Reaction mass of disodium [2,2'-(imino-kappaN)dibutanedioato-kappa2O1,O4(4-)]manganese(2-) and sodium sulphate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

one-generation reproductive toxicity

Remarks:

based on generations indicated in Effect levels (migrated information)

Type of information:

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

Adequacy of study:

key study

Study period:

September-December 2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well conducted study according to GLP

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

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

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

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

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

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

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

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

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

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

Details on mating procedure:

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

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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

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

Duration of treatment / exposure:

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

Frequency of treatment:

single daily application by gavage (parental animals)

Details on study schedule:

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

Remarks:

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

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

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

Positive control:

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

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

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

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

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

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

Oestrous cyclicity (parental animals):

Not measured

Sperm parameters (parental animals):

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

Litter observations:

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

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

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities

Postmortem examinations (parental animals):

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

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

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

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

Postmortem examinations (offspring):

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

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

ORGAN WEIGHTS: not done

HISTOPATHOLOGY: not done

Statistics:

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

Reproductive indices:

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

Offspring viability indices:

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

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

effects observed, treatment-related

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): no effects

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

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

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

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

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): no effects

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

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

GROSS PATHOLOGY (PARENTAL ANIMALS): no effects

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

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

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

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: decreased sperm motility

Clinical signs:

effects observed, treatment-related

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

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

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

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

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

ORGAN WEIGHTS (OFFSPRING): not done

GROSS PATHOLOGY (OFFSPRING): no abnormaities

HISTOPATHOLOGY (OFFSPRING): not done

OTHER FINDINGS (OFFSPRING): none

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

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

Reproductive effects observed:

not specified

Conclusions:

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

Executive summary:

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

High quality (there is sufficient data for hazard assessment).

Additional information

No reproductive toxicity studies are available for the target substance Mn(2Na)IDHA. Therefore, the data on free IDHA chelating agent and Mn (2Na) EDTA have been used to assess toxicity potential of Mn(2Na)IDHA on fertility (please refer to read-across statement). Due to the fact that Mn(2Na)IDHA can dissociate at low pH level of stomach, the toxicity results of studies conducted with inorganic manganese compounds were also taken into account.

One generation study conducted with IDHA, sodium salt

The purpose of this one-generation study was to evaluate possible effects of IDHA sodium salt on the entire reproduction process in Wistar rats (OECD 415; Eiben and Rinke, 2002, Project No. PH-32294). The substance was administered to groups of 25 male and 25 female rats each at concentrations of 0 (control), 1000, 4000 and 16000 ppm in demineralized drinking water (correspond to 0, 105.3 -140.0, 411.0 - 480.1 and 2081.4 - 2270.8 mg/kg bw in males and females, respectively). Parental F0 animals were pretreated over a period of about 10 weeks and allowed to mate over a period of up to three weeks. F1 offspring were nursed up to an age of four weeks. Clinical signs, body weights, food and water intake, mating performance, fertility, duration of pregnancy, estrus cycling and sperm parameters were examined in F0 rats. Litter size, percentage of males born and pup weight at birth as well as viability, rearing, lactation and body weight gain were studied in F1 offspring. Necropsies were done in all rats. Implantation sites were recorded in F0 females. Selected organs were weighed (F0 and F1) and histopathological evaluations were performed on some organs of F0 rats.

Mortality and clinical appearance in F0 animals were unchanged at levels of up to 4000 ppm. At 16000 ppm diarrhoea was noted in both sexes and 2/25 females exhibiting histopathologically a strong kidney damage were killed because of complications at their parturition. At 16000 ppm retarded body weights were evident in parental rats. The food intake was unchanged up to 16000 ppm. The uptake of drinking fluid was higher in treated F0 rats than in controls. At 1000 and 4000 ppm the increase in water intake is most likely a result of the enhanced salt content of the drinking fluid and not considered as adverse. At 16000 ppm, however, a correlation to diarrhoea and several morphological changes in the urinary tract described below and increase in kidney weights found in this group cannot be excluded. The reproduction parameters insemination index, mating performance, fertility index, gestation index, duration of pregnancy, life birth index, birth weights, percentages of males born, total number of pups born, prenatal loss, mean litter size at birth as well as viability, rearing and lactation index were not affected at levels of up to 16000 ppm.

The body weight development of F1 pups was retarded at 16000 ppm with the consequence that female pups exhibited reduced spleen weights. At 16000 ppm more pups were pale than in the other groups.

No test substance-related gross pathological findings were observed in F1 offspring up to 16000 ppm. The skeletal development of the offspring was unaffected. No adverse effect was seen in sperm parameters at 16000 ppm. F0 females exhibited no treatment effects on estrus cycling up to 16000 ppm.

At 16000 ppm degenerative and reparative alterations in the kidneys such as (macro-and microscopical) dilations and hyperplasias in the renal pelvis and increased amounts of renal mineralization, a reduction of male-specific hyaline droplet accumulation and basophilic tubuli occurred more frequently in male and/or female F0 rats. Slight hyperplasia of the transitional cell epithelium occurred in the urinary bladder. Furthermore, ureters of high dose females were dilated macroscopically and degeneratively changed. These changes are most likely due to the marked overload with sodium of the drinking fluid and indicate severe parental toxicity. In the pituitary of 16000 ppm females hypertrophic cells within the pars distalis and vacuolation of the pars nervosa were noted more frequently. In 16000 ppm females, pituitaries were macroscopically swollen and increased in their weights as a correlate to this. The toxicological relevance of these findings are debatable and most probably not compound specific, but due to general toxicity of this dose.

An increase in inflammatory cells of the cecum in 16000 ppm females is considered as a correlate to cecum dilations seen macroscopically and diarrhoea. Gross pathology and histopathology in remaining organs revealed no treatment-related lesions.

Thus, the concentration of 4000 ppm in the drinking fluid is established as the overall no observed adverse effect level (NOAEL) for the parent animals and reproduction parameters. The NOAEL for the fertility is 16000 ppm.

Combined oral repeated dose toxicity study with reproduction /developmental toxicity screening test with Mn(2Na)EDTA in rats

In a study, the possible effects of Mn(2Na)EDTA on reproductive performance and development, and its sub-chronic toxicity were examined in groups of 12 male and 12 female Wistar rats (OECD 408, OECD 422, Wolterbeek, 2010; Report No. V8650). Mn(2Na)EDTA was administered daily by gavage during a premating period of 10 weeks and during mating, gestation and lactation until postnatal day 4. The dose levels were 0 (tap water only), 150, 500 and 1500 mg/kg bw/day. 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”.

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 female animals treated with 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 the two groups treated with 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 lactation are unreliable. In the other groups, no statistically significantly adverse effects on sex ratio, pup survival and pup weights were found.

Treatment-related effects on epididymal sperm motility and derived parameters were observed in the male animals of the two groups treated with 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.

At necropsy no treatment related gross changes were observed in male and female animals.

Based on the results 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, and 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.

Summary results of reproductive toxicity studies conducted with inorganic manganese compounds (ATSDR, 2012; SCOEl, 2011)

Impotence and loss of libido are common symptoms in male workers afflicted with clinically identifiable signs of manganism (ATSDR, 2012). These symptoms could lead to reduced reproductive success in men. Impaired fertility (measured as a decreased number of children/married couple) has been observed in male workers exposed for 1–19 years to manganese dust (0.97 mg/m³) at levels that did not produce frank manganism. This suggests that impaired sexual function in men may be one of the earliest clinical manifestations of manganese toxicity, but no dose-response information is available; therefore, it is not possible to define a threshold for this effect. Evidence obtained in laboratory mammals indicates that exposure to high levels of manganese may adversely effect sperm quality, produce decreased testicular weights, and impair development of the male reproductive tract. No direct effect of manganese toxicity has been observed on fertility in women. Although many studies in laboratory mammals have attempted to detect effects of manganese on female fertility, only one study demonstrated the possibility that excess manganese exposure outside of pregnancy may impair future fertility (decreased number of offspring).

NOAEL of 232 and 731 mg/kg bw were established for reproductive effects in chronic two-year studies in rats and mice, respectively (ATSDR, 2012).

As overall conclusion of SCOEL (2011) is that reproductive toxicity profile for manganese and its compounds does not suggest that this aspect is key to an evaluation of occupational exposure standards (IEH 2004, cited in SCOEL, 2011). There is mentioned that there is little evidence for reproductive or developmental toxicity.

Estimation of an equivalent realistic NOAEL for reproductive toxicity for Mn(2Na)IDHA

The same as in case of repeated dose toxicity, an estimated dose level for Mn(2Na)IDHA is considered to be precautionary than the read-across from NOAEL established for Mn(2Na)EDTA or IDHA chelating agent because of possible underestimation of reproductive toxicity of manganese released from Mn(2Na)IDHA. In this regard, the lowest NOAEL of 69 mg Mn/kg bw was reported for rats established in an oral study of intermediate-duration for male reproductive toxicity (Ponnapakkam et al. 2003c, cited in ATSDR, 2012). In another oral chronic (2-year) study, no lesions on reproductive organs were observed in rats exposed to 232 mg MnSO4 /kg bw (corresponds to 84 mg manganese) (ATSDR, 2012). Thus, according to the lowest NOAEL of 69 mg Mn/kg bw, the toxicity of manganese seems to be higher than the toxicity of the chelating agent IDHA (NOAEL for fertility is 16000 ppm (corresponds to 2081 and 2270 mg/kg bw in male and females, respectively) and overall NOAEL for reproduction is 4000 ppm (corresponds to 411 and 480 mg/kg bw in males and females, respectively)) or the read-across substance Mn(2Na)EDTA (NOAEL is 500 mg/kg bw). According to equation:

4H+ + MnIDHANa2 + H2O⇔H4IDHA + 2Na+ + Mn2+

69 mg of manganese corresponds to 435 mg of Mn(2Na)IDHA: ((MW of Mn(2Na)IDHA) is 346.08 g/mol/ MW of Mn is 54.94 g/mol) x 69 g/mol). Taking into account 50 % oral absorption established for Mn(2Na)IDHA, the estimated dose would result in 870 mg/kg bw: 435 x (100 %/50%). This dose is, however, higher than the NOAEL of 500 mg/kg bw for Mn(2Na)EDTA or the NOAEL for IDHA, sodium salt of 411-480 mg/kg bw.

Therefore, 500 mg/kg bw, the NOAEL established for reproductive toxicity for the analogue chelate Mn(2Na)EDTA is considered appropriate to serve as NOAEL for reproductive toxicity for Mn(2Na)IDHA. Moreover, the NOAEL of 411 mg/kg bw/day (corresponding to 4000 ppm) for IDHA is close to the NOAEL of 500 mg/kg bw for Mn(2Na)EDTA.

Short description of key information:
- One-generation study with IDHA, sodium salt (OECD 415): 0, 1000, 4000 and 16000 ppm in drinking water, rats. NOAEL: 4000 ppm (overall toxicity and reproductive parameters); NOAEL of 16000 ppm for fertility;
- Combined study (OECD 422): gavage; 0, 150, 500 and 1500 mg/kg bw; rats; NOAEL: 500 mg/kg bw;
- ATSDR (2012) and SCOEL (2011) data on reproductive toxicity of inorganic manganese compounds: the lowest NOAELs are presented.

Justification for selection of Effect on fertility via oral route:
The NOAEL of 500 mg/kg bw established for the chelate analogue Mn(2Na)EDTA is considered to be the most appropriate for reproductive toxicity for the target substance Mn(2Na)IDHA.

Effects on developmental toxicity

Description of key information

- Developmental study  with IDHA, sodium salt (OECD 414): 0, 100, 300 and 1000 mg/kg bw; Wistar rats; NOAEL: 1000 mg/kg bw. 
- One-generation study with IDHA, sodium salt (OECD 415): 0, 1000, 4000 and 16000 ppm in drinking water, rats. NOAEL: 4000 ppm (overall toxicity, reproductive parameters and postnatal toxicity); NOAEL of 16000 ppm for teratogenicity;
- Combined study with Mn(2Na)EDTA (OECD 422): gavage; 0, 150, 500 and 1500 mg/kg bw; rats; NOAEL: 500 mg/kg bw;
- The conclusion of Dutch Health Council regarding reproductive toxicity of inorganic manganese compounds (2001): manganese substances may cause concern for human fertility; 
- ATSDR (2012) and SCOEL (2011) data on developmental toxicity of inorganic manganese compounds: the lowest NOAELs are presented. Overall conclusion: no developmental effects at the dose levels without maternal toxicity.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

277 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

High quality (there is sufficient data for hazard assessment).

Additional information

No developmental toxicity studies are available for the target substance Mn(2Na)IDHA. The data on developmental toxicity with free IDHA chelating agent and Mn(2Na)EDTA have been used to assess developmental toxicity potential of Mn(2Na)IDHA (please refer to read-across statement). The toxicity results of studies conducted with inorganic manganese compounds were also taken into account.

Developmental study conducted with IDHA, sodium salt

Groups of 25 inseminated female Wistar rats each were treated daily orally (by gavage) with IDHA, sodium salt (73.4%) solved in demineralized water from day 6 to day 19 p.c. in doses of 0, 100, 300 and 1000 mg/kg body weight (bw)/day, respectively (OECD 414; Klaus, 2002, Report No. PH-32141). The fetuses were delivered by cesarean section on day 20 of gestation. Investigations were performed on general tolerance of the test compound by the females. Findings of maternal toxicity consisted of transiently marginally impaired feed consumption and body weight gain at the 1000 mg/kg dose level. Further on treatment relationship could not be excluded for marginally reduced carcass weight and corrected body weight gain. All other parameters evaluated, i.e. mortality and clinical signs, water intake and excreta, overall body weight development including final body weight and gross pathological observations were not affected by treatment at a dose level up to and including 1000 mg/kg bw/day.With respect to intrauterine development, gestation rate, postimplantation loss and accordingly the number of fetuses, fetal sex distribution, placental weight and appearance and fetal weight were not affected by treatment with IDHA, sodium salt at a dose level up to and including 1000 mg/kg bw/day. External, skeletal and visceral evaluation of fetuses revealed neither toxicologically relevant treatment related effects at a dose level up to and including 1000 mg/kg bw/day. A teratogenic potential of IDHA, sodium salt was not evident at a dose level up to and including 1000 mg/kg bw/day.

Summarizing and evaluating all data investigated the following no-observed-effect levels (NOEL) were determined:

Maternal toxicity: 300 mg/kg bw/day

Developmental toxicity: 1000 mg/kg bw/day.

One generation study conducted with IDHA, sodium salt

The purpose of this one-generation study was to evaluate possible effects of IDHA sodium salt on the entire reproduction process in Wistar rats (OECD 415; Eiben and Rinke, 2002, Project No. PH-32294). The substance was administered to groups of 25 male and 25 female rats each at concentrations of 0 (control), 1000, 4000 and 16000 ppm in demineralized drinking water (correspond to 0, 105.3 -140.0, 411.0 - 480.1 and 2081.4 - 2270.8 mg/kg bw in males and females, respectively). Parental F0 animals were pretreated over a period of about 10 weeks and allowed to mate over a period of up to three weeks. F1 offspring were nursed up to an age of four weeks.

The body weight development of F1 pups was retarded at 16000 ppm with the consequence that female pups exhibited reduced spleen weights. At 16000 ppm more pups were pale than in the other groups. No test substance-related gross pathological findings were observed in F1 offspring up to 16000 ppm. The skeletal development of the offspring was unaffected.

Combined oral repeated dose toxicity study with reproduction /developmental toxicity screening test with Mn(2Na)EDTA in rats

In a study, the possible effects of Mn(2Na)EDTA on reproductive performance and development, and its sub-chronic toxicity were examined in groups of 12 male and 12 female Wistar rats (OECD 408, OECD 422, Wolterbeek, 2010; Report No. V8650). Mn(2Na)EDTA was administered daily by gavage during a premating period of 10 weeks and during mating, gestation and lactation until postnatal day 4. The dose levels were 0 (tap water only), 150, 500 and 1500 mg/kg bw/day. 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.

With regard to developmental toxicity, in female animals treated with 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 the two groups treated with 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 lactation are unreliable. In the other groups, no statistically significantly adverse effects on sex ratio, pup survival and pup weights were found.

Based on the results of this study (decreased number of females with liveborn pups, decreased number of (live) pups, and 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.

The conclusion of Dutch Health Council regarding reproductive toxicity of inorganic manganese compounds (2001)

The Dutch Health Council concluded that there were a few studies on manganese compounds 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.

Summary results of reproductive toxicity studies conducted with inorganic manganese compounds (ATSDR, 2012; SCOEl, 2011)

There is evidence to suggest that children exposed to high levels of manganese from environmental sources (airborne, drinking water, dietary) may develop a variety of adverse developmental effects, particularly neurological effects. Many studies suggest that children exposed to particularly high levels of manganese over a long period of time (months or years) will eventually develop one or more symptoms, including general cognitive impairment, diminished memory, attention deficit, motor impairments, aggressiveness, and/or hyperactivity. However, it is not clear from any of these studies whether other factors, perhaps environmental or genetic, are responsible for these changes in the presence of manganese, or whether manganese alone can produce these effects.

A potentially serious developmental effect of manganese was suggested by the results of a study where high infant mortality in a Bangladesh community was reported in conjunction with the presence of a local drinking water supply containing high levels of manganese (concentration up to 8.31 mg/L). Infants exposed to levels of manganese equal to or greater than those recommended by the World Health Organization (WHO) were at the highest risk of mortality prior to 1 year of age. The nature of this epidemiological study, with nutritional deficits in the population anticipated but not documented, prevents a determination that manganese alone was responsible for the high rate of infant mortality. Developmental studies involving the use of laboratory animals have detected subtle changes in growth; (e.g., diminished body weight, in animals provided with relatively high doses of manganese). These changes have been observed both when the animals were exposed while in utero or postpartum when the animals have already been born. In a developmental study, no effects on fetal survival, gross fetal malformations, or skeletal or visceral malformations or alterations were observed in rats treated with 22 mg Mn/kg bw (Grant, 1997a, cited in ATSDR). This was the lowest developmental NOAEL reported.

In conclusion, studies of manganese workers have not found increases in birth defects or low birth weight in their children. No birth defects were observed in animals exposed to manganese at dose levels without maternal toxicity. In one human study where people were exposed to very high levels of manganese from drinking water, infants less than 1 year of age died at an unusually high rate. It is not clear, however, whether these deaths were attributable to the manganese level of the drinking water. The manganese toxicity may have involved exposures to the infant that occurred both before (through the mother) and after they were born.

As overall conclusion of SCOEL (2011) is that reproductive toxicity profile for manganese and its compounds does not suggest that this aspect is key to an evaluation of occupational exposure standards (IEH 2004, cited in SCOEL, 2011). There is mentioned that there is little evidence for reproductive or developmental toxicity.

Estimation of an equivalent realistic NOAEL for developmental toxicity for Mn(2Na)IDHA

The same as in case of reproductive toxicity endpoint, the read-across from the NOAEL of 500 mg/kg bw established for the read-across substance Mn(2Na)EDTA for developmental effects (OECD 422) may underestimate the risk of developmental toxicity of manganese which became systemically available originating from the exposure to Mn(2Na)IDHA. This is, probably, because of the higher oral absorption of IDHA (37 %) comparing to 5 % for EDTA or less than 1 -2 % for EDTA complexes. According to the study report, developmental effects observed in the combined study (OECD 422) with the read-across substance Mn(2Na)EDTA were probably attributed to manganese ions. In contrast, no developmental toxicity were observed in offspring from dams treated with the IDHA, sodium salt by the highest dose level (16000 ppm or 2270.8 mg/kg bw, OECD 415). In the developmental study with IDHA, sodium salt (OECD 414), NOAEL of 1000 mg/kg bw was established for developmental toxicity. In this regard, the developmental toxicity of Mn(2Na)IDHA may be mediated by manganese from Mn(2Na)IDHA complexes, which systemically available amount is expected to be much higher than the respective amount from Mn(2Na)EDTA (due to the higher absorption of IDHA chelate. Thus, similarly to the estimation of the NOAEL for reproductive toxicity, the lowest rat NOAEL of 22 mg Mn/kg bw for developmental toxicity is considered appropriate to estimate an equivalent safe dose level for Mn(2Na)IDHA. According to equation:

4H+ + MnIDHANa2 + H2O⇔H4IDHA + 2Na+ + Mn2+

22 mg of manganese corresponds to 138.6 mg of Mn(2Na)IDHA: ((MW of Mn(2Na)IDHA) is 346.08 g/mol/ MW of Mn is 54.94 g/mol) x 22 g/mol). Taking into account 50% oral absorption established for Mn(2Na)IDHA, the estimated DNEL would be 277.2 mg/kg bw: 138.6 x (100 %/ 50%). This dose level is lower than the NOAEL of 500 mg/kg bw for Mn(2Na)EDTA and lower than 1000 mg/kg bw, NOAEL for IDHA, sodium salt for developmental effects. Therefore, 277.2 mg/kg bw is considered to be appropriate to serve as NOAEL for developmental toxicity for Mn(2Na)IDHA. The dose level is based on an equivalent safe dose level for manganese which would release in case of only full dissociation of manganese complexes (worst-case).

Justification for selection of Effect on developmental toxicity: via oral route:
No study is selected since NOAEL for Mn(2Na)IDHA is estimated based on the lowest available NOAEL of 22 mg/kg bw for inorganic manganese. The reason is that the toxicity of Mn(2Na)IDHA is believed to be mediated by manganese rather than by the chelate moiety. Additionally, IDHA (and its chelates) are expected to be absorbed more extensively due to the higher oral absorption of IDHA comparing to the absorption of EDTA and its salts.

Justification for classification or non-classification

No reproductive or developmental toxicity could be assigned for the read-across substances: the related chelate Mn(2Na)EDTA and IDHA because all the reproductive effects or effects observed in offspring were only observed in the presence of significant maternal toxicity. Effects on reproduction and effects on foetal development have not been observed at levels up to 500 mg/kg bw in the combined repeated dose toxicity study with Mn(2Na)EDTA and NOAEL for parental toxicity was also established at the same dose level of 500 mg/kg bw. Similarly, for the chelating agent IDHA, sodium salt, NOAEL of 411 -480 mg/kg bw was established for reproductive effects and general toxicity for males and females, respectively. For fertility, the highest dose level of 2081.4 - 2270.8 mg/kg bw was established in males and females, respectively. NOAEL of 1000 mg/kg bw, the highest dose level tested, was established for developmental effects in the OECD 414 study with IDHA, sodium salt. Although these toxicological data are very important for the assessment of the reproductive and developmental toxicity of Mn(2Na)IDHA, toxicity safe levels of inorganic manganese are considered more appropriate due to possible dissociation of IDHA complexes which is more extensive than that of EDTA complexes. Summarizing all available toxicological information, overall conclusion can be made for reproductive and developmental toxicity of manganese: effects were only observed in the presence of maternal toxicity or the maternal toxicity was not clear. Therefore, neither reproductive nor developmental effects can be expected at the calculated NOAEL of 435 and 139 mg/kg bw for reproductive and developmental toxicity based on the lowest NOAEL for elemental manganese. Furthermore, the NOAELs established in studies with inorganic manganese compounds serve as worst-case for the hazard assessment because inorganic salts dissociate more quickly in aquatic media (and body fluids) than the respective amounts of the chelated substance. Based on these information and taking into account the provisions laid down in Council Directive 67/548/EEC and in European regulation (EC) No. 1272/2008 classification and labelling with regard to toxicity to reproduction and development is not required for Mn(2Na)IDHA.