Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
one-generation reproductive toxicity
Remarks:
based on test guideline (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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted study according to GLP
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Deviations:
yes
Remarks:
see below
Principles of method if other than guideline:
This study was carried out as an extended OECD 422 study in which 12 animals per sex per group were exposed 10 weeks (instead of 2 weeks) prior to mating so that male fertility could be examined. In doing so the study became a one-generation test (OECD 415) rather than a combined subacute/reproscreening test (OECD 422). 12 animals/sex/group were used (at least 10 animals/sex/group) to comply to the REACH requirement for Annex VIII studies.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutshland, Sulzfeld, Germany
- Age at study initiation: 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
Gross pathological findings:
no effects observed
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. The percentages of motile (see Table below), progressive and static sperm cells as observed at 500 mg/kg bw was within the range of historical control data and, therefore, are considered of less toxicological relevance. The effects observed in groups treated with 1500 mg/kg bw (with and without extra zinc) were considered to be related to treatment.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): no effects. At 150 mg/kg bw (see Table below), the increased postimplantation loss is considered coincidentally since the increase is mainly due to only 2 animals and no effect was observed at 500 mg/kg bw. The increased postimplantation loss as observed at 1500 mg/kg bw (with and without extra zinc) is considered to be related to treatment.

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 (see for Tables section 7.5.1)
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 number 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

Table – Changes in male fertility parameters

 

0 mg/kg bw

150 mg/kg bw

500 mg/kg bw

1500 mg/kg bw

1500 mg/kg bw + extra Zn

Motile sperm cells (%)± SEM

(n=5)

76.0 ± 3.3

71.4 ± 2.5

67.0 ± 1.7*

42.8 ± 8.7*

49.2 ± 8.4*

Male fertility index (%)

92

92

92

92

100

*p<0.05


 

Table – Changes in fertility and reproductive parameters

 

0 mg/kg bw

150 mg/kg bw

500 mg/kg bw

1500 mg/kg bw

1500 mg/kg bw + extra Zn

Females pregnant

(n)

11

11

11

11

12

Females with liveborn pups (n)

11

10

11

1***

3***

Females with all stillborn pups (n)

0

0

0

2

4

Females pregnant, implants, no pups (n)

0

1

0

8**

5*

Mating index (%)

92

92

92

100

100

Female fecundity index (%)

100

100

100

92

100

Female fertility index (%)

92

92

92

92

100

Gestation index (%)

100

91

100

9

25

Duration of gestation ± SE (days)

21.36 ± 0.15

21.70 ± 0.15

21.30 ± 0.15

22.00 ± 0.00

22.00 ± 0.00*

N of corpora lutea ± SE

12.36 ± 0.54

12.55 ± 0.47

13.00 ± 0.75

13.00 ± 0.36

13.00 ± 0.54

N of implantation sites ± SE

10.64 ± 0.84

11.64 ± 0.53

11.64 ± 0.49

11.36 ± 0.47

11.58 ± 0.72

Pre-implantation loss ± SE (%)

13.44 ± 6.62

6.95 ± 3.05

9.04 ± 3.68

12.25 ± 3.60

11.22 ± 3.81

Post-implantation loss ± SE (%)

3.83 ± 1.70

23.78 ± 10.53*

8.00 ± 2.13

91.82 ± 8.18***

96.04 ± 2.42***

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

 

Table – Changes in litter and pup data

 

0 mg/kg bw

150 mg/kg bw

500 mg/kg bw

1500 mg/kg bw

1500 mg/kg bw + extra Zn

N of pups delivered (total)

112

97

118

14

19

N of pups delivered per litter ± SE

10.18 ± 0.80

9.70 ± 0. 98

10.73 ± 0.57

4.67 ± 2.33*

2.71 ± 0.71**

N of liveborn

112

97

118

9***

6***

Live birth index (%)

100

100

100

64

32

N of stillborn

0

0

0

5***

13***

Pup mortality on day 1 (%)

0

0

0

36

68

Pup weight on day 1 ± SE (g)

5.75 ± 0.19

(11 litters)

6.59 ± 0.17*

(10 litters)

5.89 ± 0.21

(11 litters)

5.67 ± 0.00

(1 litter)

3.89 ± 0.21***

(3 litters)

Pup weight on day 4 ± SE (g)

8.61 ± 0.27

(11 litters)

9.71 ± 0.32

(10 litters)

8.95 ± 0.34

(11 litters)

9.11 ± 0.00

(1 litter)

4.30 ± 0.00

(1 litter)

Clinical signs in pups during lactation (day 1-4)

-pale

-dehydration

 

 

0

0

 

 

0

0

 

 

1

0

 

 

0

0

 

 

4**

1

Macroscopic observations in stillborn and pups that died

-no abnormalities

-blood in pericardium

-atrium enlarged, right

-cannibalized

-late resorption, too small

-lost during processing

 

 

0

0

0

0

0

0

 

 

0

0

0

0

0

0

 

 

0

0

0

0

0

0

 

 

4

1

0

0

0

0

 

 

9

0

1

1

1

1

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

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:
adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The results of the study were confirmed by other studies with (metal-)chelates, see also read across document in section 13.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Although decreased sperm motility was seen at the highest dose of the structurally related substance EDTA-MnNa2 tested (1500 mg/kg bw), it did not result in effects on reproduction as there were no changes in reproductive performance in animals of these groups.

Effects on sperm were also noted in the report of the Dutch Health Council (2001) evaluating (mostly inorganic) manganese compounds other than EDTA-MnNa2 or EDTA-Mn(NH4)2. Because these effects on sperm were observed in the extended screening test with EDTA-MnNa2 using 12 male animals per group and a pre-mating period of 10 weeks, sufficient information has been obtained from this study and a similar study with EDTA-Mn(NH4)2 is therefore not warranted.

In a 2 -year feeding study on Wistar rats including reproductive and lactation experiments in four successive generations groups of 25 male and 25 female animals were exposed to EDTA-CaNa2 at dietary levels providing daily doses of approximately 50, 125, and 250 mg/kg bw (Oser et al., 1963). No significant differences in behavior or appearance nor adverse effects on the growth or on the longevity of the rats in any of the generations or among the various dose levels were reported. Evaluations of various tissues and organs (weight, histopathologic examinations) including gonads (testes) gave negative results even in the high dose group. Criteria for reproductive and lactational effects were evaluated as proportion of matings resulting in pregnancy (fertility index), proportion of pregnancies resulting in live litters (gestation index), proportion of pups that survive 4 days or longer (viability index), and proportion of rats alive at 4 days that survive to weaning. Poor responses with respect to some of the criteria of reproductive performance occurred occasionally but were not correlated with dosage or with the number of generations through which dosage continued. The overall data for two matings in the four successive generations did not give evidence for significant treatment related differences in either of these indexes. The authors concluded that the No Observed Adverse Effect Level of EDTA-CaNa2 was observed as measured by any of the usual indices of reproduction or lactation efficiency even under the stresses of repeated pregnancies and lactation. The NOAEL derived from this study is therefore at least 250 mg/kg bw/day for the parent and F1 to F3 generation.

Studies with EDTA-Na2H2 were not taken into consideration for setting a NOAEL because of methodological flaws. Studies with other metal-chelates (EDTA-MnNa2 and DTPA-FeNaH) showed effects on fertility only at very levels of 1500 mg/kg bw but not at 500 mg/kg bw. But although effects on sperm were

seen at the highest dose of EDTA-MnNa2 or DTPA-FeNaH tested (1500 mg/kg bw), it did not result in effects on reproduction as there were no changes in reproductive performance in animals of these groups.


Short description of key information:
Oral exposure of males with EDTA-MnNa2 for at least 12 weeks showed decreased sperm motility at 1500 mg/kg bw. This study together with studies of EDTA-Na2H2 and DTPA-FeNaH were used for risk assessment (for read-across justification also refer to section 13). Data from a multigeneration study on rats with EDTA-CaNa2 did not give evidence for adverse effects on reproductive performance and outcome for doses of up to 250 mg/kg bw/day. The studies with EDTA-Na2H2 were not taken into considerationfor setting a NOAEL because of methodological flaws. The study with DTPA-FeNaH showed effects only at very high levels (1500 mg/kg bw).

Justification for selection of Effect on fertility via oral route:
Study carried out with EDTA-MnNa2; GLP study

Effects on developmental toxicity

Description of key information
With regard to developmental effects, changes consisted of a decreased number of female rats with live born pups, a decreased number of (live) pups, and increased post-implantation loss in females treated at 1500 mg/kg bw (with and without additional zinc) in the presence of maternal toxicity. In rabbits, changes consisted of a slightly decreased fetal body weight and a slightly increased incidence of a skeletal variation at 100 mg/kg bw (with and without extra zinc). Retarded skeletal ossification was only seen at 100 mg/kg bw without extra zinc; the developmental effects were seen in the presence of maternal toxicity.  
Results are available on several EDTA-compounds and on DTPA-FeNaH (see also read across document in section 13). After repeated treatment of dams with several of the non-metal EDTA chelates during various periods of gestation and with the use of different routes of substance application (diet, gavage, s.c., i.m.) impaired embryo/fetal development and the induction of a pattern of gross malformations were observed during these investigations with the exception of one gavage study (Schardein et al., 1981). Gross malformations, comprised cleft palate, severe brain deformities, eye defects, micro- or agnathia, syndactyly, clubbed legs and tail anomalies. These effects were exclusively exhibited in studies using maternally toxic dose levels. With the exception of one oral (single-dose/gavage) study, during which no developmental effects were induced, the developmental effects in rats are occurring at exposure levels of approximately 1,000 mg/kg bw/day and above. This was confirmed by the studies with EDTA-MnNa2 and DTPA-FeNaH that showed developmental toxicity only at 1500 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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted study according to GLP
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
see below
Principles of method if other than guideline:
This study was carried out as an extended OECD 422 study in which 12 animals per sex per group were exposed 10 weeks (instead of 2 weeks) prior to mating so that male fertility could be examined. In doing so the study became a one-generation test (OECD 415) rather than a combined subacute/reproscreening test (OECD 422). 12 animals/sex/group were used (at least 10 animals/sex/group) to comply to the REACH requirement for Annex VIII studies.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutshland, Sulzfeld, Germany
- Age at study initiation: 10-11 weeks
- Weight at study initiation: mean weight males 171-175 g; mean weight females
- Fasting period before study: not applicable
- Housing: 4 per sex in macrolon cages, with wood shavings as bedding material, and paper strips as environmental enrichment
- Use of restrainers for preventing ingestion (if dermal): not applicable
- Diet (e.g. ad libitum): ad lib
- Water (e.g. ad libitum): ad lib
- Acclimation period: one week

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

IN-LIFE DATES: From: 16 September To: 25 December 2009
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); see for Tables section 7.5.1. In addition, decreased number of females with live born pups, and increased postimplantation loss (see Table below)
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. 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 (see Table below)
Abnormalities:
not specified
Developmental effects observed:
not specified

Table – Changes in fertility and reproductive parameters

 

0 mg/kg bw

150 mg/kg bw

500 mg/kg bw

1500 mg/kg bw

1500 mg/kg bw + extra Zn

Females pregnant

(n)

11

11

11

11

12

Females with liveborn pups (n)

11

10

11

1***

3***

Females with all stillborn pups (n)

0

0

0

2

4

Females pregnant, implants, no pups (n)

0

1

0

8**

5*

Mating index (%)

92

92

92

100

100

Female fecundity index (%)

100

100

100

92

100

Female fertility index (%)

92

92

92

92

100

Gestation index (%)

100

91

100

9

25

Duration of gestation ± SE (days)

21.36 ± 0.15

21.70 ± 0.15

21.30 ± 0.15

22.00 ± 0.00

22.00 ± 0.00*

N of corpora lutea ± SE

12.36 ± 0.54

12.55 ± 0.47

13.00 ± 0.75

13.00 ± 0.36

13.00 ± 0.54

N of implantation sites ± SE

10.64 ± 0.84

11.64 ± 0.53

11.64 ± 0.49

11.36 ± 0.47

11.58 ± 0.72

Pre-implantation loss ± SE (%)

13.44 ± 6.62

6.95 ± 3.05

9.04 ± 3.68

12.25 ± 3.60

11.22 ± 3.81

Post-implantation loss ± SE (%)

3.83 ± 1.70

23.78 ± 10.53*

8.00 ± 2.13

91.82 ± 8.18***

96.04 ± 2.42***

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

 

Table – Changes in litter and pup data

 

0 mg/kg bw

150 mg/kg bw

500 mg/kg bw

1500 mg/kg bw

1500 mg/kg bw + extra Zn

N of pups delivered (total)

112

97

118

14

19

N of pups delivered per litter ± SE

10.18 ± 0.80

9.70 ± 0. 98

10.73 ± 0.57

4.67 ± 2.33*

2.71 ± 0.71**

N of liveborn

112

97

118

9***

6***

Live birth index (%)

100

100

100

64

32

N of stillborn

0

0

0

5***

13***

Pup mortality on day 1 (%)

0

0

0

36

68

Pup weight on day 1 ± SE (g)

5.75 ± 0.19

(11 litters)

6.59 ± 0.17*

(10 litters)

5.89 ± 0.21

(11 litters)

5.67 ± 0.00

(1 litter)

3.89 ± 0.21***

(3 litters)

Pup weight on day 4 ± SE (g)

8.61 ± 0.27

(11 litters)

9.71 ± 0.32

(10 litters)

8.95 ± 0.34

(11 litters)

9.11 ± 0.00

(1 litter)

4.30 ± 0.00

(1 litter)

Clinical signs in pups during lactation (day 1-4)

-pale

-dehydration

 

 

0

0

 

 

0

0

 

 

1

0

 

 

0

0

 

 

4**

1

Macroscopic observations in stillborn and pups that died

-no abnormalities

-blood in pericardium

-atrium enlarged, right

-cannibalized

-late resorption, too small

-lost during processing

 

 

0

0

0

0

0

0

 

 

0

0

0

0

0

0

 

 

0

0

0

0

0

0

 

 

4

1

0

0

0

0

 

 

9

0

1

1

1

1

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

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:
adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The results of the study were confirmed by other studies with (metal-)chelates, see also read across document in section 13.
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

With regard to developmental effects of EDTA-MnNa2, very similar to EDTA-Mn(NH4)2, changes consisted of a decreased number of females with live born pups, a decreased number of (live) pups, and increased post-implantation loss in females treated at 1500 mg/kg bw (with and without additional zinc). Due to the low number of pups, no firm conclusion can be drawn on data on sex ratio, pup survival, pup weight and pup pathology in animals of these groups. However, the greater part of the pups that could be examined, did not show abnormalities. The Dutch Health Council concluded that there were a few studies on manganese compounds - other than EDTA-MnNa2 or EDTA-Mn(NH4)2 - that showed developmental toxicity of manganese but these effects were only observed in the presence of maternal toxicity or the maternal toxicity was not clear.Because effects on development were observed in the extended screening test using 12 rats per group and exposure during the complete pregnancy period at a high level of 1500 mg/kg bw EDTA-MnNa2 in the presence of maternal toxicity, sufficient information has been obtained from this study, and a test proposal for a full blown developmental toxicity study in rats using higher numbers of animals is therefore not warranted. The more because a full OECD 414 study with EDTA-MnNa2 in rabbits (using 22 animals per group) showed developmental effects consisting of a slightly decreased fetal body weight and a slightly increased incidence of skeletal variations at 100 mg/kg bw (with and without extra zinc) whereas retarded skeletal ossification was only seen at 100 mg/kg bw without extra zinc; and these effects were all seen in the presence of maternal toxicity.

EDTA and four of its salts were evaluated for their teratogenic potential in CD albino rats (Schardein et al., 1981). Groups of 20 females were treated by gavage during g.d. 7 to 14 with 1,000 mg EDTA/kg bw/day as well as with equimolar doses of disodium, trisodium, calcium disodium and tetrasodiumedetate. The dose level had been selected from preliminary studies with edetic acid in which there had been some evidence of both maternal and fetotoxicity under the same experimental conditions. For the dams significant drug-related reactions including diarrhea and depression of activity were reported. The former occurred in all drug groups with highest incidences for tetrasodium edetate (90%) and edetic acid (80%) and lowest incidence for calcium disodium edetate (10%). Three dams died during treatment with disodium edetate. Besides slightly decreased food intake in all test groups, treatment with all of the test compounds caused reduced weight gain in the dams during the treatment period. The mortality index of offspring in all treated groups as measured by postimplantation loss was comparable to that of the vehicle and untreated control group. None of the test compounds significantly affected litter size at term or mean fetal body weight when compared to either control. Fetuses were examined for external, visceral and skeletal anomalies. Incidental findings of skeletal anomalies did not reveal a definitive pattern regarding treatment with a particular compound. The authors stated that under these experimental conditions no teratogenic effects were evidenced even at maternally toxic doses.

In a further developmental study pregnant Sprague-Dawley rats were exposed during various periods of gestation to purified diets adjusted to either 100 or 1,000 ppm zinc (provided as zinc carbonate) and containing 2 or 3% EDTA-Na2H2 corresponding to 1000 or 1500 mg/kg bw daily intake (Swenerton and Hurley, 1971). The groups of 8 to 16 females had been set on the control diet at least 5 days before breeding and mated to normal stock-fed males. The evaluation of treatment related effects to the dams was not indicated in this study, except for the report on moderate to severe diarrhea in all females that were fed diets containing EDTA-Na2H2. While obviously complete reproductive failure occurred with the 3% EDTA-Na2H2/100 ppm zinc diet fed during g.d. 0 -21, with the 2% EDTA-Na2H2/100 ppm zinc diet reproductive outcome was essentially comparable to that of controls, however with lower mean body weight of the pups and with 7% malformed of the fullterm fetuses. Exposure to the 3% EDTA-Na2H2/100 ppm zinc diet during the period of g.d. 6 -14, and 6 -21 resulted in respectively 40% and 54% dead or absorbed fetuses, reduced number of dams with live pubs, clearly reduced mean fetal body weight and ratios of respectively 87% and 100% malformed living offspring. Gross malformations comprised cleft palate, severe brain deformities, eye defects, micro- or agnathia, syndactyly, clubbed legs and tail anomalies. The reported fetotoxic and teratogenic effects were similar to those from earlier experiments with zinc deficient diets administered to pregnant rats for various periods of during gestation (Hurley, 1966). In contrast, the live offspring of dams fed 3% EDTA-Na2H2 supplemented with 1,000 ppm zinc from g.d. 6-21 did not exhibit any malformations, and the mean number of live pups/litter and the mean fetal body weight were comparable to those of controls. The authors concluded from this study that EDTA-Na2H2 ingested during pregnancy was teratogenic, whereas supplementation with zinc prevented the detrimental effects of EDTA. It was suggested that the congenital anomalies caused by EDTA were due specifically to zinc deficiency. This was also supported by zinc analyses of fetuses (Hurley and Swenerton, 1966), where clearly lower zinc contents were found in fetuses from deficient mothers in comparison to those from zinc supplemented dams, indicating that the reported effects rather occur because of a direct lack of zinc in fetal tissues than from indirect effects of maternal metabolism on fetal development.

The toxic and teratogenic effects of EDTA-Na2H2 were studied in female CD rats following different routes of administration (dietary, gavage, s.c) during g.d. 7-14 (Kimmel, 1977). Dietary exposure to 3% EDTA-Na2H2 amounting to an average dose of 954 mg EDTA-Na2H2/kg bw/day resulted in reduced food intake, severe diarrhea and severe weight loss in the dams during treatment and produced a significant proportion of fetal deaths (about 33% resorptions/litter), significantly lower average fetal weight and gross external, internal and skeletal malformations in about 71% of the survivors. Treatment with 1,500 or 1,250 mg EDTA-Na2H2/ kg bw/day administered by gavage (respectively 625 mg/kg and 750 mg/kg twice daily) resulted in severe toxicity to the dams (7 out of 8 animals died in the 1,500 mg dose group), in particular 36% maternal deaths, significantly reduced weight gain, and diarrhea in the 1,250 mg dose group and a significantly higher proportion of (about 21%) malformed survivors. Treatment with 375 mg/kg bw administered subcutaneously produced signs of severe pain (vocalisations and shock) to the dams and resulted in 24% maternal deaths, significantly reduced food intake and maternal weight loss during the period of treatment. Fetal toxicity (about 32% resorptions/litter, significantly reduced fetal weight) and a rate of about 4% malformed survivors/litter were reported for this route of application.

EDTA-MnNa2 showed developmental effects only at 1500 mg/kg bw (key study), whereas no developmental effects were observed at 1500 mg/kg bw DTPA-FeNaH. The effects observed following treatment with EDTA-MnNa2 consisted of a decreased number of females with live born pups, decreased number of (live) pups, increased postimplantation loss. No such effects were seen at 500 mg/kg bw. Because of the higher affinity of EDTA for Zn it can also be expected that at the high level of 1500 mg/kg bw, sufficient Mn will be exchanged for Zn and as such Zn-deficiency may also occur as has occurred with the 'empty' (non-metal containing) chelates. The OECD 414 study in rabbits with EDTA-MnNa2 (other key study) indeed resulted in slightly retarded skeletal ossification in fetuses from rabbits that did not receive extra zinc. The affinity of EDTA for Ca is even lower.


Justification for selection of Effect on developmental toxicity: via oral route:
Study carried out with EDTA-MnNa2; GLP study

Justification for classification or non-classification

Effects on fertility and developmental toxicity of EDTA-MnNa2, very similar to EDTA-Mn(NH4)2, were seen in rats at a very high level of 1500 mg/kg bw - which is in excess of 1000 mg/kg bw; which was also true for the empty EDTA's (EDTA-Na4 and EDTA-H4) - in the presence of maternal toxicity. No such effects were seen at the still very high level of 500 mg/kg bw. In rabbits, slight developmental toxicity was observed at 100 mg/kg bw, whereas maternal toxicity was observed at levels of 10, 30 and 100 mg/kg bw. Because effects on fertility and development were only seen in the presence of maternal toxicity, no classification is proposed for this endpoint.