Ferrate(2-), [[N,N'-1,2-ethanediylbis[N-[(carboxy-.kappa.O)methyl]glycinato-.kappa.N,.kappa.O]](4-)]-, (OC-6-21)-

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

one-generation reproductive toxicity

Remarks:

based on generations indicated in Effect levels (migrated information)

Type of information:

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

Adequacy of study:

key study

Study period:

September-December 2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well conducted study according to GLP (as the data is used in a read-across approach, a maximal reliability score of 2 was attributed).

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

premating period was extended from 2 to 10 weeks to cover a full sperm cycle

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

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

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

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

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

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

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

VEHICLE: tap water
- Concentration in vehicle: 0, 15, 50 and 150 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 1
- Length of cohabitation: 1 week
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: not done.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: individually
- Any other deviations from standard protocol: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations of managanese measured by ICP-AES in the gavage liquids prepared on 15 September, 17 November and 8 December 2009, respectively were ‘close to intended’ for all gavage liquids at all dose levels, except for the mid-dose level liquids prepared on 15 September and 17 November 2009 (+13.6% and +11.6%, respectively).

Zinc in the diets was also measured by ICP-AES and considered to be homogeneously distributed in the diet of group 5 which was prepared on 15 September 2009. Partly due to the higher than anticipated zinc concentration in the basal diet (77.9 mg/kg instead of 52 mg/kg) the content of zinc in the diet of group 5 was higher than intended (560 mg/kg diet instead of 500 mg/kg diet).

Duration of treatment / exposure:

10 weeks pre-mating, 1 week mating, 3 weeks gestation, and 4 days lactation

Frequency of treatment:

single daily application by gavage (parentla animals)

Details on study schedule:

- F1 parental animals not mated until [...] weeks after selected from the F1 litters: not applicable as F1 animals were killed on postnatal day 4.
- Selection of parents from F1 generation when pups were [...] days of age: not applicable as F1 animals were killed on postnatal day 4.
- Age at mating of the mated animals in the study: 20-21 weeks

Remarks:

Doses / Concentrations:
0, 150, 500 and 1500 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on studies done with EDTA
- Rationale for animal assignment (if not random): computer randomization proportionately to BW

Positive control:

An additional group was included to examine differences in chelating effects of the high dose in the presence of an extra amount of dietary zinc (ca. 500 ppm instead of ca. 50 ppm), if any.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: observations outside the home cage were made once weekly; FOB and motor activity were assessed in week 8 of the pre-mating period.

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

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

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

Oestrous cyclicity (parental animals):

Not measured

Sperm parameters (parental animals):

Parameters examined:
testis weight, epididymis weight: 12 rats/group
sperm count in epididymides, sperm motility, sperm morphology: 5 rats/group
sperm count in testes, daily sperm production: 5 rats/group

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no, because this screening study was ended on day 4 post-partum

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals as soon as possible after mating
- Maternal animals: All surviving animals at or shortly aftre day 4 of lactation

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

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

HISTOPATHOLOGY:
- ovaries, uterus (12 rats/group; control and high dose groups (with and without additional zinc))
- testes, epididymides, seminal vesicles, prostate, coagulating glands (12 rats/group; control and high dose groups (with and without additional zinc))
- adrenals, axillary lymph nodes, brain, caecum, colon, femur, Peyer's patches, heart, liver, lungs, mesenteric lymph nodes, peripheral nerve, rectum, small intestines, spinal cord, spleen, stomach, thymus, thyroid, trachea/bronchi, urinary bladder (5 rats/sex/group; control and high dose groups (with and without additional zinc))
- kidneys (all animals of all groups)

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring was sacrificed at 4 days of age.
- These animals were subjected to postmortem examinations (macroscopic) externally for gross abnormalities

GROSS NECROPSY
- Gross necropsy consisted of external examinations; pups were stored in a freezer for possible skeletal analyses (not done).

ORGAN WEIGHTS: not done

HISTOPATHOLOGY: not done

Statistics:

- Clinical findings were evaluated by Fisher's exact probability test.
- Body weight, body weight gain, organ weights and food consumption data were subjected to one way analysis of variance (ANOVA).
- Fisher's exact probability test was used to evaluate the number of mated and pregnant females
and females with live pups.
- Number of corpora lutea, implantation sites, live and dead fetuses or pups were evaluated by
Kruskal-Wallis nonparametric analysis of variance.
- Mortality data and data of the pathology of parent females were evaluated by the Fisher’s exact probability test.
- Functional observational battery: one-way analysis of variance followed by Dunnett’s multiple comparison tests (continuous data), Kruskal-Wallis non-parametric analysis of variance followed by multiple comparison tests (rank order data) or Pearson chi-square analysis (categorical data).
- Motor activity data-total distance moved: one-way analysis of variance followed by Dunnett’s multiple comparison tests; habituation of activity: repeated measures analysis of variance on time blocks (each session consists of 5 time blocks of 6 minutes each).
- Sperm parameters were evaluated by ANOVA followed by Dunnett’s multiple comparison test (epididymal and testicular sperm count and numerical sperm motility parameters) or by Kruskal-Wallis non parametric ANOVA followed by Mann-Whitney U test (motility parameters expressed as a percentage and sperm morphology).

Reproductive indices:

- pre-coital time = time between the start of mating and successful copulation
- duration of gestation = time between gestation day 0 and day of delivery
- mating index= (number of females mated/number of females placed with males) x 100
- male fertility index = (number of males that became sire/number of males placed with females) x 100
- female fertility index = (number of pregnant females/number of females placed with males) x 100
- female fecundity index = (number of pregnant females/number of females mated) x 100
- gestation index = (number of females with live pups or pups/number of females pregnant) x 100
- pre-implantation loss = [(number of corpora lutea – number of implantation sites)/number of corpora lutea] x 100
- number of lost implantations = number of implantations sites - number of pups born alive
- post-implantation loss = [(number of implantation sites - number of pups born alive)/number of implantation sites] x 100

Offspring viability indices:

- live birth index = (number of pups born alive/number of pups born) x 100
- viability index day n-m= (number of pup surviving m days/number of liveborn on day n) x100
- pup mortality day n = (number of dead pups on day n/total number of pups on day n) x 100
- sex ratio day n = (number of live male fetuses or pups on day n/ number of live fetuses or pups on day n) x 100

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

effects observed, treatment-related

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): no effects

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

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS): no effects (gavage)

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS): not measured

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS): decreased sperm motility

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): no effects

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

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

GROSS PATHOLOGY (PARENTAL ANIMALS): no effects

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

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: water consumption, urinary sodium concentration, kidneys weight and histopathology

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: decreased sperm motility

Clinical signs:

effects observed, treatment-related

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

VIABILITY (OFFSPRING): reduced in the high dose groups (with and without extra zinc)

CLINICAL SIGNS (OFFSPRING): pale pups in the high dose groups (with and without extra zinc)

BODY WEIGHT (OFFSPRING): reduced in the high dose group (with extra zinc); but due to the limited numebr of pups in both high dose groups no real conclusion could be made on BW

SEXUAL MATURATION (OFFSPRING): not done, pups were necropsied on day 4 post partum

ORGAN WEIGHTS (OFFSPRING): not done

GROSS PATHOLOGY (OFFSPRING): no abnormaities

HISTOPATHOLOGY (OFFSPRING): not done

OTHER FINDINGS (OFFSPRING): none

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: decreased number of females with live born pups, decreased number of (live) pups, increased postimplantation loss

Reproductive effects observed:

not specified

Conclusions:

Based on the changes in water consumption, urinary sodium concentration, kidney weight and histopathological effects of kidneys as observed in the animals treated with the highest concentration of the test item, the No Observed Adverse Effect Level (NOAEL) for parental toxicity is 500 mg/kg body weight/day. Based on the decreased sperm motility as observed in the male animals treated with the highest concentration of the test item, the No Observed Adverse Effect Level (NOAEL) for fertility is 500 mg/kg body weight/day. Based on the decreased number of females with live born pups, decreased number of (live) pups, increased postimplantation loss as observed in the female animals treated with the highest concentration of the test item, the No Observed Adverse Effect Level (NOAEL) for developmental toxicity is 500 mg/kg body weight/day.

Executive summary:

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

Data with regard to general toxicity are reported under 'repeated dose toxicity'.

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

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

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

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

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

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

No treatment-related effects were observed in pre-coital time, mating index, female fecundity index, male and female fertility indices, duration of gestation, number of corpora lutea, number of implantation sites and pre-implanation loss.

In females animals treated wih the highest concentration of the test item (irrespectively of dietary zinc supplementation), the number of animals that delivered liveborn pups was statistically significantly decreased whereas the number of pregnant females that delivered no (live) pups and/or at which no pups were found (most probably pups were cannibalized before being found) and postimplantation loss were statistically significantly increased in these groups.

In the two groups treated wih the highest concentration of the test item (irrespectively of dietary zinc supplementation), the mean number of (live) pups delivered was statistically significantly decreased whereas the number of stillborn pups was statistically significantly increased.

In these 2 groups, due to the low number of pups, data on sex ratio, pup survival, pup weights and pathology of pups that died during lacation are unreliable. In the other groups, no statistically significantly adverse effects on sex ratio, pup survival and pup weights were found.

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

Treatment-related effects on epididymal sperm motility and derived parameters were observed in the male animals of the two groups treated wih the highest concentration of the test item (irrespectively of dietary zinc supplementation). No differences were observed in epididymal sperm count, epididymal sperm morphology and testicular sperm count between the control and treatment groups.

Both the absolute and relative weights of the kidneys of the male and females of the two groups treated wih the highest concentration of the test item (irrespectively of dietary zinc supplementation) were statistically significantly increased. At necropsy no treatment related gross changes were observed in male and female animals.

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

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

Based on the results of this study (decreased sperm motility as observed in the male animals treated with the highest concentration of the test item), the No Observed Adverse Effect Level (NOAEL) for fertility is 500 mg/kg body weight/day. The effects on sperm motility are considered a direct effect and not secondary to parental toxicity.

Based on the results of this study (decreased number of females with liveborn pups, decreased number of (live) pups, increased postimplantation loss as observed in the female animals treated with the highest concentration of the test item) the No Observed Adverse Effect Level (NOAEL) for developmental toxicity is 500 mg/kg body weight/day. The effects observed on pup development are considered a direct effect and not secondary to parental toxicity.

As there were no differences in toxic effects in the groups at 1500 mg/kg bw with and without additional zinc, it was concluded that the addition of zinc was not necessary to compensate for possible reproductive toxicity of EDTA-MnNa2, if any, due to its chelating, viz. zinc-binding properties. Instead, it was concluded that the reproductive toxicity of EDTA-MnNa2 was most probably directly due to the presence of Mn-ions. However, apparently such effects were only seen at a very high dose of 1500 mg/kg bw EDTA-MnNa2 and not at the next lower level tested of 500 mg/kg bw.   

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Well conducted study according to GLP

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Based on read across with other EDTA compounds such as EDTA-CaNa2 and EDTA-MnNa2 it is concluded that reproductive effects were not present at levels up to 250 or 500 mg/kg bw, respectively. This was also concluded by Heimbach et al. (2000) and in the RAR on EDTA-H4 and EDTA-Na4 (2004). Heimbach et al. (2000) concluded that EDTA compounds are not reproductive toxicants when fed with a nutrient sufficient diet or minimal diets supplemented with Zn. Because in this case animals were treated with EDTA-FeNa and not with an empty chelate such as EDTA-H4 or EDTA-Na4, binding of zinc will even be less.

Short description of key information:
There is no study available on EDTAFeHNa, but the potential toxicity of EDTAFeHNa was assessed by analogy with other EDTA-complex:
Studies with levels up to 250 mg/kg bw EDTA-CaNa2 did not give any indication for effects on reproduction. A study on EDTA-MnNa2 showed decreased sperm motility at a level of 1500 mg/kg bw but not at 500 mg/kg bw. In the latter study, no changes were found on female reproduction.

Effects on developmental toxicity

Description of key information

There is no study available on EDTAFeHNa, but the potential toxicity of EDTAFeHNa was assessed by analogy with other EDTA-complex:
Studies with levels up to ca. 1000 mg/kg bw EDTA-compounds did not give any indication for effects on development; higher levels revealed developmental effects including malformations. A study on EDTA-MnNa2 showed a decreased number of females with liveborn pups, a decreasd number of (live) pups and an increased post-implantation loss at a level of 1500 mg/kg bw but not at 500 mg/kg bw.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

key study

Study period:

September-December 2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well conducted study according to GLP (as the data is used in a read-across approach, a maximal reliability score of 2 was attributed).

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD 422

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

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

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

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

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

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

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

VEHICLE: tap water
- Concentration in vehicle: 0, 15, 50 and 150 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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

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

VEHICLE: tap water
- Concentration in vehicle: 0, 15, 50 and 150 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 1
- Length of cohabitation: 1 week
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: not done.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: individually
- Any other deviations from standard protocol: no

Duration of treatment / exposure:

10 weeks pre-mating, 1 week mating, 3 weeks gestation, and 4 days lactation

Frequency of treatment:

single daily application by gavage

Duration of test:

10 weeks pre-mating, 1 week mating, 3 weeks gestation, up to 4 days of lactation

Remarks:

Doses / Concentrations:
0, 150, 500 and 1500 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on studies done with EDTA
- Rationale for animal assignment (if not random): computer randomization proportionately to BW

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: observations outside the home cage were made once weekly; FOB and motor activity were assessed in week 8 of the pre-mating period.

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

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

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

URINALYSIS: on days 63-65 of the study.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No as females were allowed to litter
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities

Statistics:

- Clinical findings were evaluated by Fisher's exact probability test.
- Body weight, body weight gain, organ weights and food consumption data were subjected to one way analysis of variance (ANOVA).
- Fisher's exact probability test was used to evaluate the number of mated and pregnant females
and females with live pups.
- Number of corpora lutea, implantation sites, live and dead fetuses or pups were evaluated by
Kruskal-Wallis nonparametric analysis of variance.
- Mortality data and data of the pathology of parent females were evaluated by the Fisher’s exact probability test.
- Functional observational battery: one-way analysis of variance followed by Dunnett’s multiple comparison tests (continuous data), Kruskal-Wallis non-parametric analysis of variance followed by multiple comparison tests (rank order data) or Pearson chi-square analysis (categorical data).
- Motor activity data-total distance moved: one-way analysis of variance followed by Dunnett’s multiple comparison tests; habituation of activity: repeated measures analysis of variance on time blocks (each session consists of 5 time blocks of 6 minutes each).
- Sperm parameters were evaluated by ANOVA followed by Dunnett’s multiple comparison test (epididymal and testicular sperm count and numerical sperm motility parameters) or by Kruskal-Wallis non parametric ANOVA followed by Mann-Whitney U test (motility parameters expressed as a percentage and sperm morphology).

Indices:

- gestation index = (number of females with live pups or pups/number of females pregnant) x 100
- pre-implantation loss = [(number of corpora lutea – number of implantation sites)/number of corpora lutea] x 100
- number of lost implantations = number of implantations sites - number of pups born alive
- post-implantation loss = [(number of implantation sites - number of pups born alive)/number of implantation sites] x 100

- live birth index = (number of pups born alive/number of pups born) x 100
- viability index day n-m= (number of pup surviving m days/number of liveborn on day n) x100
- pup mortality day n = (number of dead pups on day n/total number of pups on day n) x 100
- sex ratio day n = (number of live male fetuses or pups on day n/ number of live fetuses or pups on day n) x 100

Historical control data:

Not included.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Increased water consumption, increased urinary sodium concentration, increased weight of kidneys and very slight diffuse subcortical tubular dilatation in the kidneys of animals of the high concentration groups (with and without extra zinc)

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Decreased number of females with live born pups, increased post-implantatio loss in animals of the high concentration groups with and without extra zinc).
In live pups: reduced pup viability and pale pups in high concentration groups (with and without extra zinc) , and reduced BW in the high dose group (with extra zinc) but due to the limited number of pups in both high dose groups no real conclusion could be made on BW

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

Based on the changes in water consumption, urinary sodium concentration, kidney weight and histopathological effects of kidneys as observed in the animals treated with the highest concentration of the test item, the No Observed Adverse Effect Level (NOAEL) for maternal toxicity is 500 mg/kg body weight/day. Based on the decreased number of females with live born pups, decreased number of (live) pups, increased postimplantation loss as observed in the female animals treated with the highest concentration of the test item, the No Observed Adverse Effect Level (NOAEL) for developmental toxicity is 500 mg/kg body weight/day.

Executive summary:

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

Data with regard to fertility/reproduction are presented under 'toxicity to reproduction', data on general toxicity under 'repeated dose toxicity'.

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

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

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

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

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

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

In female animals treated wih the highest concentration of the test item (irrespectively of dietary zinc supplementation), the number of animals that delivered liveborn pups was statistically significantly decreased whereas the number of pregnant females that delivered no (live) pups and/or at which no pups were found (most probably pups were cannibalized before being found) and postimplantation loss were statistically significantly increased in these groups.

In the two groups treated wih the highest concentration of the test item (irrespectively of dietary zinc supplementation), the mean number of (live) pups delivered was statistically significantly decreased whereas the number of stillborn pups was statistically significantly increased.

In these 2 groups, due to the low number of pups, data on sex ratio, pup survival, pup weights and pathology of pups that died during lacation are unreliable. In the other groups, no statistically significantly adverse effects on sex ratio, pup survival and pup weights were found.

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

Both the absolute and relative weights of the kidneys of the females of the two groups treated wih the highest concentration of the test item (irrespectively of dietary zinc supplementation) were statistically significantly increased.

At necropsy no treatment related gross changes were observed.

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

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

Based on the results of this study (decreased number of females with live born pups, decreased number of (live) pups, increased postimplantation loss as observed in the female animals treated with the highest concentration of the test item) the No Observed Adverse Effect Level (NOAEL) for developmental toxicity is 500 mg/kg body weight/day. The effects observed on pup development are considered a direct effect and not secondary to maternal toxicity.

As there were no differences in toxic effects in the groups at 1500 mg/kg bw with and without additional zinc, it was concluded that the addition of zinc was not necessary to compensate for possible reproductive toxicity of EDTA-MnNa2, if any, due to its chelating, viz. zinc-binding properties. Instead, it was concluded that the reproductive toxicity of EDTA-MnNa2 was most probably directly due to the presence of Mn-ions. However, apparently such effects were only seen at a very high dose of 1500 mg/kg bw EDTA-MnNa2 and not at the next lower level tested of 500 mg/kg bw.   

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

It was concluded that with regard to EDTA-compounds, that the mechanism by which EDTA induced developmental toxicity at high levels of EDTA was the binding of zinc by EDTA resulting in zinc deficiency during embryonic development (Heimbach et al., 2000; RAR, 2004). With regard to EDTA-MnNa2, binding of Zn will even be less. However, at a very high level of 1500 mg/kg bw EDTA-MnNa2 was developmental toxic, most probably due to the effect of manganese as other manganese compounds had also shown developmental effects. Such changes were not seen with the zinc chelate of EDTA (RAR, 2004) and are therefore also not expected for iron i.e. EDTA-FeNa. In contrast, Fe-shortage in dams (fed with 7.5 mg Fe per kg diet instead of 50 mg Fe per kg diet) resulted in greater pup mortaility, smaller pup size, and pups with larger hearts, and with smaller kidneys and spleens. The only available study on EDTA-FeNa was disregarded because of several severe limitations; however, no developmental effects were seen at 200 mg/kg bw (nominal); the effects seen at 800 mg/kg bw (nominal) such as delayed ossification were considered to be due to maternal toxicity at that level. Overall, despite all the study limitations, no teratogenic effects were observed in this study.

Justification for selection of Effect on developmental toxicity: via oral route:
Well conducted study according to GLP

Justification for classification or non-classification

As EDTA and the zinc chelate of EDTA obviously lack a specific teratogenic potential (RAR, 2004), it is expected that this applies to EDTA-FeHNa too. In addition, because malformations caused by EDTA compounds have been demonstrated at relatively high oral dose levels (i.e. 1000 mg/kg bw and above) and a steep dose response relationship can be assumed (RAR, 2004), no classification for reprotoxicity is needed.