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Repeated dose toxicity: oral

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Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1997-01-30 to 1998-06-18
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.

Lower food intake and impaired body weight development were noted at 200 mg/kg bw/day (the highest dose tested). Reversible effects on red blood cell (normochromic anaemia) and white blood cell parameters, and higher values of platelets and prothrombin activity were noted at 50 and/or 200 mg/kg bw/day. In details, lower erythrocyte count, hemoglobin concentration and hematocrit were noted for males and females treated at 200 mg/kg and for males at 50 mg/kg. A higher reticulocyte count pointing to the regenerative nature of anemia was confined to males of group 4 (200 mg/kg), and was associated with typical signs of immature erythrocytes such as higher MCV and MCH values. In addition, a minor reduction in white blood cell counts with slightly reduced values for basophils, lymphocytes and monocytes was recorded for males of the 200 mg/kg group. A higher platelet count was recorded for males at >50 mg/kg and a higher prothrombin activity was recorded for males and females of the 200 mg/kg group. Evidence of a recovery for all the above parameters was apparent after 4 weeks withdrawal from treatment.
The effects observed in the studies with the source substance Fe(Na)EDDHA clearly indicate conditions leading to IDA. It is therefore clear that these chelator-iron complexes dissociate in the body releasing free chelators that remove systemically available iron or reduce GI uptake of iron into the body. In opposite, Fe(Na)EDTA does not induce anaemia effects, but improves iron status without iron overloading effects. These facts confirm the first assumption set in the hypothesis of the read-across, allowing to exclude systemic toxicity of the organic moiety HBED and thus to ascribe the mode-of action of Fe(Na)HBED entirely to perturbation of iron metabolism induced by this moiety HBED. It means, that in such a hypothetical worst-case situation, if the Fe(Na)HBED complex dissociated in the body, certain toxicity effects could be expected relating to IDA in normal animals.

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:
1998
Report date:
1998

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

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
- Analytical purity: 100 % (UVCB); 8.16% (total iron)
- Lot/batch No.: P.201845
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
other: Sprague-Dawley derived; Tif:RAIf (SPF); hybrids of RII/1 x RII/2
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Animal Production, Novartis Pharma AG, 4332 Stein, Switzerland
- Age at start of acclimation period: approximately 5 weeks old
- Weight range at acclimation period: 149.5-185.8 g (males), 134.7-165.6 g (females)
- Housing: in groups of 5 animals, in macrolon cages type 4 with wire mesh tops and standardised granulated soft wood bedding
- Diet: pelleted, certified standard diet (Nafag No. 8900 FOR GLP), provided ad libitum (exception: food was withheld overnight prior to blood collection)
- Water: tap water, ad libitum
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 55 ± 10 %
- Air changes: 16-20 air changes/hour
- Photoperiod: 12 hours dark / 12 hours light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5 % CMC and 0.1 % Tween 80 in distilled water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Suspensions of the test article in the vehicle at the appropriate concentrations were freshly prepared every day immediately prior to the dosing of the animals and administered within about 2 hours.

VEHICLE
- Justification for use and choice of vehicle: standard procedure
- Amount of vehicle: 10 mL/kg bw of suspension were applied
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
1.Analyses of the test article in the vehicle were performed in a previously conducted study (941102) at the analytical laboratories of RCC Umweltchemie AG, 4452 Itingen / Switzerland. In the tested range (0.1 - 100 mg/mL), homogeneous distribution of the test article in the vehicle was recorded. The content of the test article in the vehicle was in agreement with the nominal concentrations. CGA 65047 F (A-5787 A) was stable in the vehicle over a period of 4 hours at room temperature.
2. Control analyses of the test article in the vehicle were carried out at all dose levels on samples collected once per experimental weeks 1, 4, 8 and 13. Samples were analysed by RCC Umweltchemie AG, 4452 Itingen, Switzerland (RCC Project Number 651688). The analytical procedure for content determination yielded values close to the targeted concentrations and within the limits of acceptance.
Duration of treatment / exposure:
90 days
Frequency of treatment:
once per day, 7 times per week
Doses / concentrations
Remarks:
Doses / Concentrations:
5, 50 and 200 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
Main Experiment: 10 animals
Recovery: 10 animals (control and high dose group only)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose levels were selected based on the results of a preceding 28-day dose range finding toxicity study, project no. 941102 (CIBA-GEIGY, 1996):
The treatment with CGA 65047 F (A-5787 A) at doses of 0, 50, 2 00 and 1000 mg/kg body weight by daily gavage resulted in impaired body weight development of rats treated at 200 and 1000 mg/kg body weight and correspondent lower food intake. Animals treated at 1000 mg/kg body weight consumed markedly more water. All animals did not show signs of overt toxicity and survived to scheduled sacrifice. Laboratory investigations revealed an anemia at 200 and 1000 mg/kg body weight without erythropoietic response. At the 1000 and 200 mg/kg dose levels, the kidney was revealed as target organ by histopathological examination, by blood chemistry data and by organ weight evaluation. In addition, organ weight evaluation showed changes in relative weights of heart (males at 200 and males and females at 1000 mg/kg), adrenals (males at 50, 200 and 1000 mg/kg), and spleen (females at 1000 mg/kg). However, in the absence of corroborative findings, the experimental relevance of these findings was considered equivocal.
- Rationale for animal assignment (if not random): computer-generated random numbers
- Rationale for selecting satellite groups: to investigate reversibility of the effects.
- Post-exposure recovery period in satellite groups: 4 weeks
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
- Time schedule: twice daily for mortality and once daily for general observations.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: at least weekly

BODY WEIGHT:
- Time schedule: once weekly

FOOD CONSUMPTION:
- Food consumption was determined cagewise on a weekly basis and mean daily diet consumption calculated as g food/kg bw/day.

WATER CONSUMPTION
- Time schedule for examinations: weekly, determined cagewise

OPHTHALMOSCOPIC EXAMINATION:
- Time schedule for examinations: during the acclimation period (all animals), at the end of the treatment and recovery periods (control and high dose group only)
- Dose groups that were examined: all animals during the acclimation period, only high dose and control group animals on day 87 (treatment period) and day 115 (recovery period)

HAEMATOLOGY:
- Time schedule for collection of blood: at the end of the treatment and recovery periods
- Anaesthetic used for blood collection: ether
- Animals fasted: food was withheld overnight prior to blood sampling
- How many animals: all animals
- Parameters examined: erythrocyte count, haematocrit, mean corpuscular volume, red cell volume distribution width, haemoglobin concentration, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, haemoglobin concentration distribution width, prothrombin time, methaemoglobin concentration and leukocyte, neutrophil, eosinophil, basophil, lymphocyte, monocyte, large unstained cells, reticulocyte and thrombocyte counts

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: at the end of the treatment and recovery periods
- Animals fasted: food was withheld overnight prior to blood sampling
- How many animals: all animals
- Parameters examined: glucose, urea, creatinine, total bilirubin, total protein, albumin, globulin, cholesterol, triglycerides, sodium, potassium, calcium, chloride and inorganic phosphorus concentration, and A/G ratio and aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and gamma-glutamyl transpeptidase activity

URINALYSIS:
- Time schedule for collection of urine: at the end of the treatment and recovery periods
- Metabolism cages were used for collection of urine
- Animals fasted: food was withheld overnight prior to blood sampling
- Parameters examined: urine volume, relative density, pH-value, urine colour and protein, glucose, ketone, bilirubin, erythrocyte, leukocyte and urobilinogen content
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY:
The following organs or tissues were examined microscopically:
spleen, mesenteric lymph node, axillary lymph node, femur with joint, bone marrow (femur), trachea, lung, heart, aorta, submandibular salivary gland, liver, pancreas, oesophagus, stomach, small intestine (duodenum, ileum, jejunum), large intestine (caecum, colon, rectum), kidney, urinary bladder, testis, epididymis, uterus, vagina, ovary, adrenal gland, thyroid with parathyroid gland, thymus, peripheral nerve (sciatic nerve), brain (including medulla, pons, cerebral and cerebellar cortex) and any organ with gross lesions.
Other examinations:
ORGAN WEIGHT
The weights of the following organs were determined at necropsy:
brain, heart, liver, kidneys, adrenals, thymus, ovaries or testes, spleen and thyroid
Statistics:
Each treated group was compared to the control group either by Lepage's or Wilcoxon's two-sample test and tested for increasing or decreasing trends from control up to the respective dose group by Jonckheere's test for ordered alternatives.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY
During this study neither changes to the behavior nor relevant clinical signs were observed.
There was no mortality during this study which was attributed to the treatment with the test article.
In the third treatment week one female animal (no. 86) died. However, there were no indications of a testarticle-related occurrence.

BODY WEIGHT AND WEIGHT GAIN
Decreased mean body weights were noted from weeks 5 and 8 onwards at 200 mg/kg bw/day in male and female rats, respectively. The mean body weight gains were decreased by the end of the treatment period in animals treated with 200 mg/kg bw/day. During the recovery period, body weight gain in animals previously treated with 200 mg/kg bw/day was higher as compared to controls.
The body weight development in the other treated groups was not influenced by treatment.

FOOD CONSUMPTION
Food consumption and food consumption ratios were decreased at 200 mg/kg bw/day in both sexes (decreased food consumption of 13% in males and of 8% in females, compared to the controls). During the recovery period food intake improved.
The mean food consumption of the other treated groups was not influenced by treatment.

WATER CONSUMPTION
The mean water consumption was not influenced by treatment.

OPHTHALMOSCOPIC EXAMINATION
The examinations of the eyes (lids and surrounds, conjunctiva, pupillary reflex, cornea, sclera, anterior chamber, lens, vitreous, and fundus) towards treatment end (day 87) and towards recovery end (day 115) did not reveal any treatment-related findings.

HAEMATOLOGY
At the end of the treatment period, a normochromic anaemia with lower erythrocyte count, haemoglobin concentration and haematocrit was observed in males and females at 200 mg/kg bw/day and males at 50 mg/kg bw/day. A higher reticulocyte count associated with higher MCV and MCH values and reduced white blood cell, basophil, lymphocyte and monocyte counts were confined to males at 200 mg/kg bw/day. A higher platelet count was recorded for males at 50 and 200 mg/kg bw/day and a higher prothrombin activity was recorded for males and females at 200 mg/kg bw/day. Evidence of reversibility for all the above parameters was apparent after the recovery period.

CLINICAL CHEMISTRY
Several clinical chemistry parameters including creatinine, urea, protein, globulin, cholesterol and sodium concentration were increased at 50 and/or 200 mg/kg bw/day. Lower potassium levels were noted in males at 200 mg/kg bw/day. All values were similar to the control group values after the 4-week recovery period.

URINALYSIS
Excretion of more acidic red-brown (males) or yellow-brown (females) discoloured urine was observed at 200 mg/kg bw/day. More acidic urine was also excreted by males at 50 mg/kg bw/day. By the end of the recovery period, the colour and pH of the urine excreted by male and females previously treated at 200 mg/kg bw/day was similar to that of control group animals.

ORGAN WEIGHTS
The mean carcass weights were decreased at the end of the treatment period at 200 mg/kg bw/day. Males at 200 mg/kg bw/day showed an elevated mean heart to body weight ratio which still was higher than the concurrent control value at the end of the recovery period.

GROSS PATHOLOGY and HISTOPATHOLOGY
There were no test item related findings.

Effect levels

Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: haematology parameters (anaemia) The NOAEL was estimated based on the LOAEL of 50 mg/kg bw/day applying an assessment factor of 5.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

CHEMICAL ANALYSIS OF DOSE FORMULATIONS

The calculated overall mean contents of the test item in the dose formulations used for the low, mid and high dose groups were 96.9, 104.2 and 101.0 % of the nominal concentrations, respectively (RCC project no. 651688). In a previous 28 -day dose range finding study with the same test item and vehicle, the stability and homogeneity of dose formulations were within the limits of acceptance (RCC project no. 393322).

Estimation of NOAEL:

A more realistic NOAEL was estimated from the LOAEL of 50 mg/kg bw/day applying an assessment factor of 5. This method is applicable and scientifically justified for this test, as only slight adverse effects were observed at 50 mg/kg bw/day (haematology parameters - anaemia). To take the relatively large concentration gap between 5 (clear NOEL) and 50 mg/kg bw/day into account, 5 mg/kg bw/day was not taken as NOAEL, but extrapolated form the LOAEL. The guidance on information requirements and CSA, R.8 (ECHA, 2008 -2010) recommends a factor between 3 and 10 for extrapolation from LOAEL to NOAEL. An assessment factor of 5 seems to be approprate and conservative enough for the current study as only slight effects were observed at the LOAEL of 50 mg/kg bw/day. Consequently a NOAEL of 10 mg/kg bw/day is calculated for this study and used for DNEL and PNEC(oral) derivation.

Applicant's summary and conclusion

Conclusions:
NOEL of 5 mg/kg bw was established in this study (for males and females). NOAEL of 10 mg/kg bw was derived based on LOAEL of 50 mg/kg bw.
Executive summary:

In a subchronic toxicity study (Novartis Crop Protection AG, 1998), FeNaEDDHA in 0.5 % CMC and 0.1 % Tween 80 in distilled water was administered to 10 Sprague-Dawley derived rats/sex/dose level by oral gavage (10 mL/kg bw) at dose levels of 5, 50 or 200 mg/kg bw/day for a period of 90 days. Male and female animals of the concurrent control group were treated with the vehicle only. Additional 10 animals/sex of the high dose and control group were kept on control diet for a 4 -week recovery period before sacrifice. Treatment with the test item resulted in lower food intake and impared body weight development of rats treated at 200 mg/kg bw/day. Reversible effects on the red blood cell (normochromic anaemia) and white blood cell parameters, and higher values of platelets and prothrombin activity were noted at 50 and/or 200 mg/kg bw/day. In addition, there were changes of blood chemistry and urine parameters concerning the liver and kidneys. The body weight relative heart weight was increased in males at 200 mg/kg bw/day. Under the conditions of this study, the NOAEL for FeNaEDDHA when administered by daily oral gavage for three months was 10 mg/kg bw/day (estimated from the LOAEL).

This subchronic toxicity study in the rat is acceptable and satisfies the guideline requirement for a subchronic oral study (OECD 408).

Estimation of NOAEL:

A more realistic NOAEL was estimated from the LOAEL of 50 mg/kg bw/day applying an assessment factor of 5. This method is applicable and scientifically justified for this test, as only slight adverse effects were observed at 50 mg/kg bw/day (haematology parameters - anaemia). To take the relatively large concentration gap between 5 (clear NOEL) and 50 mg/kg bw/day into account, 5 mg/kg bw/day was not taken as NOAEL, but extrapolated form the LOAEL. The guidance on information requirements and CSA, R.8 (ECHA, 2008 -2010) recommends a factor between 3 and 10 for extrapolation from LOAEL to NOAEL. An assessment factor of 5 seems to be approprate and conservative enough for the current study as only slight effects were observed at the LOAEL of 50 mg/kg bw/day. Consequently a NOAEL of 10 mg/kg bw/day is calculated for this study.