Ferrate(1-), [[N,N'-1,2-ethanediylbis[N-[[2-(hydroxy-.kappa.O)phenyl]methyl]glycinato-.kappa.N,.kappa.O]](4-)]-, sodium (1:1), (OC-6-13)-

Developmental toxicity / teratogenicity

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:

1995-01-10 to 1995-03-30

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
[Describe why the read-across can be performed (e.g. common functional group(s), common precursor(s)/breakdown product(s) or common mechanism(s) of action]

The underlying hypothesis for the read-across is that Fe(Na)HBED, and Fe(Na)EDDHA have the same mode of action based on their ability to chelate, remove or add iron to body causing perturbation of body’s iron balance leading, possibly, to iron deficiency anaemia (IDA) effects. The target and the source substances are six-dentate chelates which ligands (here called also chelators) have the same functional groups (= donor groups: carboxylic, amine and phenolic, each double), that bind iron (central metal atom).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
[Provide here, if relevant, additional information to that included in the Test material section of the source and target records]

The typical purity of the marketed target substance Fe(Na)HBED is in the range of 78-88 % (w/w) whereby the typical concentration of the main component sodium [2,2'-(ethane-1,2-diylbis{[2-(hydroxy-kO)benzyl]imino-kN})diacetato-kO(4-)]ferrat(1-) is 81 % and water 5-9 % (average 7 %). Sodium chloride (19 %) is specified as impurity. As mentioned above, sodium chloride will not affect validity of the read-across statement, since the percentages of sodium and chloride ions (both are macro elements) are negligible to cause toxicity effects in living organisms.

In contrast, Fe(Na)EDDHA is UVCB substance, containing except the structures of complexes also considerable amounts of polycondensation products as well as by-products remaining after the synthesis reaction:

Product [%]
Fe[o,o]EDDHANa 34.2
Fe[o,p]EDDHANa 4.4
Fe[p,p]EDDHANa 2.3
Fe polycondensate Na 13.3
NaCl 24.7
NaNO3 13.4
KCl 1.3
Moisture content 5.9
Sum 99.5

Molecular weight of Fe-polycondensate chelate of 678-680 g/mol was determined by HPLC-MS analyses (plase see RA). The substances with such a high molecular weight have difficulties to pass cell membrane in the gut according to ECHA guidance on Toxicokinetics (Chapter R.7C, section R. 7.12; 2014). They can be transported by pinocytosis or per sorption, but, if the polycondensates are very hydrophilic (negative log Kow, similar to the main components), the absorption is likely to be limited. Therefore, no extensive absorption into systemic circulation is expected for polycondensates. Their affinity to iron is not determined experimentally, but if absorption into systemic circulation is negligible, no remarkable concern can be attributed to the polycondensates as potential sequesters of iron from the body.

The amount of sodium, and chloride ions from NaCl, KCl and Na ions from NaNO3 are comparable to the amounts in the Fe(Na)HBED. Thus, no considerable differences in the toxicological activity of the target and the source substance related to these ions can be expected. Nitrate, however, is present only in the source substance. Its impact on the toxicological activity is not expected to be considerable, since only a small amount of nitrate originates from the source substances which are negligible with regard to the toxicity profile.
Water content is also similar by the target and the source substances.
According to published literature, commercial EDDHA consists of mixture of positional OH isomers: orto-orto (o-o), orto-para (o-p) and para-para (p-p) (Lucena, 2003). EDDHA o-o and o-p can form stable iron chelates while “p-p-EDDHA was completely unable to form iron cheltes” (Lucena, 2003). Since the source substances contain low amount of p-p isomers, they are not toxicologically relevant. No differences in the binding capacity were reported between o-o and o-p isomers (Lucena, 2003, Yunta et al., 2003b). The iron binding capacity of the chelator EDDHA is 36.89 (mean of meso and racemic forms). The stability constant of Fe(Na)HBED (39.01) (Ma et al., 1994) is however higher than those of its analogues. The higher Fe(III) affinity of Fe(Na)HBED relative to that of Fe(Na)EDDHA is due to a more favourable steric orientation of donor groups (Ma and Martell, 1993).
Based on this information, the presence of secondary components of the UVCB source substance i.e. polycondensates, geometrical isomers (o-o, o-p and p-p) as well as nitrates, that are different from the monoconstituent target substance Fe(Na)HBED, is considered not to influence the toxicological activity of the main components o-o Fe(Na)EDDHA.


3. ANALOGUE APPROACH JUSTIFICATION
[Summarise here based on available experimental data how these results verify that the read-across is justified]

Please refer to section 13 of the IUCLID file where the read-across statement is attached.

In a developmental study according to OECD 414 conducted with source substance Fe(Na)EDDHA, there were no treatment-related clinical signs or incidences of mortality. The body weight gain was reduced at 500 mg/kg bw/day for the period of days 6 -16 of gestation, and reduced food consumption was also noted at this dose level. No adverse effects on pregnancy and no embryo-/foetotoxic effects were observed. There was no indication of any teratogenic potential. The established NOELs were 100 mg/kg bw/day for maternal toxicity and 500 mg/kg bw/day for developmental toxicity and teratogenicity.
As evident from the results of the available studies, impaired developmental and post natal findings occur only at higher or at the same dose levels where maternal toxicity is observed and therefore those deviations are considered as a consequence of this maternal systemic toxicity. Thus, it could be assumed that a link exists between developmental toxicity and iron deficiency conditions.

4. DATA MATRIX
Data matrix on the source substance used in this robust study summary is described in greater details in the read-across statement attached to the section 13 of the IUCLID file.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OTS 798.4900 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Acetic acid, oxo-, sodium salt, reaction products with ethylenediamine and phenol, iron sodium salts

EC Number:

283-044-5

EC Name:

Acetic acid, oxo-, sodium salt, reaction products with ethylenediamine and phenol, iron sodium salts

Cas Number:

84539-55-9

Molecular formula:

non specified (UVCB substance)

IUPAC Name:

Acetic acid, oxo-, sodium salt, reaction products with ethylenediamine and phenol, iron sodium salts

Details on test material:

- Test article: CGA65047 SG 100 (A-5787 A)
- Physical state: granules
- Lot/batch No.: P.201845
- Storage condition of test material: room temperature
- Analytical purity: 100 % (UVCB)

Test animals

Species:

rat

Strain:

other: Sprague-Dawley derived; Tif:RAIf (SPF); hybrids of RII/1 x RII/2

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Animal Production, WST-455, CIBA-GEIGY Limited, 4332 Stein, Switzerland
- Age at mating: minimum 8 weeks
- Weight range on gestation day 0: 176.8-231.5 g (females)
- Diet: pelleted, certified standard diet (Nafag No. 890, tox; Nafag, Nähr- und Futtermittel AG, Gossau, Switzerland), provided ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 50 ± 20 %
- Air changes: about 16 air changes/hour
- Photoperiod: 12 hours dark / 12 hours light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5 % (w/w) aqueous solution

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
Test substance-vehicle mixtures were prepared with a high speed homogeniser. Homogeneity of the mixtures during administration was maintained with a magnetic stirrer.

VEHICLE
A 0.5 % (w/w) aqueous solution of sodium carboxymethyl cellulose (CMC) was used
- Concentration in vehicle: 0, 0.5, 10.0 and 50.0 mg/mL mixture
- Amount of mixture: 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of test substance-vehicle mixtures were taken once before (samples taken from top, middle and bottom of the container) and once after dosing (sample taken from middle of the container) and analysed for content, homogeneity and stability of the test item in the vehicle.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1/3
- Length of cohabitation: 3-6 hours
- The mating procedure was repeated for non-pregnant females
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

from day 6 to day 15 of gestation

Frequency of treatment:

1x/day

Duration of test:

21 days

No. of animals per sex per dose:

24 rats

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dose levels were selected based on the results of a previous range finding study no. 941104 in pregnant rat:
"Virgin female albino rats, approximately two months old (Stock: Tif: RAI f (SPF), hybrids of RII/1 x RII/2) were used for this study. Four groups of eight mated females per group were treated by gavage with the test substance mixed in a 0.5% aqueous solution of sodium carboxymethylcellulose (CMC) in a volume of 10 mL/kg actual body weight, at daily doses of 0, 10, 500 and 1000 mg/kg body weight (groups 1, 2, 3, and 4, respectively), from day 6 through day 15 post coitum.
Maternal body-weight gain/weights and feed consumption were dose-dependent ly reduced in groups 3 and 4 during treatment (days 6 to 15 p.c). At cesarean section, one control animal (no. 8) and one mid dose group animal (no. 18) were not pregnant. All other dams had viable fetuses. Pre- and post-implantation losses, number of live fetuses per litter and fetal weights were not affected by treatment. There were no dead or aborted fetuses. Mean carcass weight and net body weight change from day 6 were dose dependently reduced in groups 3 and 4. At necropsy, kinked tail was observed in one control fetus.
In summary, in the present preliminary study, CGA 65047 F, A-5787 A was toxic to dams at 500 and 1000 mg/kg/day and was not toxic to embryos or fetuses at doses up to 1000 mg/kg/day. There was no indication of teratogenesis".

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS:
- Time schedule: daily (incl. mortality)

BODY WEIGHT:
- Time schedule for examinations: daily

FOOD CONSUMPTION
- Time schedule for examinations: days 6, 11, 16 and 21

POST-MORTEM EXAMINATIONS:
- Sacrifice on gestation day 21
- Organs examined: main organs of the thoracic and abdominal cavities, in particular the genitals

Ovaries and uterine content:

The ovaries and uterine content was examined after termination.
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: uterine content assessment (live and dead foetuses, early and late losses)

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [approximately half per litter]
- Skeletal examinations: Yes: [approximately half per litter]
- Head examinations: Yes: [all per litter]

Statistics:

Statistical analysis of continuous data was performed using the Analysis of Variance Procedure (ANOVA) followed by Dunnett's t-test in case of a significant result in the ANOVA. Categorical data were analysed using Chi-Square test followed by Fisher's Exact test in case of a significant result in the Chi Square test. Non-parametric data were analysed during the Kruskal-Wallis non-parametric analysis of variance test followed by Mann-Whitney U-test.

Indices:

No indices were calculated

Historical control data:

Historical control data were included in the report.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
There was a reduction in mean food consumption and body weight gain during the second half of the dosing period at 500 mg/kg bw/day.

Effect levels (maternal animals)

1

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOEL

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:no effects

Details on embryotoxic / teratogenic effects:
There was no indication for test item related embryotoxicity or teratogenic effects under the conditions of this study.

Effect levels (fetuses)

Dose descriptor:

NOEL

Effect level:

500 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Maternal data

Clinical Signs and Mortality

There were no treatment-related clinical signs or treatment-related deaths in this study.

Body weights

There were no significant differences in mean maternal body weights between groups at any time during the study.

Maternal body weight gain was statistically significantly reduced in the 500 mg/kg group during the second half of the dosing period (days 11 to 16 p.c.). Body weight gain was unaffected by treatment in the 5 and 100 mg/kg groups.

Food consumption

At 500 mg/kg, there was a statistically significant reduction in food consumption during the second half of the dosing period (days 11 to 16 p.c.). At 5 and 100 mg/kg, values for food consumption were similar to those of the control group throughout the study.

Maternal Survival and Pregnancy Status

Of the 24 mated animals per group, three, one and three were not pregant in the low, mid and high dose groups, respectively. Thus the number of pregnant animals per group was 24, 21, 23 and 21, respectively. One dam in the mid dose group was sacrificed moribund on day 11 p.c. At necropsy, the number of dams per group with viable fetuses was therefore 24, 21, 22, and 21, respectively.

Cesarean Section Data

Preimplantation losses, numbers of implantation sites, and early and late postimplantation losses were comparable between groups. There were no dead or aborted fetuses. The number of live fetuses per litter and fetal weights were not affected by treatment.

Uterine and Carcass Weights

Mean carcass weight and gravid uterus weights were not affected by treatment. Net body weight gain was significantly reduced in the 500 mg/kg group.

Maternal Postmortem Examination

No macroscopic findings were noted at necropsy of the dams on day 21 p.c.

Fetal Data

There were no treatment-related external fetal abnormalities.

Fetal Visceral Examination

No treatment-related findings were observed.

Fetal Skeletal Examination

There were no treatment-related skeletal malformations.

Summary of All Fetal Observations

Fetal sex ratios and mean fetal body weights were not affected by treatment. Incidental malformations were seen in two control fetuses (no. 13/3: runt, and no. 5/1: umbilical hernia) and one low dose group fetus (no. 33/3: generalized edema, cleft lower jaw and cleft palate). The overall incidence of external, visceral and skeletal anomalies and variations were not affected by treatment.

Chemical Analysis

Administration / Treatment 1:

The mean concentrations of the test item in the vehicle were 88.1 % (0.5 mg/mL) , 94.0 % (10 mg/mL) and 93.3 % (50 mg/mL) of the nominal concentrations for the low mid and high dose groups, respectively. The homogeneity varied in the range from -2 % to 4 % of the mean concentration. The test item was stable in the vehicle.

Administration / Treatment 2:

The mean concentrations of the test item in the vehicle were 94.9 % (0.5 mg/mL) , 93.9 % (10 mg/mL) and 94.1 % (50 mg/mL) of the nominal concentrations for the low mid and high dose groups, respectively. The homogeneity varied in the range from -1 % to 1 % of the mean concentration. The test item was stable in the vehicle.

Applicant's summary and conclusion

Conclusions:

Evidence of maternal toxicity, in terms of reduced body weight gain and reduced food consumption, was observed in the high dose group (500 mg/kg). No adverse effects on pregnancy or fetal parameters were observed. On the basis of the results obtained in this study, the no observed effect level (NOEL) of CGA 65047 SG 100, (A-5787 A) was 100 mg/kg body weight/day for the dams and 500 mg/kg body weight/day for the progeny. There was no indication of teratogenic potential.

Executive summary:

In a developmental toxicity study (CIBA-GEIGY, 1995), FeNaEDDHA was administered once daily to groups of 24 mated female Sprague-Dawley derived rats by oral gavage at dose levels of 5, 100 or 500 mg/kg bw/day (10 mL/kg bw) from day 6 through day 15 of gestation. Control group females received the vehicle, 0.5 % (w/w) CMC in distilled water, only. The nominal concentrations, homogeneity and stability of the test item in the vehicle were confirmed by chemical analysis of dose formulations. All dams were sacrificed on day 21 of the gestation period and foetuses removed for examination. In dams, there were no treatment-related clinical signs or incidences of mortality. The body weight gain was reduced at 500 mg/kg bw/day for the period from days 6 -16 of gestation, and reduced food consumption was also noted at this dose level. No adverse effects on pregnancy and no embryo-/foetotoxic effects were observed. There was no indication of teratogenic potential. On the basis of these results, the NOEL was 100 mg/kg bw/day for maternal toxicity and 500 mg/kg bw/day for develomental toxicity and teratogenicity.

The developmental toxicity study in the rat is classified as acceptabel and satisfies the requirements of test guideline OECD 414.