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)-

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 test type (migrated information)

Type of information:

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

Adequacy of study:

supporting study

Study period:

1996-03-13 to 1997-01-07

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)EDDHMA 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)EDDHMA 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]EDDHMANa 65.2
Fe[o,p]EDDHMANa Unknown*
Fe[p,p]EDDHMANa Unknown*
Fe polycondensate Na Unknown*
NaCl 14.0
Moisture content 4.1
Further components Unknown*
Sum 83.3-Unknown
*Only for the ortho-ortho isomer was an analytical method known, for other isomers not. Therefore only quantitative information for this o-o-isomer is given (Notox, 1997)

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 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. Water content is also similar by the target and the source substances.
According to published literature, commercial EDDHMA consists of mixture of positional OH isomers: orto-orto (o-o), orto-para (o-p) and para-para (p-p) (Lucena, 2003). Isomers o-o and o-p can form stable iron chelates while p-p are 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 EDDHMA is 36.59 (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)EDDHMA 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 and geometrical isomers (o-o, o-p and p-p), that are different from the monoconstituent target substance Fe(Na)HBED, is considered not to influence the toxicological activity of the main component Fe(Na)EDDHMA.


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

Fe(Na)EDDHMA was tested in an one-generation reproductive toxicity study (NOTOX, 1997), in which the test material produced systemic toxicity in parental animals at the highest (750 mg/kg bw) and at the mid (200 mg/kg bw) doses. The primary effect of treatment was poor physical condition, resulting in premature mortality, growth reduction and reduced food consumption in male and female animals at 750 mg/kg bw/day. These signs of test item-related toxicity were seen with reduced severity at 200 mg/kg bw/day in males only. Thus, the NOAEL for systemic toxicity in parental animals was 50 mg/kg bw/day and the NOAEL for reproductive performance/fertility was 200 mg/kg bw/day due to slight decrease in the conception indices and a minimal delay in precoital time at 750 mg/kg bw/day. In the offspring, increased postnatal loss and reduced viability were noted during PND 0 -4 at 200 and 750 mg/kg bw/day, and with lower incidence at 50 mg/kg bw/day. The poor physical condition seen among parental females of the high dose group might have been responsible for these findings, but this is less clear for females of the 50 and 200 mg/kg dose groups (see below).
The source substance Fe(Na)EDDHMA is not reproductive or developmental toxicants. The toxicity effects in parental animals and in the offspring observed in the one-generation study with Fe(Na)EDDHMA are reported to be related to macrocytic anaemia as concluded by the study director. The reduced viability and postnatal losses that were observed in offspring in the one-generation study were also reported in rat’s offspring which mothers were fed iron deficient diets (Li et al., 2014; Zheng et al., 2001). Thus, these effects could be a consequence of IDA induced by the chelates.
Please refer also to section 13 of the IUCLID file where the read-across statement is attached.

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.34 (One-Generation Reproduction Toxicity Test)

Deviations:

no

GLP compliance:

yes

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 Wiga, Sulzfeld, Germany
- Age at study initiation: (P) Males: 4 weeks, Females 12 weeks
- Weight at study initiation (pre-mating period): (P) Males: 165-218 g, Females: 265-330 g
- Housing: individually, in polycarbonate cages containing purified sawdust bedding (Woody SPF, Type BK10/20 or during lactation Type 3-4 was supplied by B.M.I., Helmond, The Netherlands). During mating, 1 female was caged together with 1 male in suspended stainless steel cages with wire mesh floors.
- Diet: standard pelleted laboratory animal diet (Carfil Quality BVBA, Type R-03-18-K, Oud Turnhout, Belgium), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 °C
- Humidity: 55 %
- Air changes: 15 air changes/hour
- Photoperiod: 12 hours dark/12 hours light

IN-LIFE DATES: From: 13-Mar-1996 (males), 08-May-1996 (females) To: 09-Aug-1996 (males), 08-Aug-1996 (females)

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations of the test item in water (w/w) were prepared daily immediately prior to dosing. Formulations were stirred prior to and during dosing procedures.

VEHICLE
- Justification for use and choice of vehicle (if other than water): distilled water
- Stability in vehicle: Stated as being stable for at least 96 hrs (other NOTOX project)
- Concentration in vehicle: not reported
- Amount of vehicle (if gavage): 5 mL/kg b.w./day. Actual dose volumes were calculated according to the latest body weight.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: overnight for a maximum of 7 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After 7 days of unsuccessful pairing replacement of first male by another male with proven fertility
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged individually in a cage with sawdust bedding material.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of dose formulations were collected during the beginning of treatment, at an intermediate time and during the end of the dosing period. Samples of the highest and lowest concentrations were analysed to check stability (first analysis only) and homogeneity. Accuracy of preparation was determined for all concentrations.

Duration of treatment / exposure:

Parental males were treated until days 122 to 138 of the treatment period.
Parental females were treated until days 48 to 65 post coitum.

Frequency of treatment:

1x/day

Details on study schedule:

- Age at mating of the mated animals in the study: 16 weeks

Remarks:

Doses / Concentrations:
0, 50, 200 and 750 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

28 rats

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose level selection was based upon the information obtained from previous oral toxicity studies using rats, i.e. a 28-day oral toxicity study (LSR 87/AKB005/802) and a teratogenicity study (NOTOX 158759).

Positive control:

none

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
- Time schedule: once daily for clinical signs, twice daily for incidences of mortality.
The time of onset, degree and duration were noted. Clinical signs were recorded under NOTOX Project 173565, which has been generated for the purpose of reporting only. Animals showing pain, distress or discomfort, which was considered not transient in nature or which was likely to become more severe, were sacrificed for humane reasons. The time of death was recorded precisely as possible. Cage debris of pregnant females was examined to detect abortion or premature birth. Signs of difficult or prolonged parturition were recorded. In the clinical signs tables, findings for each animal are presented as highest weekly scores.

BODY WEIGHT:
- Time schedule: weekly for males and females
Mated females were weighed on days 0, 7, 14 and 20 of pregnancy and days 1, 7, 14 and 21 of the lactation period.
Live pups of one litter were weighed individually according to sex on the morning after birth (day 1) and on days 4, 7, 14 and 21 of the lactation period.

FOOD CONSUMPTION
- Time schedule: weekly for males and females
During the mating period analysis of food consumption was suspended.
Food consumption of mated females was measured during days 0-7, 7-14 and 14-20 of pregnancy and weekly thereafter starting on day 1 of the lactation period.

OTHER:
The females were allowed to litter normally. The day that partirition began was designated day 0 of lactation. Deficiencies in maternal care, such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pubs or apparently inadequate lactation or feeding, were recorded.

Oestrous cyclicity (parental animals):

No data on estrous cyclicity are given in the report.

Sperm parameters (parental animals):

No data on sperm parameters are given in the report.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum
- Litters were adjusted to 4 pups/sex or as near as possible; excess pups were killed and discarded. Litters of less than 8 pups remained intact.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
duration of gestation, 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:
For external and internal abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: all surviving animals (F0-males were killed between days 122 to 138 of the treatment period)
- Maternal animals: all surviving animals (F0-females were killed between days 48 to 65 post coitum)

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

HISTOPATHOLOGY
The following tissues were prepared and examined microscopically: cervix, coagulation gland, epididymides, ovaries, pituitary, prostate, seminal vesicles, testes, uterus and vagina.

Postmortem examinations (offspring):

SACRIFICE
- F1-offspring were killed on the day of culling the litters (day 4 of lactation) or when weaned on day 21 of lactation.
- These animals were subjected to postmortem examinations as follows:
GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. All animals were sexed.

Statistics:

For assumed normally distributed variables, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control group.
The Steel-test (many-to-one rank test) was applied instead of the Dunnett-test if the data were assumed not to be normally distributed.
The exact Fisher-test was applied if the variables could be dichotomised without loss of information.
All tests were two-tailed.

Reproductive indices:

Fertility index, conception index, gestation index, sex ratio (F1)

Offspring viability indices:

Live birth index, viability index, weaning index and overall survival index

Clinical signs:

effects observed, treatment-related

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:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
In the 750 mg/kg bw/day group, 1/28 males and 4/28 females died during the study. At this dose level, hunched posture, piloerection and emaciated and/or pale appearance were noted. A few males of the 200 mg/kg bw/day group showed hunched posture.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Body weight gain and food consumption were decreased in animals of both sexes at 750 mg/kg bw/day and in males at 200 mg/kg bw/day. This decrease was dose-related. During the lactation period, body weight gain of females at 750 mg/kg bw/day showed a marked increase. Body weight ratios were decreased during the first 5 weeks of treatment for males and during the first 3 weeks for females at 750 mg/kg bw/day.

Food consumption of males receiving 50 mg/kg/day and of females receiving 50 or 200 mg/kg/day remained in the same range as controls, before and after correction for the body weight.
Food consumption was decreased with statistical significance compared to controls, in males receiving 200 mg/kg/day from day 57 of treatment until termination and in males receiving 750 mg/kg/day from the beginning until the end of treatment. Following correction for the body weight, relative food consumption of males receiving 750 mg/kg/day showed a statistically significant decrease in comparison with control males between days 1 to 36 of treatment.
The food consumption of females treated at 750 mg/kg/day was noted as decreased during the premating period and until the end of the gestation period. During the period of lactation the food consumtion of females treated at 750 mg/kg/day was similar as control females. Relative food consumption was decreased during the premating period and during the first week of the gestation period only. In all cases the difference with control females was statistically significant.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)

A slight delay of the precoital time in the 750 and 200 mg/kg dose groups was observed.
For all males and females that were paired, mating could be confirmed, resulting in 100 percent mating for each dose group. The fertility and conception indices of the 200 and 50 mg/kg dose groups were comparable with the control group. In the 750 mg/kg dose group these indices were considered to be slightly low, although the difference with controls did not achieve a level of statistical significance.
The gestation index was 100% in all treatment groups and were not affected by treatment with FeEDDHMANa.
In the absence of concomitant histopathological findings, a slight decrease of the fertility and conception indices was noted at 750 mg/kg bw/day.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no test item-related macroscopic findings at necropsy.

HISTOPATHOLOGY (PARENTAL ANIMALS)
There was no histopathological evidence of toxicity or infertility.

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: systemic toxicity: based on clinical signs, incidences of mortality, and changes in body weight and food consumption at higher dose levels

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: reproductive toxicity, fertility: based on a slight decrease in the conception indices and a minimal delay in precoital time at 750 mg/kg bw/day.

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

VIABILITY (OFFSPRING)
Adverse effects on the F1-offspring consisted of an increased mortality rate during postnatal days 0-4 seen across the treatment groups (please refer to the table 1 in the section "Any other information on results incl.tables". The majority of the post natal loss was attributable to a few litters of each group, including 1, 3 and 3 litters in the low, mid and high dose groups, respectively. For further details, please refer to the table 2 in the section "Any other information on results incl. tables".

CLINICAL SIGNS (OFFSPRING)
There were no unexpected clinical signs seen among pups of any dose group. Signs that were more frequently observed among the pups of all treatment groups during the first litter check (FLC), post partum phase and last litter check (LLC), consisted of hypothermia, no milk in the stomach and small appearance. These signs normally precede the death of non-viable pups and were considered not to represent a distinct toxic effect caused by the test substance.

BODY WEIGHT (OFFSPRING)
Reduced body weights were noted during days 4 to 21 of lactation in pups of the high dose group (750 mg/kg bw/day).

GROSS PATHOLOGY (OFFSPRING)
There were no test item related macroscopic findings.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Reproductive effects observed:

not specified

Comparison of the relative number of live and dead pups during the various phases of lactation (i.e. first litter check, post partum days 0-4 and post partum days 5-21) revealed a statistically significant increase in post natal loss during days 0-4 in all treatment groups. The Viability Indices of these groups were correspondingly low. However, it was noted that the number of pups found dead at the first litter check (FLC) of control dams, were relatively high in comparison with the treatment groups. In accordance with these findings a relatively low Live Birth Index was noted in control litters. The following number of decedents among the F^-offspring was noted when calculating the total number of deaths during days 0-4 Post partum including the FLC:

Table 1: TOTAL DEAD PUPS DURING FLC & DAYS 0-4 P.P.

	GROUP 1 0 MG/KG	GROUP 2 50 MG/KG	GROUP 3 200 MG/KG	GROUP 4 750 MG/KG
Litters affected	5	8	9	6
Total dead pups	21	24	44	42
Mean	0.9	0.9	1.7	2.2
N (litters)	24	27	26	19

When comparing the total number of dead pups over the first 4 days of lactation, including the FLC, the mean number of dead pups per litter in the 50 mg/kg dose group equals the number in the control group. At the 200 and 750 mg/kg dose level, the mean number of deaths per litter revealed a dose-related increase when compared to the control group. A similar pattern was seen in the Overall Surviving Indices of the treatment and control groups. However, it must be considered that 2 females of the 750 mg/kg dose group died on the first day of lactation and that therefore 26 of 29 of their pups were killed in extremis.

After culling of all litters to 4 male and 4 female pups (if practically possible), the low mortality rate seen among pups of the treatment groups was comparable to that of the control group. This was also reflected by the comparable Weaning indices in control and treated groups.

Table 2: POSTNATAL LOSS AND VIABILITY
Endpoint	Dose Group [mg/kg bw/day]
	0	50	200	750
Dams with total litter loss at FLC [n]	1	0	0	0
(Pups lost [n])	(13)
Dead pups at FLC [n]	19	3	5	3
(Litters [n])	(5)	(3)	(3)	(1)
Postnatal loss PND 0-4 [n]	2	21	39	39
(Litters [n])	(1)	(7)*	(7)	(6)
Viability index (%)	99.4	94.9	90.4	85.4
FLC: first litter check
*: including 1 female with total pup loss (15/15)

CHEMICAL ANALYSIS

For the nominal concentrations of 0, 10, 40 and 150 mg/g, the concentrations analysed were in agreement with the concentrations prepared in this study. The test item was found to be mixed homogeneously through the vehicle and to be stable for 4 hours at ambient temperature.

Conclusions:

Based on the mortality observed in animals of the high dose group, and the reduced body weight gain and food consumption seen in animals of the mid- and high-dose groups, a parental No Observed Adverse Effect Level (NOAEL) of 50 mg/kg was established. Due to the increased post natal loss and reduced viability index in the treatment groups observed on post natal Days 0-4, the developmental NOAEL could, in fact, not be established. However, based on the results of the 28-day and 90-day study with FeEDDHMANa, it cannot be excluded that anaemia and/or impaired renal function may have been present that finally caused the observed litter losses. On the other hand, the finding in the low dose group consisted of only one female with total litter loss (15 pups) and 6 other females with 1 dead pup/litter which was within normal limits and might have occurred by chance. Therefore, a developmental NOAEL of 50 mg/kg might be considered; effects at higher levels were considered to be closely related to (subclinical) maternal toxicity.

Executive summary:

In a one-generation reproduction toxicity study (NOTOX B.V., 1997), the structural analogue FeEDDHMANa (read across) in distilled water was administered to 28 Wistar rats/sex/dose level by single oral gavage (5 mL/kg bw) at dose levels of 50, 200 or 750 mg/kg bw/day. A concurrent control group was treated with the vehicle only. Treatment commenced 10 weeks prior to mating for males and 2 weeks prior to mating for females and continued for both sexes until at least the end of the lactation period. Pregnant females were allowed to litter normally. On day 4 of lactation, each litter was adjusted to 4 males and 4 females or as near as possible. The surviving offspring was euthanised as soon as possible after weaning.

The primary effect of treatment with the test item on parental animals was poor physical condition, resulting in premature mortality, growth reduction and reduced food consumption in male and female animals at 750 mg/kg bw/day. These signs of test item-related toxicity were seen with reduced severity at 200 mg/kg bw/day in males only. Thus, the NOAEL for systemic toxcity in parental animals was 50 mg/kg bw/day under the conditions of this study.

In the offspring, increased post natal loss and reduced viability were noted during PND 0 -4 at 200 and 750 mg/kg bw/day, and with lower incidence at 50 mg/kg bw/day. With special regard to the low incidence and unusual distribution pattern of findings noted at 50 mg/kg bw/day, the NOAEL for developmental toxicity was 50 mg/kg bw/day under the conditions of this study.

Based on a slight decrease in the conception indices and a minimal delay in precoital time at 750 mg/kg bw/day, the NOAEL for reproductive performance/fertility was 200 mg/kg bw/day.

This study is acceptable and satisfies the guideline requirement for a one-generation reproduction toxicity study (OECD 415).

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

good quality

Additional information

In a one-generation reproduction toxicity study (NOTOX B.V., 1997), the structural analogue Fe(Na)EDDHMA (CAS 84539 -53 -7) in distilled water was administered to 28 Wistar rats/sex/dose level by single oral gavage (5 mL/kg bw) at dose levels of 50, 200 or 750 mg/kg bw/day (according to the OECD TG 415). A concurrent control group was treated with the vehicle only. Treatment commenced 10 weeks prior to mating for males and 2 weeks prior to mating for females and continued for both sexes until at least the end of the lactation period. Pregnant females were allowed to litter normally. On day 4 of lactation, each litter was adjusted to 4 males and 4 females or as near as possible. The surviving offspring was euthanized as soon as possible after weaning.

The primary effect of treatment with the test item on parental animals was poor physical condition, resulting in premature mortality, growth reduction and reduced food consumption in male and female animals at 750 mg/kg bw/day. These signs of test item-related toxicity were seen with reduced severity at 200 mg/kg bw/day in males only. Thus, the NOAEL for systemic toxicity in parental animals was 50 mg/kg bw/day under the conditions of this study.

In the offspring, increased post natal loss and reduced viability were noted during PND 0 -4 at 200 and 750 mg/kg bw/day, and with lower incidence at 50 mg/kg bw/day. With special regard to the low incidence and unusual distribution pattern of findings noted at 50 mg/kg bw/day, the NOAEL for developmental toxicity was 50 mg/kg bw/day under the conditions of this study. Based on a slight decrease in the conception indices and a minimal delay in precoital time at 750 mg/kg bw/day, the NOAEL for reproductive performance/fertility was 200 mg/kg bw/day.

Short description of key information:
For the assessment of toxicity to reproduction/fertility of Fe(Na)HBED, the study conducted with the read-across substance Fe(Na)EDDHMA (CAS 84539-53-7) was used. The read-across approach from this structural analogue is considered justified since, in addition to the very similar physico-chemical properties of this substance with the target substance, there are marked similarities with respect to their toxicological profiles as evidenced by: no acute toxicity, no skin or eye irritating properties, no mutagenic effects (please refer to read-across justification attached to the IUCLID file under section 13).
In the one-generation reproduction toxicity study performed with the test item Fe(Na)EDDHMA in rats, the NOAEL for reproductive performance/fertility was established at 200 mg/kg bw/day based on a slight decrease in the conception indices and a minimal delay in precoital time noted at the high dose level of 750 mg/kg bw/day. In agreement with the read-across approach, this NOAEL is considered to represent a reliable key value for chemical safety assessment of Fe(Na)HBED.

Justification for selection of Effect on fertility via oral route:
Guideline and GLP study conducted with the structurally related analogue Fe(Na)EDDHMA (CAS 84539-53-7).

Effects on developmental toxicity

Description of key information

In a developmental oral toxicity study (CIBA-GEIGY Limited, 1995) with the related substance Fe(Na)EDDHA in rats, the NOEL for developmental effects was established at the high dose level of 500 mg/kg bw/day based on the absence of embryo-/foetotoxic or teratogenic effects.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

key study

Study period:

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OTS 798.4900 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

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

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".

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.

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.

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOEL

Effect level:

500 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects: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.

Dose descriptor:

NOEL

Effect level:

500 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

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.

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.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

good quality

Additional information

In a developmental toxicity study (CIBA-GEIGY, 1995; Report No. 941105), the related substance Fe(Na)EDDHA (CAS 84539 -55 -9) 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 (according to the OECD TG 414). 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 teratogenicity 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 developmental toxicity and teratogenicity.

Justification for selection of Effect on developmental toxicity: via oral route:
Guideline and GLP study conducted with the structurally related analogue Fe(Na)EDDHA (CAS 84539-55-9)

Justification for classification or non-classification

Effects on reproductive performance/fertility and developmental toxicity were assessed by means of a read-across approach using a developmental study according to the OECD TG 414 conducted with the structural analogue Fe(Na)EDDHA and one generation toxicity study according to test guideline OECD 415 conducted with the structural analogue Fe(Na)EDDHMA. The test item Fe(Na)EDDHA caused no developmental toxicity and no teratogenicity in the rat in the developmental toxicity study according to test guideline OECD 414. Only a slight change in reproductive performance was noted in the one-generation study at 1000 mg/kg bw/day. In the absence of concomitant histopathological findings, this slight change in reproductive performance was considered to be secondary to the poor clinical condition of the animals at this dose level. Similarly, developmental effects were seen in this study at dose levels at pronounced maternal toxicity only.

Accordingly, Fe(Na)HBED is not subject to classification for toxicity to reproduction and developmental effects according to the Regulation 1272/2008/EC.