN-93M diet at constant concentrations by mixing in mechanical mixers at TNO.
- Storage temperature of food: Dietary iron was found to be stable after storage for 7 days at room temperature in an open container and after 7
days or 5 weeks in a refrigerator in a closed container and after 5 weeks in a freezer in a closed container.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The content, stability and homogeneous distribution of iron in the test diets was analyzed by means of atomic absorption spectrometry (AAS). The content of each of the six diets was measured three times during the study.

The iron content was close to the intended values in 17 of the 18 diet samples measured during the experiment. In the one case that the criteria for 'close to intended' were not met (10% of the intended concentration), a concentration of 61 mg Fe per kg diet was found, which was 13% lower than the intended 70 mg Fe/kg.
Duration of treatment / exposure:
31 or 61 days
Frequency of treatment:
daily via the diet
Remarks:
Doses / Concentrations:
FeSO4 or FeEDTA were mixed with these diets to obtain diets containing 35, 70 or 140 mg Fe/kg diet. ± FeEDTA = FENaEDTA.3H2O 264, 528, 1056 mg/kg diet (mol weight Fe 55.847, mol weight EDTA-FeNa 3H2O 421.096)
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
The mean intake of iron was 2.81, 5.67, 11.19 mg/kg bw/day for the -low, -mid and high-dose, respectively. ± FeEDTA = FENaEDTA.3H2O 21, 43, 84 mg/kg bw/day (mol weight Fe 55.847, mol weight EDTA-FeNa 3H2O 421.096)
Basis:
actual ingested
No. of animals per sex per dose:
40 male rats, 20 were sacrificed after 31 days and the remaining were sacrificed after 61 days
Control animals:
other: - 10 male animals were sacrificided at the beginning of the experiment in order to establish baseline levels of the analytical parameters to be measured. No other data were collected for these animals. No further control group was included.
Details on study design:
- Dose selection rationale: no data
- Rationale for animal assignment (if not random): randomized by computer
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): no data
Positive control:
Not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each animal was observed daily and all abnormalities, signs of ill-health or reactions to treatment were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each animal was recorded once during the acclimatization period, at initiation of treatment, and once weekly thereafter. In addition, the animals were weighed on the day of scheduled autopsy in order to determine their correct organ to body weight ratios.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
Food consumption was measured per animal twice weekly, over periods of 3 or 4 days, by weighing the feeders. The results were expressed in g per animal per day.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes
The efficiency of food utilization was calculated and expressed in g weight gain per g food consumed.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At autopsy
- Anaesthetic used for blood collection: Yes; No data on identity
- Animals fasted: Yes
- How many animals: All
- Parameters examined: hemoglobin, packed cell volume, red blood cell count, total white blood cell count, differential white blood cell count, prothrombin time and thrombocyte count. The following parameters were calculated: mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At autopsy
- Animals fasted: Yes
- How many animals: all
- Parameters examined: alkaline phosphatase activity (ALP), aspartate aminotransferase activity (ASAT), alanine arninotransferase activity (ALAT), gamma glutamyl transferase activity (GGT), total protein, albumin, ratio albumin to globulin, urea, creatinine, glucose, bilirubin (total), cholesterol (total), triglycerides, phospholipids, calcium (Ca), sodium (Na), potassium (K), chloride (Cl) and inorganic phosphate

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
- Intake of iron from FeEDTA or FeSO4
- Determination of iron and total iron binding capacity (TIBC) in blood plasma
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
After completion of the treatment periods, the animals were killed on several successive working days, in such a sequence that the average time of killing was approximately the same for each group. The animals were killed by exsanguination from the abdominal aorta under ether anesthesia
and then examined macroscopically for pathological changes.
The adrenals, brain, caecum, colon, heart, kidneys, liver, oesophagus, rectum, small intestines (duodenum, ileum, jejunum), spleen, stomach, testes and thymus, were excised, examined for gross lesions and preserved in a neutral aqueous phosphate buffered 4% solution of formaldehyde (10% solution of formalin). The organs that are italicised were weighed prior to preservation.
HISTOPATHOLOGY: Yes
Samples of liver, spleen and all gross lesions were embedded in paraffin wax, sectioned at 5 µm and stained with haematoxylin and eosin and with Perl's Prussian blue for iron. The stained sections, except the Perl's Prussian blue stained sections of gross lesions, were examined by light microscopy by an experienced pathologist.
Fresh samples of the liver, spleen and kidneys of all animals were analyzed for non-heme iron content by the bathophenanthroline reaction and the results were expressed as µg Fe/g tissue (wet weight). The analysis was performed according to the method described by Whittaker et al. (1997). Before analysis of study samples, the method was validated for the different tissues under investigation.
Other examinations:
None
Statistics:
Body weights were analyses by one-way analysis of covariance (covariate: body weight on day 0) followed by Dunnett's multiple comparison tests, food consumption and food conversion efficiency were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison tests, red blood cell and coagulation variables, total white blood cell counts, absolute differential white blood cell counts, clinical chemistry values, plasma iron, TIBC and organ weights were analyzed by one-way ANOVA followed by Dunnett's multiple comparison tests, relative differential white blood cell counts were analyzed by Kruskal-Wallis nonparametric ANOVA followed by Mann-Whitney U-tests, histopathological changes were analyzed by Fisher's exact probability test and non-heme iron analyses in tissue were analyzed by two-way ANOVA followed by Student's t-tests. All tests were two-sided. Probability values of P < 0.05 were considered significant.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
Hematology
After 31 days of feeding, the mean corpuscular hemoglobin was statistically significantly higher in groups receiving the high dose of FeSO4 and the mid and high dose of FeEDTA in comparison to the group receiving the low dose of FeSO4. The mean corpuscular hemoglobin concentration was statistically significantly higher in the group receiving the high dose of FeEDTA in comparison to the group receiving the low dose of FeS04. No other statistically significant differences in red blood cell or coagulation variables were observed after 31 days of feeding.
After 61 days of feeding, no statistically significant differences in red blood cell or coagulation variables were observed. After 31 days of feeding, the absolute number of eosinophils was statistically significantly higher in the high-dose FeEDTA group in comparison to the lowand mid-dose FeEDTA groups and the mid-dose FeSO4 group. The percentage of eosinophils was statistic all significantly higher in the high-dose FeEDTA group in comparison to the low-dose FeEDTA and the mid-dose FeSO4 groups. No other statistically significant differences in white blood cell counts were observed.
After 61 days of feeding, no statistically significant differences in white blood cell counts were observed.

Clinical chemistry values in plasma
The following statistically significant differences were observed:

At day 32
Alkaline phosphatase activity was decreased in the mid-dose FeEDTA group in comparison to the low-, mid- and high-dose FeSO4-groups. At day 62, this difference was no longer present. The concentration of total bilirubin was higher in the mid-dose FeSO4 group in comparison to the low-dose FeEDTA and FeSO4 - groups. At day 62, this difference was no longer present. Sodium and chloride concentrations decreased with
increasing dietary levels of iron (from both sources). At day 62, the difference in chloride concentrations was no longer present.

At day 62
Total protein concentrations were lower in the FeEDTA groups than in the FeSO4 groups and albumin concentrations were lower in the mid- and high-dose FeEDTA groups than in the low-dose FeEDTA group and the low- and mid-dose FeSO4 groups. The calcium concentration was lower in the high-dose FeEDTA group than in the low-dose FeSO4 and FeEDTA groups and the mid-dose FeSO4 group. Sodium concentrations were lower in the high-dose FeEDTA and FeSO4groups than in the low-dose FeSO4 group.

Total iron binding capacity in blood plasma
After 31 days of feeding, no statistically significant differences were observed in TIBC along the groups.
After 61 days of feeding, a statistically significantly higher TIBC was observed in the low-dose FeSO4 group compared to the other groups. Furthermore, the TIBC was statistically significantly lower in the high dose FeEDTA group than in the low-dose FeEDTA and the mid-dose FeSO4 groups.

Iron disposition: non-heme iron in liver, spleen and kidney
A statistically significant increase in non-heme iron concentration in the liver with increasing dietary iron concentrations after 31 and 61 days of feeding was found. Independently from the dose level, feeding FeEDTA resulted in statistically significantly lower non-heme iron concentrations in the liver compared to FeSO4 after 31 and 61 days of feeding. After 31 days of feeding, the non-heme iron concentrations in the spleen had increased statistically significantly with increasing dietary iron concentrations. Independently from the dose level, feeding FeEDTA resulted in statistically significantly lower non-heme iron concentrations in the spleen compared to feeding FeSO4 for 3 1 days. After 61 days of feeding, the effect of the dose level had disappeared, but feeding FeEDTA still resulted in statistically significantly lower non-heme iron concentrations in the spleen, compared to FeSO4. After 31 and 61 days of feeding, the non-heme iron concentrations in the kidneys had increased statistically
significantly with increasing dietary iron concentrations, but no statistically significant differences in non-heme iron concentrations between FeEDTA or FeSO4 feeding were observed.

Microscopic examination of Prussian blue-stained sections
After 31 days of feeding, no positive Prussian blue reaction was observed in the liver.
After 61 days of feeding, a positive Prussian blue reaction in the liver was observed in 11, nine and nine animals of the low-, mid and high-dose FeEDTA groups, respectively, and in six, 15 and 14 animals of to the low-, mid- and high-dose FeSO4 groups, respectively. In animals with a positive reaction, the blue staining was found predominantly in the Kupffer cells. The incidences of blue staining in hepatocytes and Kupffer cells of livers of the FeEDTA exposed animals was not influenced by the dose. In the mid- and high-dose FeSO4 groups, the incidences of blue staining in the Kupffer cells were higher than those in the low-dose FeSO4 group.
After 31 days of feeding, the spleen of all animals, low Fe-group animals included, demonstrated a positive Prussian blue reaction. Blue staining was mainly confined to the red pulp, and the degree (intensity of the staining and the number of positive cells) varied from
very slight to slight. The incidence of slight blue staining tended to be somewhat higher in the high-dose FeSO4 and FeEDTA groups compared to the low Fe-groups. No differences were observed between the FeSO4 and FeEDTA groups maintained on diets with comparable iron content.
After 61 days of feeding, a positive reaction in the spleen was observed in all animals. Compared to animals exposed for 31 days, the degree of staining in the spleen of animals exposed for 61 days was somewhat increased, and the staining was extended from red pulp only to red pulp
and marginal sinus, especially in animals with moderate blue staining. A distinct shift from (very) slight to moderate degree of blue staining was observed in the high-dose FeSO4 group when compared to the low-dose FeSO4 group, and in the mid- and high-dose FeEDTA groups
when compared to the low-dose FeEDTA group.
Dose descriptor:
NOAEL
Effect level:
> 11.2 mg/kg bw/day (actual dose received)
Based on:
element
Sex:
male
Basis for effect level:
other: No test item / dose response related toxicological effects observed; 11.2 mg Fe per kg bw corresponds with 421/56 x 11.2 = 84 mg EDTA-FeNa.3H2O per kg bw.
Critical effects observed:
not specified
Conclusions:
- It was concluded that, with respect to iron parameters in blood plasma, feeding FeSO4 or FeEDTA up to dietary concentrations of 140 mg Fe/kg, did not result in excessive iron loading.
- In the present study, the dietary exposure to iron from FeSO4 or FeEDTA did not result in toxicologically relevant changes in clinical signs, body or organ weights, food consumption, red and white blood cell or coagulation variables or cliincal chemistry or in pathology.
Executive summary:

A study was performed to provide data on the disposition, accumulation and toxicity of sodium iron EDTA in comparison with iron (II) sulfate in rats on administration via the diet for 31 and 61 days. Clinical signs, body weights, food consumption, food conversion efficiency, hematology, clinical chemistry and pathology of selected organs were used as criteria for disclosing possible harmful effects. Determination of iron and total iron binding capacity in blood plasma and non-heme iron analysis in liver, spleen and kidneys were used to assess the disposition and accumulation of iron originating from sodium iron EDTA or iron (II) sulfate.

It was concluded that, under the conditions of the present study, iron is accumulated from the diet in liver, spleen and kidneys in a dose-dependent manner, and iron derived from FeEDTA is taken up and/or accumulated less efficiently in liver and spleen than iron from FeSO4.

Moreover, feeding iron up to 11.5 and 11.2 mg/kg body weight/day, derived from FeSO4 and FeEDTA, respectively, did not result in tissue iron excess nor in any other toxicologically significant effects. The latter value corresponds to 84 mg EDTA-FeNa.3H2O per kg bw.

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
April-August 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted study under GLP
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See section 13
Reason / purpose:
reference to same study
Reason / purpose:
read-across source
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
for histopathology (except for sex organs) 5 animals/sex/group were used
GLP compliance:
yes (incl. certificate)
Limit test:
no
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: 5 weeks (females), 6 weeks (males)
- Weight at study initiation: mean weight males ca. 170 g; mean weight females ca. 106 g
- 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, reaching a minimum of 19.2 degrees C
- Humidity (%): at least 45% and not exceeding 65%. During several periods, humidity was outside the limits reaching a minimum of 43% and a maximum of 96% during a short period
- Air changes (per hr): ca. 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 7 April to 4 August 2010
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Weekly, one bottle of test formulation per dose level was prepared. Preparation of the test formulations was performed one day before the first day of the dosing period and at weekly intervals thereafter until completion of the dosing phase of the study. The different concentrations of the test substance in tap water were prepared by stirring on a magnetic stirrer for at least 1h. The pH of the test formulations of groups 2, 3 and 4 were set between pH 6-7 using sodium carbonate (Na2CO3). 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 in the dark. On each subsequent day, one aliquot for each group was removed from the refrigerator and allowed to equilibrate to ambient temperature. All aliquots were continuously stirred on a magnetic stirrer during the entire administration period in order to maintain the homogeneity of the test substance in the vehicle.
Sodium carbonate was added to all three test formulations to adjust the acidity of the formulations to pH 6-7: it appeared that the amount of test substance used for the preparation of the test solution for the high-dose group did not dissolve unless the pH was adjusted. On the first 2 days of the study, animals of the low- and mid-dose groups were treated with test formulations without the addition of sodium carbonate. From Day 2 onwards, all animals of the low-, mid- and high-dose groups were treated with test formulations with added sodium carbonate. In week 2 of the study, the pH of the test formulations of the low-, mid- and high-dose groups were marginally higher (resp 7.03, 7.05 and 7.06). This also applied for week 8 of the study, regarding test formulations of the low- and mid-dose groups (resp 7.18 and 7.01).

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:
To determine the homogeneity and content of DTPA-FeNaH in gavage liquid, iron was used as a marker for the test item. Iron concentrations in gavage liquid were determined using inductively coupled plasma atomic emission spectroscopy (ICP-AES).
The concentrations of iron measured in the gavage liquids prepared on 15 April 2010, 15 June 2010 and 06 July 2010 were ‘close to intended’ (relative difference < 10 %) for all gavage liquids at all dose levels, except for the low-dose level gavage liquids prepared on 15 April 2010 (+10.7%) and the low-, mid- and high-dose level gavage liquids prepared on 06 July 2010 (+10.1%, +11.0% and +13.6%, respectively).
Duration of treatment / exposure:
10 weeks pre-mating, 16 days mating, 3 weeks gestation, and 4 days lactation
Frequency of treatment:
single daily application by gavage
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 and EDTA-MnNa2
- Rationale for animal assignment (if not random): computer randomization proportionately to BW
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 during 10 weeks

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: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of week 10, 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: end of week 10, 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: No

Sacrifice and pathology:
SACRIFICE
- Male animals: All surviving animals as soon as possible after mating (at least 13 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)
- adrenals, brain, heart, kidneys, liver, spleen, thymus (5 rats/sex/group)

HISTOPATHOLOGY:
- ovaries, uterus (12 rats/group; control and high dose group
- testes, epididymides, seminal vesicles, prostate, coagulating glands (12 rats/group; control and high dose group
- adrenals, axillary lymph nodes, brain, caecum, colon, femur, Peyer's patches, heart, kidneys, 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 group

Other examinations:
See at reproduction and developmental toxicity
Statistics:
- Clinical findings were evaluated by Fisher's exact probability test.
- Body weight, body weight gain, food consumption and organ weights data were subjected to one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison tests.
- Haematology and clinical chemistry parameters were subjected to one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison tests.
- Mortality data and data of the pathology of parent animals were evaluated by the Fisher’s exact probability test.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
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):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
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:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY: Soft faeces were observed in males and females of the high-dose groups in various weeks of the pre-mating period . This finding was most pronounced in the first week of the study. Soft feaces were also observed in some rats of the mid-dose group in the first week of the study only. The findings in the high-dose group occasionally reached the level of statistical significance. The repeated occurrence of soft faeces in the high-dose group was considered treatment-related.
One female of the low-dose group (no. 33) was found dead on day 64 of the pre-mating period. In the fourth week of the study, one female of the mid-dose group (no. 57) showed a hunched body position, piloerection and was thin and weak. On Day 32 of the pre-mating period it was found dead. On Day 3 of the gestation period, a female of the high dose group (no. 85) was killed because it was thin and showed piloerection and abnormal respiration (rales, dyspnoea, decreased frequency). The death of females nos 33 and 85 is likely to have been caused by misdosing. The cause of death of
female no. 57 of the mid-dose group could not be examined at necropsy because its organs were already autolytic.

BODY WEIGHT AND FOOD CONSUMPTION: The mean body weight of males of the high-dose group was decreased when compared to the control group, reaching the level of statistical significance as from Day 35 of the study. In addition, the mean body weight change of males of the high-dose group was statistically significantly decreased at various time periods (Days 0-7, 28-49, 63-70). These observed effects on body weight were considered to be related to treatment. Food consumption was not affected by treatment.

TEST SUBSTANCE INTAKE: no effects (gavage)

WATER CONSUMPTION: not measured

OPHTHALMOSCOPIC EXAMINATION: not measured

HAEMATOLOGY: Prothrombin time was increased in males of the high-dose group. Haemoglobin concentration, mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) were statistically significantly increased in males of the high-dose group when compared to controls.

CLINICAL CHEMISTRY: Alkaline phosphatase activity (ALP) showed a dose-related and statistically significant decrease in males of the mid- and high-dose groups and in females of all treatment groups. In males of the high-dose group, alanine amino transferase activity (ALAT) was statistically
significantly decreased when compared to controls. The chloride concentration in males of the high-dose group was statistically significantly
decreased.

URINALYSIS: not measured

NEUROBEHAVIOUR: no treatment-related effects

ORGAN WEIGHTS: The relative weights of the kidneys and liver of male and female animals of the high-dose group were statistically significantly increased. These findings were considered treatmentrelated. The absolute weight of the epididymides was decreased in males of the mid- and high-dose
group. The relative weight of this organ was decreased in males of the high-dose group but not in males of the mid-dose group. Therefore, only the decreased weight of the epididymides of males of the high-dose group was considered to be related to treatment.

GROSS PATHOLOGY: no effects

HISTOPATHOLOGY (NON-NEOPLASTIC): no treatment-related effects

HISTOPATHOLOGY (NEOPLASTIC): no changes

HISTORICAL CONTROL DATA: not needed

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: see 'Remark'
Critical effects observed:
not specified

Most of the effects observed were confined to animals of the high-dose group and comprised soft faeces (both sexes), decreased body weight gain (males), prolonged prothrombin time (males), increased haemoglobin concentration (males), decreased ALAT activity and chloride concentration (males) and increased relative weights of kidneys and liver (both sexes). The soft faeces, and decreased body weight gain and decreased male fertility (see also at section 7.8.1) may point at zinc deficiency at the high concentration level. Since DTPA is a chelating agent like EDTA, DTPA depletes metals including zinc.

Alkaline phosphatase (ALP) activity in blood showed a dose-related decrease in males of the mid- and high-dose group and in females of all treatment groups. The toxicological relevance of this finding is not clear as increased serum ALP activity is generally considered to be a toxic effect. It has been shown that a decrease in ALP levels might also be the consequence of zinc deficiency or depletion of zinc.

The log K binding values for DTPA are as follows (for comparison the values of EDTA are also given):

DTPA: 16.2 (Fe(II)), 18.2 (Zn), 28.0 (Fe(III))

EDTA: 14.3 (Fe(II)), 16.5 (Zn), 25.1 (Fe(III))

[ A.E. Martell, R.M. Smith, NIST Critically selected stability constants of metal complexes; NIST standard reference database 46, Version 7.0, 2003)]

This means that DTPA (like EDTA) has the highest affinity for Fe(III), followed by Zn and then Fe(II). The gut enterocytes have the ability to convert Fe(III) to Fe(II) prior to absorption, so the Fe(III) is released, then converted to Fe(II) for which DTPA has a lower affinity so it picks up whatever else for which it has a higher affinity, such as zinc. Thus, the release of the Fe leaves the DTPA open to bind zinc either in the gut or subsequent to absorption (the absorption of DTPA is estimated to be ca. 5%, as with EDTA). This zinc chelation then causes the effects on male reproductive organs (see below) and also on the liver enzymes. In addition, ALP is an enzyme with 2 zinc atoms attached, one bound tightly and one not so much. Therefore, any chelator of zinc present in blood could remove some of the zinc or prevent sufficient zinc from being available to the enzyme. It is therefore concluded that the effect on serum ALP activity is most probably due to effects on zinc homeostasis. In addition, the removal of zinc has caused a decrease in ALP activity, it does not necessarily mean that the ALP level as such had decreased.

The toxicological significance of a decrease in serum ALP activity is not clear. On the one hand it can be expected that a sufficient deficiency of this enzyme in cells could lead to issues with DNA repair, cell replication, and other processes requiring the dephosphorylation of proteins, nucleotides, etc. On the other hand, it is not clear in how far a decrease in serum ALP activity reflects decreased cellular ALP activity. There are a number of diseases associated with decreased serum ALP levels such as severe anemia, hypothyroidism, malnutrition, hypophosphatasia, and chronic myelogenous leukemia. Most probably these diseases have resulted in decreased ALP activity rather than that decreased ALP activity has caused these diseases. In addition, it can be concluded that the body can cope with a decrease in serum ALP activity to a certain level, since in the present study there were no other effects observed at the mid- and low–dose level. As such it can be argued that the decrease in serum ALP activity is a treatment-related effect, but non-adverse.

Conclusions:
It is concluded that at the low and mid dose the decrease in serum ALP activity is a treatment-related effect that is of questionable toxicological significance, and most probably not adverse in itself. Based on the above considerations, the No Observed Adverse Effect Level (NOAEL) for parental toxicity 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 substance DTPA-FeNaH following subchronic exposure, and on reproductive performance of Wistar rats (see also section 7.8.1) and the development of pups (see also section 7.8.2) following daily oral administration at concentrations of 0, 150, 500 or 1500 mg/kg bw of the test substance by gavage to male and female rats during a pre-mating period of 10 weeks, during mating (16 days), and during gestation and lactation until postnatal Day 4 (PN Day 4).

The homogeneity and content of the test substance in the gavage solutions were confirmed by analysis.

Males and females of the high-dose group showed soft faeces in various weeks of the premating period. Daily clinical observations during the gestation and lactation period did not reveal any treatment-related changes in the animal’s appearance, general condition or behaviour. Neurobehavioural observations and motor activity assessment did not indicate any neurotoxic potential of the test substance. Mean body weights were decreased in males of the high-dose group from week 5 onwards. There were no treatment-related effects on female body weights during the entire study. No treatment-related effects were observed on food consumption of male and female animals during the entire study.

In males of the high-dose group, prothrombin time was increased, and haemoglobin concentration, mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) were statistically significantly increased. Alkaline phosphatase activity (ALP) showed a dose-related decrease in males of the mid- and high-dose group and in females of all treatment groups. In males of the high-dose group, alanine amino transferase activity (ALAT) and chloride concentration were decreased.

The relative weights of the kidneys and liver were increased in both sexes of the high-dose group. The relative weight of the epididymides was decreased in males of the high-dose group. Macroscopic examination at necropsy, and microscopic examination of organs and tissues did not reveal any treatment-related changes.

Based on the results of this study, viz. soft faeces (both sexes), decreased body weight gain (males), prolonged prothrombin time (males), increased haemoglobin concentration (males), decreased ALAT activity and chloride concentration (males) and increased relative weights of kidneys and liver (both sexes) as observed in animals treated with the highest concentration of the test substance, the No Observed Adverse Effect Level (NOAEL) for parental toxicity is 500 mg/kg body weight/day.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
The in-life phase of the study was conducted between 05 December 2012 (first day of treatment) and 15 January 2013 (final necropsy).
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: A sufficient number of male and female Wistar Han:RccHan:WIST strain rats were obtained from Harlan Laboratories U.K. Ltd.
- Age at study initiation: Approximately twelve weeks old.
- Weight at study initiation: At the start of treatment the males weighed 302 to 346g, the females weighed 191 to 217g.
- Fasting period before study: None.
- Housing: Initially, all animals were housed in groups of four in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet (e.g. ad libitum): The animals were allowed free access to food (a pelleted diet was used).
- Water (e.g. ad libitum): The animals were allowed free access to water. Mains drinking water was supplied from polycarbonate bottles attached to the cage.
- Acclimation period: Six days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Set to achieve target values of 21 ± 2°C.
- Humidity (%): Set to achieve target values of 55 ± 15%.
- Air changes (per hr): At least fifteen air changes per hour.
- Photoperiod (hrs dark / hrs light): Low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness.

Route of administration:
oral: gavage
Vehicle:
other: Distilled water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was prepared at the appropriate concentrations as a solution in Distilled water.
Formulations were prepared weekly and stored at 4ºC in the dark.

The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 5 ml/kg of Distilled water.

The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at regular intervals.


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each test item formulation were taken and analysed for concentration of Sodium Glucoheptanate.

The concentration of Sodium Glucoheptonate in the test item formulations was determined by direct injection into a mass spectrometer (MS) using an external standard technique.

The results indicate that the prepared formulations were within acceptable ranges for the purpose of this study.
Duration of treatment / exposure:
The test item was administered for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females).
Frequency of treatment:
Once daily.
Remarks:
Doses / Concentrations:
0 (control), 30, 100 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
12 males and 12 females per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were based on the results of a 14-day range-finding study.

Chronological Sequence of Study:
i) Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
ii) Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioural toxicity.
iii) On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iv) Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
v) On completion of the pre-pairing phase (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.
vi) Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded during this period.
vii) At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.
viii) Blood samples were taken from five males from each dose group for haematological and blood chemical assessments on Day 42. The male dose groups were killed and examined macroscopically on Day 43.
ix) Blood samples were taken from five randomly selected females from each dose group for haematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically. Any female which did not produce a pregnancy was also killed and examined macroscopically.
Positive control:
None.
Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS:
All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, up to thirty minutes after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before dosing, soon after dosing, and one hour after dosing at weekends (except for females during parturition where applicable). All observations were recorded.

FUNCTIONAL OBSERVATIONS:
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

BEHAVIOURAL ASSESSMENTS:
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait, Hyper/Hypothermia, Tremors, Skin colour, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behaviour, Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation.

FUNCTIONAL PERFORMANCE TESTS:
Motor Activity: Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time each day, under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute period
and also during the final 20% of the period (considered to be the asymptotic period).

Forelimb/Hindlimb Grip Strength: An automated meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).

Sensory Reactivity:
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988). Grasp response, Touch escape, Vocalisation, Pupil reflex, Toe pinch, Blink reflex , Tail pinch, Startle reflex, Finger approach.

BODY WEIGHT:
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until mating was evident. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION:
During the maturation period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum.

Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-mating phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

WATER CONSUMPTION:
Water intake was measured daily during the first two weeks of the study.

LABORATORY INVESTIGATIONS:
Haematological and blood chemical investigations were performed on five males and five females selected from each test and control group prior to termination (Day 42 for males and Day 4 post partum for females). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination. Animals were not fasted prior to sampling.

HAEMATOLOGY:
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:
Haemoglobin (Hb)
Erythrocyte count (RBC)
Haematocrit (Hct)
Erythrocyte indices
- mean corpuscular haemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular haemoglobin concentration (MCHC)
Total leucocyte count (WBC)
Differential leucocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic) - Methylene blue stained slides were prepared but reticulocytes were not assessed
Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/l).

BLOOD CHEMISTRY:
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea
Calcium (Ca++)
Glucose
Inorganic phosphorus (P)
Total protein (Tot.Prot.)
Aspartate aminotransferase (ASAT)
Albumin
Alanine aminotransferase (ALAT)
Albumin/Globulin (A/G) ratio (by calculation)
Alkaline phosphatase (AP)
Sodium (Na+)
Creatinine (Creat)
Potassium (K+)
Total cholesterol (Chol)
Chloride (Cl-)
Total bilirubin (Bili)
Bile acids (Bile)






Sacrifice and pathology:
PATHOLOGY:
Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 26 post coitum.

For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution.

All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

ORGAN WEIGHTS:
The following organs were dissected free from fat and weighed before fixation from five selected males and five selected females from each dose grroup.
Adrenals, Prostate, Brain, Seminal vesicles, Epididymides, Spleen, Heart, Testes, Kidneys, Thymus, Liver, Thyroid (weighed post-fixation with Parathyroid), Ovaries, Uterus (weighed with Cervix)


HISTOPATHOLOGY:
Samples of the following tissues were removed from five selected males and five selected females from each dose group.
Adrenals
Ovaries
Aorta (thoracic)
Pancreas
Bone & bone marrow (femur including stifle joint)
Bone & bone marrow (sternum)
Pituitary
Prostate
Brain (including cerebrum, cerebellum and pons)
Oesophagus
Caecum
Rectum
Coagulating gland
Salivary glands (submaxillary)
Colon
Sciatic nerve
Duodenum
Seminal vesicles
Epididymides
Skin (hind limb)
Eyes
Spinal cord (cervical, mid-thoracic and lumbar)
Gross lesions
Heart
Spleen
Ileum (including peyer’s patches)
Stomach
Jejunum
Thyroid/parathyroid
Kidneys
Trachea
Liver
Testes
Lungs (with brochi)
Thymus
Lymph nodes (cervical and mesenteric)
Urinary bladder
Mammary gland
Uterus/Cevix
Muscle (skeletal)
Vagina

Tissues were despatched to the histology processing site for processing. Microscopic examination was conducted by the Study Pathologist.
Other examinations:
REPRODUCTION SCREENING:
MATING:
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation.

PREGNANCY AND PARTURITION:
Each pregnant female was observed at approximately 0830, 1230 and 1630 hours and around the period of expected parturition. Observations were carried out at approximately 0830 and 1230 hours at weekends and public holidays. The following was recorded for each female:
i) Date of pairing
ii) Date of mating
iii) Date and time of observed start of parturition
iv) Date and time of observed completion of parturition

LITTER DATA:
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.
For each litter the following was recorded:
i) Number of offspring born
ii) Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii) Sex of offspring on Days 1 and 4 post partum
iv) Clinical condition of offspring from birth to Day 5 post partum
v) Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data)

PHYSICAL DEVELOPMENT:
All live offspring were assessed for surface righting reflex on Day 1 post partum.
Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; Statistical analysis was performed on the following parameters:
Grip Strength, Motor Activity, Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Corpora Lutea, Implantation Sites, Implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offspring Surface Righting, Haematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights



Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Treatment at dose levels of 30, 300 and 1000 mg/kg bw/day did not produce any convincing toxicological effects of treatment.
Dose descriptor:
NOEL
Remarks:
reproductive toxicity
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects were observed for reproduction.
Critical effects observed:
not specified
Conclusions:
The oral administration to rats of Sodium Glucoheptanate (EC 250-480-2) (incorporating a test item correction factor for 50.5% w/w purity minus 49.5% w/w test item water content) to rats by gavage, at dose levels of 30, 300 and 1000 mg/kg bw/day did not produce any convincing toxicological effects of treatment and on this basis the ‘No Observed Adverse Effect Level’ (NOAEL) and possible ‘No Observed Effect Level’ (NOEL) for systemic toxicity for either sex was considered to be 1000 mg/kg bw/day.

No treatment-related effects were observed for reproduction, the ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was therefore considered to be 1000 mg/kg bw/day.
Executive summary:

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development) and is designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 22 March 1996).

This study was also designed to be compatible with the Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods:

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 30, 300 and 1000 mg/kg bw/day (incorporating a correction factor for 50.5% purity). A control group of twelve males and twelve females was dosed with vehicle alone (Distilled water).

Clinical signs, behavioural assessments, body weight change and food and water consumption were monitored during the study.

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 4 post partum. Haematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose

group.

Adult males were terminated on Day 43, followed by the termination of all females and offspring on Day 5 post partum. Any female which did not produce a pregnancy was terminated on or after Day 25 post coitum. All animals were subjected to a gross

necropsy examination and histopathological evaluation of selected tissues was performed.

Results:

The study did not produce any convincing toxicological effects of treatment and on this basis the ‘No Observed Adverse Effect Level’ (NOAEL) and possible ‘No Observed Effect Level’ (NOEL) for systemic toxicity for either sex was considered to be 1000 mg/kg bw/day.

No treatment-related effects were observed for reproduction, the ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was therefore considered to be 1000 mg/kg bw/day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Well performed GLP studies
System:
other: prolonged prothrombin time (males), increased haemoglobin concentration (males), decreased ALAT activity and chloride concentration (males) and increased relative weights of kidneys and liver (both sexes) at 1500 mg/kg bw.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

As general toxicity was only generally seen at levels above 1000 mg/kg bw, no classification needed according to GHS.