Reaction mass of disodium [2,2'-(imino-κN)dibutanedioato-κ2O1,O4(4-)]calcium(2-), sodium chloride and aspartic acid disodium salt

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

migrated 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.

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.