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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

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
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 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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
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
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
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
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

Examinations

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.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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)

Effect levels (maternal animals)

open allclose all
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

Results (fetuses)

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)

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

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 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.