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Toxicological information

Toxicity to reproduction

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Administrative data

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
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Chemical name: Ethylenediaminetetraacetic acid, manganese disodium complex
Purity: 92.3%
Batch no: CFC 9380
Expiry date: 31 August 2012

Test animals

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

Administration / exposure

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
Doses / concentrations
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.

Examinations

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

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
effects observed, treatment-related
Reproductive performance:
no effects observed

Details on results (P0)

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)

Effect levels (P0)

open allclose all
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

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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

Effect levels (F1)

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

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.