Reaction mass of iron chelate of sodium salt N-[1,2 dicarboxyethyl] D,L aspartic acid and sodium chloride

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

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

Adequacy of study:

key study

Study period:

2001-10-21 to 2002-03-18

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: A reliable GLP compliant guideline study conducted with the read-across substance chelating agent IDHA.

Justification for type of information:

Please see attached file.

Cross-reference

Reason / purpose for cross-reference:

read-across: supporting information

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

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

Adequacy of study:

key study

Study period:

1960-1962

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Please see attached file.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

no guideline followed

Principles of method if other than guideline:

Four groups of 25 male and 25 female rats were fed diets containing 0, 50, 125 and 250 mg/kg bw/
dy CaNa2EDTA for two years. Feeding was carried on through four successive generations. Rat w
ere mated after 12 weeks' feeding and allowed to lactate for three weeks with one week's rest before
producing a second litter. Ten male and 10 female rats of each group (F1 generation) and similar F2
and F3 generation groups were allowed to produce two litters. Of the second litters of F1, F2, and F3
generations only the control and the 250 mg/kg bw/dy groups were kept until the end of two-years' st
udy on the F0 generation. This scheme permitted terminal observation to be made on rats receiving
test diets for 0, 0.5, 1, 1.5 or 2 years in the F3, F2, F1 and F0 generations, respectively.

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

CaNa2-EDTA was prepared as a white, drum-dried flake material in the dihydrate form, for purpose as food additive.
Specifications: Assay: 99.0% minimum by weight as the dihydrate (by Th(NO3)4 titration-method); free EDTA less than 100 ppm (by Cal-Red method); unche;ated calcium, less than 1000 ppm (by Cal-Red method); pH (1-15% water solution) 6.5 - 7.5

In this studies , a 25% solution of calcium EDTA was used. Two samples wereprepared and stored in polyethylene bottles at room temperature. Portion for use in th experiment work were withdrawn as needed. Analytical studies demonstrated that those solutions were stable throught the period covered by this work.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

FDRL colony (derived from the Wistar rats more than 30 year ago).

Sex:

male/female

Details on test animals or test system and environmental conditions:

200 weanling rats were assigned to the threetest groups and one control group, of 25 males and 25 females ech. The rats were haused individually in raised-bottom cages, fresh water being availableat all times. The basal diet was a mixture of natural foods suplemented with inorganic salts and vitamis. Its composition resembles the food consumption pattern of the USA population with respect to the ratios of milk, meat, and grain components. Vitamins and minerals were presented at levels adequate for normal growth and development. The test material was added at such levels as to provide 50, 125, and 250 mg CaNa2EDTA (anhydrous basis) per kg bw of rat per day. Because f the initially high and gradually diminishing ratio of food intake to body weight during the period between weaning and maturity, adjustments of the proportion of test material in the diet were made biweekly up to the eleventh week. Since the food intake of rats at this time is normally stabilized at approximately 50 g / kg bw, the concentration of test material was kept constant for the remainder of the study.

One aspect of the extended study included reproduction and lactation experiments. Offspring bred from the dams were raised on their respective parents' diets and carried through similar short-term studies as in the initial generation.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

The test material was added at such levels as to provide 50, 125, and 250 mg CaNa2EDTA (anhydrous basis) per kg bw of rat per day. Because f the initially high and gradually diminishing ratio of food intake to body weight during the period between weaning and maturity, adjustments of the proportion of test material in the diet were made biweekly up to the eleventh week. Since the food intake of rats at this time is normally stabilized at approximately 50 g / kg bw, the concentration of test material was kept constant for the remainder of the study.

One aspect of the extended study included reproduction and lactation experiments. Offspring bred from the dams were raised on their respective parents' diets and carried through similar short-term studies as in the initial generation.

The study was continued without change of dietary treatment for the remaider of the 2-yers period.

Details on mating procedure:

After approximately 13 weeks (when the ras were about 120 days of age and sexually mature) matings were set up with male and two females per cage. As pregnancy was recognized (visually, by palpation, or by weight increments) the dam was transferred to an individual cage. If pregnancy was not established by the third week, the male was replaced. A female was regarded as infertile and matings were discontinued after two successive mating failures. In successive matings the maleswere rotated among females within their respective test group.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

the verification of dose was carred out by weighting of food portion and tested substance.

Duration of treatment / exposure:

2 yers/ exposure: with food

Frequency of treatment:

every day

Details on study schedule:

F0: After approximately 13 weeks (when the ras were about 120 days of age and sexually mature) matings were set up.

F1: Ten rats of each sex selected from as many litters as possible and representative of the average weight within the litters were assigned to the F1 generation group. They were raised to maturity in accordance with the same program as the parent generation. Similarly, groups of rats from second litters of the F1 generation and, in turn, the F2 and F3 generation, were each carried through the production of two litters.
When the F0 rats reached 2 years on test, the entire study was terminated.

Dose / conc.:

0 mg/kg bw/day

Remarks:

control group

Dose / conc.:

50 mg/kg bw/day

Dose / conc.:

125 mg/kg bw/day

Dose / conc.:

250 mg/kg bw/day

No. of animals per sex per dose:

25 males abd 25 females in each group (per dose)

Control animals:

yes, plain diet

Details on study design:

200 weanling rats were assigned to the threetest groups and one control group, of 25 males and 25 females ech. The rats were haused individually in raised-bottom cages, fresh water being availableat all times. The basal diet was a mixture of natural foods suplemented with inorganic salts and vitamis. Its composition resembles the food consumption pattern of the USA population with respect to the ratios of milk, meat, and grain components. Vitamins and minerals were presented at levels adequate for normal growth and development. The test material was added at such levels as to provide 50, 125, and 250 mg CaNa2EDTA (anhydrous basis) per kg bw of rat per day. Because f the initially high and gradually diminishing ratio of food intake to body weight during the period between weaning and maturity, adjustments of the proportion of test material in the diet were made biweekly up to the eleventh week. Since the food intake of rats at this time is normally stabilized at approximately 50 g / kg bw, the concentration of test material was kept constant for the remainder of the study.
The body weights of all rats and food intake of a representative sampling of 10 rats of each sex per group were recorded weekly. The efficiency of food utilization was calculated for 12 -week period which constitued the short-term phase of experiment. At 6 and 12 weeks, following determiantion were made on 1/4 of the rats of each group: Blood hemoglobin levels, red and white cell counts, differential white cell counts, prothrombin time, blood sugar and nonprotein nitrogen, and serum calcium levels. Ueine was examined for albumin and reducing sugar, and the sediment was examined microscopically. two rats of each sex per group were sacreficed for histopatologic axamination at 12 weeks.
Survivous of the short-term experiment (approximately 23 rats of each sex per group) were continued without change of dietary treatment for the remaider of the 2-year period. Growth records were maintained, but food consuption records were discontinued. All rats were inspected daily for physical condition; the hematologic, blood chmical, and urinary examinations were repeated at 1, 1.5, and 2 yers.

After approximately 13 weeks (when the ras were about 120 days of age and sexually mature) matings were set up with male and two females per cage. As pregnancy was recognized (visually, by palpation, or by weight increments) the dam was transferred to an individual cage. If pregnancy was not established by the third week, the male was replaced. A female was regarded as infertile and matings were discontinued after two successive mating failures. In successive matings the maleswere rotated among females within their respective test group.
Ten rats of each sex selected from as many litters as possible and representative of the average weight within the litters were assigned to the F1 generation group. They were raised to maturity in accordance with the same program as the parent generation. Similarly, groups of rats from second litters of the F1 generation and, in turn, the F2 and F3 generation, were each carried through the production of two litters.

The rats selected from each generation for breeding were continued on their respective diets for a 12-week feeding period, as described for the F0 (parent) generation. Following the weaning of the second litters in the descendant generation rats at the 50- and 125-mg/kg dosage levels were sacrified and examined grossly post mortem, but the control anf highest dosage level groups were continued without change in dietary treatment until about the end of the 2-year study. This scheme permited termminalobservation to be made on rats receiving test diets for 0.5, 1, 1.5, or 2 years, in the F3, F2, F1, and F0 generations, respectively.
All animals that died, or were sacrified at the specified periods were autopsed and weights were taken of livers, kidneys, spleens, hearts, adrenals, thyroids, and gonads. As stated above, representative animals in the F0 groups had been sacrificed at 12 weeks for histopatologic examination. Again at the end of the year, two males and two females at each dose level were sacrificed ans examined for gross pathologic changes. At both periods livers and kidneys of rats at the lower dose levels were examined microscopically. At the end of the study, 15 or more major organs and tissues were likewise examined in 10 or more rats of each sex in the 250 mg/kg and control groups.
Beccause the sequestring action of CaNa2-EDTA suggested the possibility of interference with mineral metabolism, certain additional examinations were made. These included determination of the ash content of the tibias of rats in the highest dosage and control groups; microscopic exammination of the jaws of representative animals (at 20 x magnfication) for evidence of dental caries; xanthine oxidase determinations in the liver (because molybdenum is a component of this enzyme); and cabonic anhydratase determinations in serum (because zinc is a component of this enzyme).

Positive control:

None

Parental animals: Observations and examinations:

Daily observation for physical conditin and behaior. The body weights of all rats and food intake of a representative sampling of 10 rats of each sex per group were recorded weekly. The efficiency of food utilization was calculated for 12 -week period.
At 6 and 12 weeks, following determiantion were made on 1/4 of the rats of each group: Blood hemoglobin levels, red and white cell counts, differential white cell counts, prothrombin time, blood sugar and nonprotein nitrogen, and serum calcium levels. Urine was examined for albumin and reducing sugar, and the sediment was examined microscopically. two rats of each sex per group were sacreficed for histopatologic axamination at 12 weeks.
Two rats of each sex per group were sacrificed for histopatologic examination at 12 weeks.
Survivors of the short-term experiment (approximately 23 rats of each sex per group) were continued without change of dietary treatment for the remaider of the 2-year period. Growth records were maintained, but food consumption records were discontinued. All rats were inspected daily for physical condition; the hematologic, blood chemical, and urinary examinations were repeated at 1, 1.5, and 2 years.

Oestrous cyclicity (parental animals):

No data

Sperm parameters (parental animals):

No data

Litter observations:

The same as for parents generation.

Postmortem examinations (parental animals):

Two rats of each sex per group were sacrificed for histopatologic examination at 12 weeks. Again at the end of the year, two males and two females at each dose level were sacrificed and examined for gross pathologic changes. At both periods livers and kidneys of rats at the lower dose levels were examined microscopically. At the end of the study, 15 or more major organs and tissues were likewise examined in 10 or more rats of each sex in the 250 mg/kg and control groups.

Postmortem examinations (offspring):

The rats selected from each generation for breeding were continued on their respective diets for a 12-week feeding period, as described for the F0 (parent) generation. Following the weaning of the second litters in the descendant generation rats at the 50- and 125-mg/kg dosage levels were sacrificed and examined grossly post mortem, but the control of highest dosage level groups were continued without change in dietary treatment until about the end of the 2-year study. This scheme permited terminal observation to be made on rats receiving test diets for 0.5, 1, 1.5, or 2 years, in the F3, F2, F1, and F0 generations, respectively.

Statistics:

Duncan multiple range and multiple F test

Reproductive indices:

Fertility Index (FI), the proportion of matings resulting in pregnancy
Gestation Index (GI), the proportion of pregnancies resulting in live litters

Offspring viability indices:

Viability Index (VI), the proportion of rats born that survive 4 days or longer
LActation Index (LI), the proportion of rats alive at 4 days that surive to weaning

Clinical signs:

no effects observed

Description (incidence and severity):

No siginificant abnormalities or differences in behavior or appearance of the rats in any of the various dose levels were observed in short or long term.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

The only deaths in the short-term studies were:
- 3 control males died in 12th week
- 1 female in the 50 mg/kg test group died in 12th week
- 1 female in the 125 mg/kg groupdied un 8th week.
No deadth occured in 250 mg/kg group.

At 1.5 years survival in all groups ranged from 62 to 86%. Subsequent losses due to death or to sacrifice of moribund rats reduced the groups to approximately half the original size at the 2-year point. In the last quarter of the 2-year period deaths were somewhat more frequent than has been observed previously in these laboratories, possibly because the entire rat colony was moved to new quarters during this period. Comparison of the test and control groups revealed no significant effects of the dietary treatments on the longevity of the rats.That the small diferences in mortality among thevarious groups were not the result of the dietary treatment is apparent from the response of the 250 mg group in which the average survival of the combined sexes was 61% compared to 45% for the control.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The growth of the experimental groups was as good as or better that of the control groups of the same sex. Growth in all groups proceeded at normal rate, plateauing at about 1 year. From the 2 -year responses in the F0 generation it can be seen that up to the 76th week the growth responses within all sexes at all levels were essentialy equel. During the final half-year the average body weights varied somewhat more because of premortal losses and deaths, but no significant variations occured in the intergroup relationsships.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Description (incidence and severity):

Please see description for body weight and weight changes above

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Short-term
No evidence of interference with hematopoetic process was observed. The hemoglobin levels, hematocrit, and red blood cell counts at all levels of dosage were normal. The leucocyte counts showde no trend with increasing dietary level of the test material, the ranges for all F0 groups being 12,400-18,700 and 8700-14,000for the males and females,respectively.

Long-term
The hemoglobin, hematocrit, and red blood cell counts all fell within normal ranges up to 1 year. Following this there was a slight downward trend in hemoglobin and red cells with advancing age in all groups, including the controls, but there were no dose-related differences. The minimum average values for the males and females, respectively, were 13.2 and 12.4 g hemoglobin per 100 ml, and 7.71 and 6.68 million red cell / cm3. The total and differential leucocyte counts likewise disclosed no effects attributable to the test material. In all groups, including control, the proportion of polymorphonuclear neutrophiles increased with advancing age, but even at the 250-mg dose level the values are similar to those found in the controls.

Prothrobin times, determined at 78 and 104 weeks in both the control and 250-mg groups, were normal, the averages ranging drom 15 to 19 second.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Short-term
The levels for blood sugar, blood NPN, and serum calcium were normal regardless of dietary treatment; in F0 eneration the averages ranged from 95 to113, 33 to 44, and 9.7 to 11.1 mg/100 ml, respectively.

Long-term
the ranges for average blood sugar, nonprotein nitrogen, and serum calciumvalues of all groups over the entire 2-year period were 92 to 114 mg, 31 to 49 mg, and 9.7 to 11.4 mg/100 ml, respectively, all being within normal limits.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Short-term
The findings were negative with respect to the urinary albumin and sugar, at all levels of dosage. A normal assortment of microscopical elements (a few leucocytes, occasional squamous or round epithelial cells, and a moderate number of crystals) was seen, but not to any greater extend in the test grous than in the controls.

Long-term
Urinary findings were essentially normal throughout, the only findings worthy of mention being the slight to moderate (and only occasionally marked) senile albuminuria in 1,5- to 2-year-old rats and the sporadic occurrence of oxalate crystals in all groups early in the test but not subsequently.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No siginificant abnormalities or differences in behavior of the rats in any of the various dose levels were observed in short or long term.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In histopatologic examinations of F0 gerenation rats sacrificed at 2 years, principal attention was directed to the highes dose level and the control groups. In the examinations of approx. 15 organs and tissues of 10 males and 10 females in each dose groups, a few organs revealed changes of such a nature and incidence as to suggest a possible relationship to dosage. Hence, the organs in which those deviations were found, namely, liver, pituitary, and sdrenal glands, were subsequently examined in the 50- and 125-mg/kg groups and in additional animals the 250-mg/kg and control groups.
The total incidennce of liver pathology for the combined sexes was not significantly greater in the 250-mg/kg group (13/40) than in the controls (11/40). This comparison, as well as he similar incidence at the two lower dosage, is the basis for the conclusion that these hepatic changes are not related to dosage. In the anterior pituitaries, focal hyperplasia was seen occasionally in the medulla, occured with a frequency which was not correlated to increasing dosage. Thus, it may be concluded that those changes were not causally related to test dosage.
The remaining organs examined histopatologiccally included kidneys, pancreas, heart, spleen, lungs, marrow, stomach, small and large intestines, gonads, thyroid, parathyroid, lymps nodes, spinal cord, and tibias. In none of those organs (including the few spleens of elevated weight) were significant morphological deviations observed.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Exept for mammary tumors which are fairly common in females with a history of continous breeding, the character and number of tumors obsered indicated them to be of an incidental nature. They occured with a frequency comparable to that usually seen in this colony.

Other effects:

no effects observed

Description (incidence and severity):

The test and

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Poor response with respect to some of the criteria of reproductive performance occured occasionally for no discernible reason. However, that they were not causally related to the test material may be inferred from the fact that they were not correlated with dosage or with number of generations throught which dosage continued.

Dose descriptor:

dose level:

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Clinical signs:

no effects observed

Description (incidence and severity):

No significant abnoralities of difference in behavior or appearance of rats in any of the generations or among the various dose levels were observed.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

No mortality observed in short-term study period (first 12 weeks). At 1.5 years survival in all groups ranged from 62 to 86%.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Growth data for F1, F2 and F3 generation rats in the control and highest dosage test groups was as good as or better than that of the control group.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Description (incidence and severity):

Growth data for F1, F2 and F3 generation rats in the control and highest dosage test groups was as good as or better than that of the control group.

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Short-term
No evidence of interference with the hematopoetic process was observed.
Long-term
The hemoglobin, hematocrit, and red blood cell counts all fell within normal ranges up to 1 year.
Following this there was a slight downward trend in hemoglobin and red cells with advancing age in
all groups, including the controls, but there were no dose-related differences. The minimum averag
e values for the males and females, respectively, were 13.2 and 12.4 g hemoglobin per 100 ml, and
7.71 and 6.68 million red cell / cm3. The total and differential leucocyte counts likewise disclosed no
effects attributable to the test material. In all groups, including control, the proportion of polymorph
onuclear neutrophiles increased with advancing age, but even at the 250-mg dose level the values ar
e similar to those found in the controls.
Prothrobin times, determined at 78 and 104 weeks in both the control and 250-mg groups, were
normal, the averages ranging from 15 to 19 second.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

The ranges for average blood sugar, nonprotein nitrogen, and serum calciumvalues of all groups over
the entire 2-year period were 92 to 114 mg, 31 to 49 mg, and 9.7 to 11.4 mg/100 ml, respectively, all
being within normal limits.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urinary findings were essentially normal throughout, the only findings worthy of mention being the slig
ht to moderate (and only occasionally marked) senile albuminuria in 1,5- to 2-year-old rats and the
sporadic occurrence of oxalate crystals in all groups early in the test but not subsequently.

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No significant differences were found for the liver, kidneys, spleen, heart, adrenals, gonadas, or thyroid
glands. Please see table

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

By virtue of their diverse charakter and sporadic distribution among the groups, the gross phatologic
findings were considered not to be causally related to test dosage. Pulmonary changes were typical
of the respiratory infection common in laboratory rats and their frequency in the test groups was, for
the most part, less than in the controls. Liver abnormalities also occured at least as frequently in the
control as in the test groups.
Exept for mammary tumors which are fairly common in females with a history of continous breeding,
the character and number of tumors obsered indicated them to be of an incidental nature. They
occured with a frequency comparable to that usually seen in this colony.
Microscopically, no important aberration were evident in the liver, kidneys, gastrointestinal tract, and
tibias of the four rats in each group selected for ascrifice either at 12 weeks or at 1 year. Especially
noteworthy is the fact thah in the 250-mg/kg group, in which 13 organs and tissues of each rat were
examined, the findings were consistently negative.

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The total incidennce of liver pathology for the combined sexes was not significantly greater in the 250
-mg/kg group (13/40) than in the controls (11/40). This comparison, as well as he similar incidence at
the two lower dosage, is the basis for the conclusion that these hepatic changes are not related to
dosage. In the anterior pituitaries, focal hyperplasia was seen occasionally in the medulla, occured wi
th a frequency which was not correlated to increasing dosage. Thus, it may be concluded that those
changes were not causally related to test dosage.
The remaining organs examined histopatologiccally included kidneys, pancreas, heart, spleen, lungs,
marrow, stomach, small and large intestines, gonads, thyroid, parathyroid, lymps nodes, spinal cord,
and tibias. In none of those organs (including the few spleens of elevated weight) were significant
morphological deviations observed.

Other effects:

no effects observed

Behaviour (functional findings):

no effects observed

Developmental immunotoxicity:

not examined

Dose descriptor:

dose level:

Generation:

F1

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Clinical signs:

no effects observed

Description (incidence and severity):

No significant abnoralities of difference in behavior or appearance of rats in any of the generations or
among the various dose levels were observed.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

No mortality observed in short-term study period (first 12 weeks). At 1.5 years survival in all groups
ranged from 62 to 86%.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Growth data for F1, F2 and F3 generation rats in the control and highest dosage test groups was as g
ood as or better than that of the control group.

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 specified

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No evidence of interference with hematopoetic process was observed. The hemoglobin, hematocrit, and red blood cell counts all fell within normal ranges up to 1 year.
Following this there was a slight downward trend in hemoglobin and red cells with advancing age in
all groups, including the controls, but there were no dose-related differences. The minimum averag
e values for the males and females, respectively, were 13.2 and 12.4 g hemoglobin per 100 ml, and
7.71 and 6.68 million red cell / cm3. The total and differential leucocyte counts likewise disclosed no
effects attributable to the test material. In all groups, including control, the proportion of polymorph
onuclear neutrophiles increased with advancing age, but even at the 250-mg dose level the values ar
e similar to those found in the controls.
Prothrobin times, determined at 78 and 104 weeks in both the control and 250-mg groups, were
normal, the averages ranging drom 15 to 19 second.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

The ranges for average blood sugar, nonprotein nitrogen, and serum calciumvalues of all groups over
the entire 2-year period were 92 to 114 mg, 31 to 49 mg, and 9.7 to 11.4 mg/100 ml, respectively, all
being within normal limits.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Short-term
The findings were negative with respect to the urinary albumin and sugar, at all levels of dosage and in all generations. A
normal assortment of microscopical elements (a few leucocytes, occasional squamous or round epit
helial cells, and a moderate number of crystals) was seen, but not to any greater extend in the test
grous than in the controls.
Long-term
Urinary findings were essentially normal throughout, the only findings worthy of mention being the slig
ht to moderate (and only occasionally marked) senile albuminuria in 1,5- to 2-year-old rats and the
sporadic occurrence of oxalate crystals in all groups early in the test but not subsequently.

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No significant differences were found for the liver, kidneys, spleen, heart, adrenals, gonadas, or thyroid
glands. Please see table

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

By virtue of their diverse charakter and sporadic distribution among the groups, the gross phatologic
findings were considered not to be causally related to test dosage. Pulmonary changes were typical
of the respiratory infection common in laboratory rats and their frequency in the test groups was, for
the most part, less than in the controls. Liver abnormalities also occured at least as frequently in the
control as in the test groups.
Exept for mammary tumors which are fairly common in females with a history of continous breeding,
the character and number of tumors obsered indicated them to be of an incidental nature. They
occured with a frequency comparable to that usually seen in this colony.
Microscopically, no important aberration were evident in the liver, kidneys, gastrointestinal tract, and
tibias of the four rats in each group selected for ascrifice either at 12 weeks or at 1 year. Especially
noteworthy is the fact thah in the 250-mg/kg group, in which 13 organs and tissues of each rat were
examined, the findings were consistently negative.

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The total incidennce of liver pathology for the combined sexes was not significantly greater in the 250
-mg/kg group (13/40) than in the controls (11/40). This comparison, as well as he similar incidence at
the two lower dosage, is the basis for the conclusion that these hepatic changes are not related to
dosage. In the anterior pituitaries, focal hyperplasia was seen occasionally in the medulla, occured wi
th a frequency which was not correlated to increasing dosage. Thus, it may be concluded that those
changes were not causally related to test dosage.
The remaining organs examined histopatologiccally included kidneys, pancreas, heart, spleen, lungs,
marrow, stomach, small and large intestines, gonads, thyroid, parathyroid, lymps nodes, spinal cord,
and tibias. In none of those organs (including the few spleens of elevated weight) were significant
morphological deviations observed.

Other effects:

no effects observed

Behaviour (functional findings):

no effects observed

Developmental immunotoxicity:

not examined

Dose descriptor:

dose level:

Generation:

F2

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

food efficiency

haematology

clinical biochemistry

urinalysis

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

Remarks on result:

not determinable due to absence of adverse toxic effects

Reproductive effects observed:

no

Conclusions:

Two-year feeding studies in rats showed no significant deviations from normal physiological responses nor any evidence of interference with mineral metabolism in rats at levels of calcium EDTA (CaNa2 EDTA) up to 250 mg/kg bw.

Executive summary:

In 2 -year feeding studies with rats receiving diets containing calcium EDTA (CaNa2 EDTA) at levels to provide 50, 125, or 250 mg/kg bw, no adverse effects on growth or food efficiency were observed. Hematologic examinations concluded periodically, and determination of prothrombin time , blood sugar, NPN, and serum calcium were likewise normal throughout the test period. Responses similar to those seen in the paren generation were observed in the rats of the three succeeding generations maintained on the same diet. Under the stresses of repeated pregnancies and lactation, no adverce effect of CaNa2 EDTA was observed as measured by any of the usual indexes of reproduction or lactation efficiency. At autopsy neither gross examination nor weights of the major organs discolsed any siginificant differences between the test and the control groups. The histopathologic findings likewise revealed no consistent or dose-related effects.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

yes

Remarks:

there were no further treatment of F1 weanlings

Principles of method if other than guideline:

The conduct of this study complied with recommendations published by the OECD Guideline for Testing Chemicals No. 416 with the exception that there were no further treatment of F1 weanlings. The conduct of this study includes also recommendations of OECD Guideline for Testing Chemicals No. 415 (adopted 1983).

GLP compliance:

yes

Remarks:

the OECD Principles of Good Laboratory Practice as revised in 1997 (ENV/MC/CHEM(98)17) and the German Principles of Good Laboratory Practice (Bundesgesetzblatt Part I No. 21 of May 14, 2001).

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

144538-83-0

Cas Number:

144538-83-0

IUPAC Name:

144538-83-0

Constituent 2

Reference substance name:

2-(1,2-Dicarboxy-ethylamino)-succinic-acid tetra- Na-salt

IUPAC Name:

2-(1,2-Dicarboxy-ethylamino)-succinic-acid tetra- Na-salt

Constituent 3

Reference substance name:

Imminodisuccinic acid, tetrasodium salt

IUPAC Name:

Imminodisuccinic acid, tetrasodium salt

Test material form:

other: water solution

Details on test material:

- Name of test material (as cited in study report): IDS, Na-Salt (Iminodisuccinat, Na-Salt)
- Molecular formula (if other than submission substance): C8H7NNa408
- Molecular weight (if other than submission substance): 337 g/mol
- Smiles notation (if other than submission substance): C(C(C(=O)[O-])NC(CC(=O)[O-])C(=O)[O-])C(=O)[O-].[Na+].[Na+].[Na+].[Na+]
- InChl (if other than submission substance): InChI=1S/C8H11NO8.4Na/c10-5(11)1-3(7(14)15)9-4(8(16)17)2-6(12)13;;;;/h3-4,9H,1-2H2,(H,10,11)(H,12,13)(H,14,15)(H,16,17);;;;/q;4*+1/p-4
- Structural formula attached as image file (if other than submission substance): see Fig.
- Substance type: chelating agent
- Physical state: powder (colorless to light yellow)
- Analytical purity: 73.4%
- Purity test date: April 9, 2001
- Lot/batch No.: FA 36610 (reserve sample is stored)
- Expiration date of the lot/batch: April 3, 2003
- Stability under test conditions: at least 15 days (at animal room conditions)
- Storage condition of test material: at room temperature

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Cri: (Wi) WU BR
- Source: Charles River Deutschland Sulzfeld, Germany (delivered on October 16, 2001)
- Age at study initiation: 6 - 7 weeks
- Weight at study initiation: (P) Males: 142 (124-168) g; Females: 123 (106- 139) g
- Fasting period before study:
- Housing: During the acclimatization period and study rats were housed singly under conventional conditions in Makrolon® cages. During the mating periods females were co-housed overnight with their males.
- Diet (e.g. ad libitum): ad libitum (NAFAG 9441 W10 Pellets, from January 02 or 04 2002 onwards declared as PROVIMI KLIBA 3883.015)
- Water (e.g. ad libitum): ad libitum (tap water during the acclimatization period and demineralized water throughout the study).
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2°
- Humidity (%): 55 ± 5
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: October 24, 2001 (first day of treatment) To: March 18, 2002 (last day of treatment)

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

IDS, Na-Salt was blended with demineralized water (see Table 2). The amounts of test item were calculated on the basis of the real content (73.4%) of IDS, Na-Salt. As a rule the drinking fluids were prepared once a week.

Analytical investigations on stability (at least over 15 days) of the test item in demineralized water preparations were done prior to the study or at study begin. Since the administration medium was a clear fluid homogeneity was not necessary to investigate. Four contents checks were done on mixtures given to the animals. Reserve samples from each mixture were stored at least for 8 weeks at about -20°C.

Details on mating procedure:

- M/F ratio per cage: 1/1 (overnight from about 4 p.m. to 8 a.m.)
- Length of cohabitation: maximum of 12 times during the three-week mating period
- Proof of pregnancy: [sperm in vaginal smear] referred to as day 0 of pregnancy
- F0 females found sperm-positive after the first mating day but were shown to be not pregnant were co-housed again over one week with the same male without checking insemination or measuring body weight and water intake during possible further pregnancy.

Females which exhibited marked weight gains although insemination had not been established were not further co-housed. No duration of pregnancy could be determined for these animals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Stability and homogenity of test material for toxicological investigations: study No. N00/0054/03 LEV and
- Content of test substance in formulations: study No. N00/0054/07 LEV.

Duration of treatment / exposure:

The test substance was administered to parental (P) animals prior (10 weeks) to and during their mating (up to 21 days), during the resultant pregnancy (about 22 days), and through the weaning of their F1 offspring (28 days).

Frequency of treatment:

continuously throughout the whole exposure period

Details on study schedule:

The FO animals were pretreated with the compound for about 10 weeks up to the cohabitation period. Within the weeks 6 - 8 of this premating period investigations on estrus cycle were performed. During the following mating period the first male was co-housed with the first female FO animal within the group and so on over night at a maximum of 12 times during the three-week mating period. Inseminated females were not further co-housed. Insemination was established by investigating vaginal smears prepared in the morning. After a gestation period of about 22 days litters were born and the dams were allowed to rear them. If necessary, four days after birth the F1 litters were reduced (= culled) to eight pups according to random lists. If possible, four male and four female pups remained per litter. Pups found in a moribund state at day 4 were excluded from lactation immediately. This was done to investigate possible malformations and to prevent cannibalism during the further rearing period. The remaining F1 pups were raised to an age of four weeks and then necropsied. FO females were killed and necropsied when 28 day old F1 animals had been weaned. FO males were killed after the mating period partly in the course of spermatological investigations

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 1000, 4000 and 16000 ppm
Basis:
nominal in water
corresponds to 0, 105.3 - 140, 411 - 480.1 and 2081.4 - 2270.8 mg/kg bw for males and females, respectively.

No. of animals per sex per dose:

25

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale:
The dose selection is based on results of a subchronic toxicity study where 0, 5000, 10000 or 15000 ppm IDS, Na-Salt were given over 13 weeks in their diet (EIBEN, R. BAYER Report PH-32188, 2002). In this study no compound-related toxic effects were evident. Due to difficulties arising from analytical recovery of the test compound in the diet, it was decided to administer IDS, Na-Salt in the one-generation study via drinking fluid instead of the diet.
To prove the tolerance of IDS, Na-Salt in the drinking fluid for rats a 4-week study was performed where 0, 10000 or 15000* ppm IDS, Na-Salt were ingested with the drinking water (EIBEN, R. BAYER Report PH-32189, 2002). In this study 10000 ppm were tolerated without any effects. At 15000 ppm there was an increase in water intake in males and enhanced kidney weight in both sexes. Thus the concentration levels of 0, 1000, 4000 and 16000* ppm was be used in the one generation study.
* this concentration was thought to result in a dose >2000 mg/kg body weight per day in the planned one-generation study.

- Rationale for animal assignment (if not random): randomization
- Other:

Positive control:

None

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (once daily on weekends and public holidays)
Any findings e.g. prolonged parturition, morbidity and mortality noticed during this cage side observation were recorded. The persistence of those cage side noticeable clinical signs, which had been already noted during weekly detailed clinical observation were recorded daily but not reported separately.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: On all F0 parental animals a detailed clinical observation was done at least once weekly as routine at their cage change in the week. All clinical symptoms were recorded. This investigation includes the evaluation of the general state of health, behavior, condition of the fur, and the orifices as well as excretory products were examined and recorded. During gestation periods females were clinically examined on day 0, 7, 14, 20, and during lactation on dayO, 4, 7, 14, 21, and 28.

BODY WEIGHT: Yes
- Time schedule for examinations: All FO animals were weighed at the start of the study (first day of dosing). The FO males were weighed at weekly intervals up to necropsy. Female FO animals were weighed weekly until week 10 (= end of premating period). After insemination had been established, the female animals were weighed on postcoital days 0, 7, 14 and 20; and on days 0, 4, 7, 14, 21 and 28 after birth of their pups. F0 animals were weighed at the day of necropsy to permit calculations of the relative organ weights. As a rule females not inseminated during mating periods were weighed weekly (data not shown).
In F0 males water intake was measured weekly up to necropsy on a seven day basis except during mating period, whereas food intake was measured in the premating period only. In F0 females food and water intake was measured in the same way during the premating period.
During gestation period water intake was measured from day 0 to 7, 7 to 14 and 14 to 20 p.c. and during lactation from day 0 to 4 and 4 to 7.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: daily
The food and water intake was recorded by weighing the quantity of food/water provided and back-weighing the amount which remained unconsumed.
From these primary data the following was calculated:
a) daily food/water intake per animal
b) mean daily food/water intake per animal
c) mean daily food/water intake per kg body weight
d) mean daily test substance intake per kg body weight

Averaged for each premating period
e) mean food/water intake per animal and day
f) mean food/water intake per kg body weight and day
g) cumulative food/water intake per animal
h) cumulative food/water intake per kg body weight
i) mean test substance intake per animal and day
j ) mean test substance intake per kg body weight and day
k ) cumulative test substance intake per animal
I) cumulative test substance intake per kg body weight

OTHER:

Oestrous cyclicity (parental animals):

Vaginal smears were taken daily during the week 6-8 of premating period (19 days). All females per dose level were used. The vaginal smears were examined microscopically whether large serrated cells indicating estrus had occurred. These data were used to characterize the estrus cycle length and to determine if females were cycling properly.

Sperm parameters (parental animals):

Spermatological investigations were performed in all living F0 males of the 0 and 16000 ppm group.
Parameters examined in P male parental generations: testis weight, epididymis weight, sperm count in epididymides, sperm motility, sperm morphology.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring shortly after birth (on postpartum day 0), day 4 (before and after reduction), 7, 14, 21, and 28.: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical and behavioural abnormalities.

GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead. Pups that were found dead at birth, that died during lactation as well as those killed (with carbon dioxide) in moribund condition were macroscopically inspected after opening the body cavities, with particular attention on the organs of reproduction, except in case of autolysis or cannibalism.
A lung flotation in water was performed during the necropsy of pups found dead on the day of the first litter inspection to determine whether pups had breathed at birth or not. Macroscopically changed organs were fixed in 10% formalin.

Postmortem examinations (parental animals):

SACRIFICE
Parent animals that died or were killed in moribund condition (under diethyl ether narcosis) during the study were necropsied and macroscopically examined.
- Male animals: All surviving animals. F0 males were killed as scheduled under carbon dioxide narcosis when they were not required for further treatment. They were necropsied and macroscopically examined in the same way. In some cases this was done during the course of spermatological investigations.
- Maternal animals: All surviving animals After the F1 pups had been weaned, the dams were anesthetized with carbon dioxide and killed by exsanguination and examined for gross pathology.

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs/organ specimen of the FO animals were fixed in 10 % neutral buffered formalin solution: Adrenals, brain, liver, spleen, urinary bladder, pituitary gland (in situ at head), thyroid glands with parathyroids, trachea, larynx and esophagus as well as vagina, uterus (with cervix), ovaries, oviducts, mammary gland with skin, epididymides, coagulation glands, seminal vesicles, prostate, preputial glands, urethra, tattooed ears and all organs/organ specimen exhibiting macroscopic changes. The testes (if sperm analysis was done one organ only) and kidneys were fixed in Davidson's solution.
Organ weight determinations of the brain, pituitary gland (fixed), liver, kidneys, adrenals, spleen, thyroid (fixed, only one organ), uterus, seminal vesicles with coagulation glands, prostate, epididymides (only left organ), testes and ovaries were done during the scheduled necropsy.
The fixed organ samples except physical identifier, trachea, urethra with preputial glands and head were investigated histopathologically.
All these organ specimen were evaluated in rats of the 0 and 16000 ppm group. Pituitary gland, kidneys, urinary bladder, fixed organs of decedents and gross lesions were evaluated in all groups.

Postmortem examinations (offspring):

SACRIFICE

Scheduled Necropsies
The pups selected for litter reduction were killed with carbon dioxide on postpartum day 4. Weanlings were killed under carbon dioxide anesthesia by cervical dislocation on postpartum day 28. Both, pups selected for culling as well as weanlings were examined for macroscopical alteration.
When the F1 rats were weaned (day 28) they were anesthetized with carbon dioxide, killed by exsanguination and necropsied.

- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. Gross lesions, testes, epididymides, seminal vesicles, coagulation glands, prostate, ovaries/oviducts, vagina, uterus/cervix, liver, urinary bladder, kidneys and adrenals were fixed in 10% formalin from one male and one female from five litters/group.

ORGAN WEIGTHS
The body weights and the weights of brain, spleen, thymus and uterus were determined in the first male and first female living F1 weanling per litter.

Statistics:

The following methods were used to test for statistical significance:
a. The Dunnett-Test in connection with a variance analysis for
- Body weights of parent animals
- Organ weights of parent animals
b. The Kruskal-Wallis-Test with a Steel-Test for food/water consumption data These calculations were performed using SAS®routine on a HP 3000 computer system.
c. For parametric data of the reproductive parameters the two-tailed Fisher's exact probalitity test (in case of a positive CHI-SQUARE test with a significance levels of a = 5%) was used. A Fisher's exact CHI-SQUARE test was also used for sperm motility and morphology data.
d. For nonparametric data of the reproductive parameters the Dunnett-Test (in case of a positive ANOVA test with a significance levels of a = 5%) was used.
These calculations were preformed using a HP Vectra PC connected with an Alpha computer TASC (Grosse) system.
The sperm and spermatid count data were evaluated with the t-test using the Excel program.
The mean pup weight of each individual litter was used as the basis for calculating the pup weight means of the dose groups. The litter size calculation was based on the number of female animals with living pups.
Results of organ weights of F1 weanlings were evaluated statistically using a student t-Test on Excel-basis and calculations described by Keller, F 1982: Statistik fur naturwissenschaftliche Berufe. Incidences of clinical symptoms (parental and pup generation) were not evaluated statistically.

Reproductive indices:

Insemination index (%) = (No. of sperm positive females*/ No. of females co-housed with a male) x 100

Fertility index (%) = (No. of pregnant females/ No. of sperm positive females*) x 100

Gestation index (%) = (No. of females with live pups / No. of pregnant females) x 100

Rearing index (%) = (No. of females reared a litter up to weaning/ No. of females born a litter) x 100
* including pregnant females that were not sperm positive.

Offspring viability indices:

Live birth index (%)§ = (No. of live pups at birth/ total No. of pups born) x 100
Viability index (%)§ = (No. of live pups on day 4 pre-culling/ No. of live pups born) x 100
Lactation index (%)§ = (No. of live pups after three/four weeks No. of live pups after four days (after culling)** x 100

** moribund pups that died during the course of culling were not included.
§ Index calculation from litter means.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

diarrhoea (the highest dose group) and two dead dams

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

reduced (the highest dose group)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

reduced (the highest dose group)

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

treatment-related alterations in urinary tract and kidneys in the highest dose group

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Test substance intake: The compound intake values correspond to the theoretical dose intervals; slightly increase in the highest dose group

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No test substance-related effects on the appearance, health or behavior were observed in male or female F0 animals at levels of up to 4000 ppm. All 16000 ppm 18/25 males and 11/25 females showed diarrhoea.
In the 16000 ppm group two females (No. 180 and 200) were killed unscheduled. Both of them exhibited strong clinical signs of bad conditions. Female No. 180 exhibiting 12 unborn living pups was killed on day 20 p.c. and female No. 200 had delivered one stillborn and showed several implantation sites.
The necropsy findings of these animals did not indicate other changes that could be taken as a death cause (see Pathology Report). However, for both dams unspecific complications in parturition most probably due to the treatment are assumed. Therefore, a slight increase in mortality is stated for the 16000 ppm group in females

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
The body weight development of male and female FO animals receiving up to 4000 ppm IDS, Na-Salt were not toxicologically relevantly reduced. Significancies occurring among the body weight means of 4000 ppm males (week 2-4) are too small to reflect an adverse effect on growth.
F0 males ingesting 16000 ppm exhibited significantly reduced body weights nearly throughout the whole study (maximal -11.5% in week 2), whereas females ingesting 16000 ppm showed significant reduced body weights during lactation only (maximal -8.7% on day 14 p.p. see in the tables under week 17).
The mean daily food intake per animal and per kg body weight, as well as the corresponding cumulative consumption figures for each study group averaged over the whole premating period. The food intake values were not remarkable changed in treated F0 males and females receiving up to 16000 ppm. Sporadically occurring significances among the means calculated from the weekly food intake are based on very slight deviations to control values, which were in week 2 unexpectedly high for males.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
The mean daily water intake per animal and per kg body weight, as well as the corresponding cumulative consumption figures for each study group averaged over the whole premating period. The water intake values per animal and per body weight were dose-independently higher and wide spreading in treated F0 males and females than in controls and gained statistical significancies in all dose groups nearly during the whole study duration.
From 1000 to 4000 ppm the intake of IDS, Na-Salt corresponds roughly to the theoretical dose intervals. Concerning 16000 ppm rats a somewhat higher compound intake than expected from the theoretical dose factor was evident.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Estrus staging was done over 19 days in week 6-8 before F0 rats were co-housed for mating. As can be seen from Table 6 there was no adverse effect on the estrus cycle parameters up to 16000 ppm.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
In males receiving 16000 ppm there were no remarkable changes in motility and morphology of sperms (Table 4). The mean frequency of sperm abnormalities in samples of the 16000 ppm group was comparable with that at 0 ppm.
The male No. 16 (0 ppm) had no sperms. In male No. 90 (16000 ppm) the number of sperms was too low for the evaluation of motility and morphology. The number of spermatids and sperms counted in the right epididymis or testis was not affected (Table 5). In summary there were no statistical significances among the sperm parameters. Therefore, no sperm analyses were done in 1000 and 4000 ppm males.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
The calculated indices of insemination, fertility and gestation, as well as the mean duration of pregnancy per group are listed in the Table 8.The insemination, fertility and gestation indices as well as the mean duration of pregnancy did not differ to a toxicologically relevant extent from the pertinent control data at levels of up to 16000 ppm. The somewhat low gestation index is covered by historical control data and considered to be incidental.
There were some F0 females (0-3-3-0 with ascending dose) which had been found to be sperm-positive after the first day of co-housing, but failed to become pregnant. According to experience this could happen, if an inexperienced male, co-housed with a female for the first time, inseminated the female outside the estrus. This assumption is obviously correct, since only two (0-1-1-0) of these animals had no pups when remated with the same male for one week following the three week co-housing period.An overview over the mating performance of the F0 animals is given in Table 9. Only those mated females were included, in which the mating date could be determined by detection of sperms in the vaginal smear or vaginal plug. The mating performance was not toxicologically changed by the treatment at levels of up to 16000 ppm.

ORGAN WEIGHTS (PARENTAL ANIMALS)
There were mostly significantly higher absolute and relative kidney weights in 16000 ppm rats (Table 3.).
The absolute liver weights were about 10% significantly reduced (p<0.01) at 16000 ppm in males most probably due to the reduced body weights. The pituitary weights were not remarkable changed up to 16000 ppm in males and 4000 ppm in females. At 16000 ppm slightly higher absolute and relative means were calculated for females, which were both p<0.01. The weights of the remaining organs were not changed in a toxicologically relevant manner. Significancies occurring for these organs base on very small and/or dose independent deviations from control values.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There no remarkable gross pathological findings were made at necropsy of male or female F0 animals at levels of up to 4000 ppm. High dose rats exhibited some treatment-related organ changes such as dilation of renal pelvis (males:1-1-1-4 females: 1-0-0-7) as well as swollen pituitaries (0-0-0-4), dilated ceca (0-0-1-9) and dilated ureters (0-0-0-5) in females.
Concerning uterine implantation sites counted during necropsy no toxicologically relevant discrepancies between the numbers of implantation sites and those of delivered pups occurred, if controls and treated pups are compared (Table 2.). Accordingly there was no significant change in prenatal loss up to 16000 ppm.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No remarkable organ changes were found up to 4000 ppm. Treatment-related alterations were found in the urinary tract of males and/or females at 16000 ppm (see Table 7). These were degenerative and reparative changes in the kidneys, especially basophilic tubuli, tubular dilations and hyperplasias in the renal pelvis, pelvis dilations and increased amounts of renal mineralization indicating an altered pH of the urine. Males revealed additionally a reduction of male-specific hyaline droplet accumulation.
Slight hyperplasia of the transitional cell epithelium occurred in the urinary bladder. In general, females were much more affected than males, as could be shown by degenerative changes in the renal cortico-medullary junction and in the ureters (only gross findings examined) which occurred exclusively in this gender. Apart from a slightly increased number of inflammatory cells in a few females, no further alteration was seen corresponding to the gross lesion of dilated ceca.
In the pituitary gland 15/25 females of the high dose group showed slightly hypertrophic cells within the pars distalis. Additionally, several females of this group revealed an increased vacuolation of the pars nervosa.
No treatment-related changes were observed in the reproductive organs of either high dose males or females.

OTHER FINDINGS (PARENTAL ANIMALS)

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

overall parental toxicity and reproduction

Effect level:

4 000 ppm (nominal)

Based on:

dissolved

Remarks:

in drinking water

Sex:

male/female

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

16 000 ppm (nominal)

Based on:

dissolved

Remarks:

in drinking water

Sex:

male/female

Basis for effect level:

other: no adverse effects on sperm parameters, estrous cycles, reproductive organs and reproduction performance in all dose groups

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

pups in the highest dose group were pale

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

slightly depressed pup weights during lactation (sporadical significance)

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

slightly reduced absolute spleen weights (-13%) in 16000 ppm females, which were statistical significant

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

VIABILITY (OFFSPRING)
The life birth indices, the total number of born pups, the mean litter size and the percentages of males born were not changed toxicologically relevant up to 16000 ppm (Table 10). The viability, rearing and lactation indices of the treatment groups were not significantly different from those of the 0 ppm group (Table 12).

CLINICAL SIGNS (OFFSPRING)
No remarkable clinical signs were observed in F1 pups during the four week lactation period at levels of up to 4000 ppm. At 16000 ppm more pups were pale than in other groups. Malformations were not observed.

BODY WEIGHT (OFFSPRING)
As shown in Table 11 the birth weights of pups of the treatment groups were not significantly reduced. During lactation the weight of pups at 1000 and 4000 ppm was not affected. At 16000 ppm slightly depressed pup weights were obvious in both sexes, which showed sporadically statistical significance.

ORGAN WEIGHTS (OFFSPRING)
The Table 13 summarizes the calculated means per group. There were no remarkable changes in organ weight means up to 4000 ppm. Significantly reduced absolute and/or relative organ weights found in these groups were of no toxicological relevance because a dose dependence is absent. There were slightly reduced absolute spleen weights (-13%) in 16000 ppm females, which were statistical significant.

GROSS PATHOLOGY (OFFSPRING)
In F1 pups necropsied during the lactation period up to weaning no macroscopical alteration showing a treatment-related distribution was observed up to 16000 ppm. No skeletal deviations were determined in the F1 pups, which died before day four p.p., were killed in the process of litter reduction on postpartum day four, or were necropsied unscheduled during lactation at levels of up to 16000 ppm.

Effect levels (F1)

1

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Generation:

F1

Effect level:

4 000 ppm (nominal)

Based on:

dissolved

Remarks:

in drinking water

Sex:

male/female

Basis for effect level:

other: based on retarded body weight development in the highest dose group (16000 ppm) with the consequence that female pups exhibited reduced spleen weights. At 16000 ppm more pups were pale than in the other groups.

2

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

16 000 ppm (nominal)

Based on:

dissolved

Remarks:

in drinking water

Sex:

male/female

Basis for effect level:

other: No test substance-related gross pathological findings were observed in F1 offspring up to 16000 ppm. The skeletal development of the offspring was unaffected.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 2. Evaluation of Implantation Sites in F0 Females

Dose	No. of Implantation	No. of Pups at Birth	Prenatal Loss
ppm	Sites		%
0	283	255	9.9
1000	308	261	15.3
4000	293	266	9.2
16000	264	247	6.4

Table 3. Organ Weights of F0-Rats

Absolute Organ Weights of F0-Rats (mg)
Dose (ppm)	BW (g)	Brain	Pituitary	Adrenals	Thyroids§	Liver	Spleen	Kidneys	Testes	Epididymides §	Seminal vesicle	Prostate
males
0	437	1868	11	59	13	16176	738	2849	3369	819	1496	1074
1000	453	1958++	13++	47++	15++	17123	730	2837	3539	740+	1534	1093
4000	428	1912	13++	46++	14	15998	687	2789	3283	698++	1405	1006
16000	413+	1863	11	62	13	14701++	734	2966	3408	754+	1596	1011
									Ovaries	Uterus
females
0	241	1765	13	55	11	11096	498	1873	125	484
1000	244	1776	14	55	12	11295	564	1894	124	499
4000	251	1763	15++	58	12	12187	563	2003	126	479
16000	242	1755	20++	60	12	11638	471	2431++	115	571
Relative Organ Weights of F0 Rats (mg/ 100 g body weight)
Dose (ppm)	BW (g)	Brain	Pituitary -	Adrenals	Thyroids§	Liver	Spleen	Kidneys	Testes	Epididymides§	Seminal vesicle	Prostate
males
0	437	428	3	13	3	3694	168	651	771	187	343	246
1000	453	433	3ns	10++	3+	3775	161	626	784	164++	340	241
4000	428	449+	3++	11++	3+	3725	161	651	759	163++	330	234
16000	413+	452++	3ns	15+	3ns	3554	178	718++	825	183	387+	245
									Ovaries	Uterus
females
0	241	736	5	23	5	4595	206	779	52	202
1000	244	732	6ns	23	5	4603	233	777	51	205
4000	251	703	6+	23	5	4846	225	798	50	190
16000	242	729	8++	25	5	4803	195	1008++	48	237

§ Unilateral determination

+ difference against control for p ≤ 0.05% significant

++ difference against control for p ≤ 0.01% significant

Table 4. Evaluation of Sperm Motility and Morphology (F0)

	Sperm Motility
Dose (ppm)	1^stmin	5^thmin	Difference	Abnormal Sperms
	%	%		%
0	80	69	-11	0.65
16000	77	69	-8	0.50

Table 5. Evaluation of Sperm and Spermatid Counts (F0)

Evaluation of Sperm and Spermatid Counts
	Mean Number of
Dose (ppm)	Spermatids per mg Testis	Sperms per mg Epididymis
0	52361	887819
16000	49826	910798

Table 6. Evaluation of the Estrus Cycle in F0 Females

Dose (ppm)	Mean Cycle Length (in Days)	Mean No. of Cycles (in 19 Days)	No. of Females (Cycling Normally)
0	4.88	2.88	20
1000	4.40	3.12	20
4000	4.53	3.04	20
16000	4.84	2.83	20

Applicant's summary and conclusion

Conclusions:

NOAEL for overall parental toxicity and reproduction is 4000 ppm (corresponds to 411.0 and 480.1 mg/kg bw in males and females, respectively).
NOAEL for fertility is 16000 ppm (corresponds to 2081.4 and 2270.8 in males and females, respectively).
NOAEL for systemic toxicity of F1 is 4000 ppm.
NOAEL for developmental toxicity is 16000 ppm.

Executive summary:

The purpose of this one-generation study was to evaluate possible effects of IDS, Na-Salt on the entire reproduction process in Wistar rats. IDS, Na-Salt was administered to groups of 25 male and 25 female rats each at concentrations of 0 (control), 1000, 4000 and 16000 ppm in demineralized drinking water (correspond to 0, 105.3 -140.0, 411.0 - 480.1 and 2081.4 - 2270.8 mg/kg bw in males and females, respectively). Parental F0 animals were pretreated over a period of about 10 weeks and allowed to mate over a period of up to three weeks. F1 offspring were nursed up to an age of four weeks. Clinical signs, body weights, food and water intake, mating performance, fertility, duration of pregnancy, estrus cycling and sperm parameters were examined in F0 rats. Litter size, percentage of males born and pup weight at birth as well as viability, rearing, lactation and body weight gain were studied in F1 offspring. Necropsies were done in all rats. Implantation sites were recorded in F0 females. Selected organs were weighed (F0 and F1) and histopathological evaluations were performed on some organs of F0 rats.

Mortality and clinical appearance in F0 animals were unchanged at levels of up to 4000 ppm. At 16000 ppm diarrhoea was noted in both sexes and 2/25 females exhibiting histopathologically a strong kidney damage were killed because of complications at their parturition. At 16000 ppm retarded body weights were evident in parental rats. The food intake was unchanged up to 16000 ppm. The uptake of drinking fluid was higher in treated F0 rats than in controls. At 1000 and 4000 ppm the increase in water intake is most likely a result of the enhanced salt content of the drinking fluid and not considered as adverse. At 16000 ppm, however, a correlation to diarrhoea and several morphological changes in the urinary tract described below and increase in kidney weights found in this group cannot be excluded. The reproduction parameters insemination index, mating performance, fertility index, gestation index, duration of pregnancy, life birth index, birth weights, percentages of males born, total number of pups born, prenatal loss, mean litter size at birth as well as viability, rearing and lactation index were not affected at levels of up to 16000 ppm.

The body weight development of F1 pups was retarded at 16000 ppm with the consequence that female pups exhibited reduced spleen weights. At 16000 ppm more pups were pale than in the other groups.

No test substance-related gross pathological findings were observed in F1 offspring up to 16000 ppm. The skeletal development of the offspring was unaffected. No adverse effect was seen in sperm parameters at 16000 ppm. F0 females exhibited no treatment effects on estrus cycling up to 16000 ppm.

At 16000 ppm degenerative and reparative alterations in the kidneys such as (macro-and microscopical) dilations and hyperplasias in the renal pelvis and increased amounts of renal mineralization, a reduction of male-specific hyaline droplet accumulation and basophilic tubuli occurred more frequently in male and/or female F0 rats. Slight hyperplasia of the transitional cell epithelium occurred in the urinary bladder. Furthermore, ureters of high dose females were dilated macroscopically and degeneratively changed. These changes are most likely due to the marked overload with sodium of the drinking fluid and indicate severe parental toxicity. In the pituitary of 16000 ppm females hypertrophic cells within the pars distalis and vacuolation of the pars nervosa were noted more frequently. In 16000 ppm females, pituitaries were macroscopically swollen and increased in their weights as a correlate to this. The toxicological relevance of these findings are debatable and most probably not compound specific, but due to general toxicity of this dose.

An increase in inflammatory cells of the cecum in 16000 ppm females is considered as a correlate to cecum dilations seen macroscopically and diarrhoea. Gross pathology and histopathology in remaining organs revealed no treatment-related lesions.

Thus, the concentration of 4000 ppm in the drinking fluid is established as the overall no observed adverse effect level (NOAEL) for the parent animals and reproduction parameters. The NOAEL for the fertility is 16000 ppm.