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)-

Repeated dose toxicity: dermal

Administrative data

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

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

Adequacy of study:

key study

Study period:

1996-01-26 to 1995-03-03

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.

FeNaEDDHA in distilled water was administered to the skin (clipped fur) of 5 Sprague-Dawley derived rats/sex/dose level at dose levels of 10, 100 or 1000 mg/kg bw/day for a period of 28 days (5 days per week basis). Male and female animals of the concurrent control group were treated with the vehicle only. Dermal treatment with the test item resulted in no mortality, no relevant clinical signs, no changes in food consumption, no effects on haematology and clinical chemistry parameters and no gross findings. A transient slight body weight loss was noted in females at 1000 mg/kg bw/day during the first week of treatment. There was an increase in adrenal weight in males at 1000 mg/kg bw/day. Microscopically, the skin application sites of females at 1000 mg/kg bw/day revealed epidermal hyperkeratosis associated with an increased severity of acanthosis. In 2/5 males at 1000 mg/kg bw/day centrilobular hypertrophy of hepatocytes was noted. Based on the slight effects on the liver and skin and due to the increased adrenal weight noted at 1000 mg/kg bw/day under the conditions of this study, the NOEL was established at 100 mg/kg bw/day.

This results support assumption that the chelates are not expected to be absorbed significantly into the stratum corneum following dermal exposure, based on the physico-chemical properties: very high water solubility (34.55 g/L) and the negative LogPow (-1.96). Accordingly, their systemic toxicity via the skin has been proven to be low (no mortality after dermal application of 2000 mg/kg bw of the structurally similar analogues Fe(Na)HBED and Fe(Na)EDDHA in rats (please refer to section 2.7.2 read-accros stataement and section 7.2.3. of IUCLID file).

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:

1996

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPP 82-2 (Repeated Dose Dermal Toxicity -21/28 Days)

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)
- Analytical purity: 100 % (UVCB)
- 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: Laboratory Animal Breeding, Pharmaceutical Division, CIBA-GEIGY Limited, 4332 Stein, Switzerland
- Age at study initiation: young adult, based on body weight ranges
- Weight range at acclimation period: 213.9-228.1 g (males), 211.8-235.7 g (females)
- Housing: individually, in macrolon cages type 3 with wire mesh tops and granulated soft wood bedding
- Diet: pelleted, certified standard diet Nafag No. 890 (NAFAG AG, Gossau, SG, Switzerland), provided ad libitum (exception: food was withheld overnight prior to blood removal performed at the end of the treatment period)
- Water: tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 55 ± 15 %
- Air changes: approximately 15 air changes/hour
- Photoperiod: 12 hours dark / 12 hours light

Administration / exposure

Type of coverage:

occlusive

Vehicle:

water

Details on exposure:

TEST SITE
- Area of exposure: dorsal area of the trunk
- Coverage: at least 10 % of the body surface area
- Type of wrap: gauze patches, aluminium foil and adhesive but non-irritating tape
- Time intervals for shavings or clipplings: once weekly

REMOVAL OF TEST SUBSTANCE
- Washing: with lukewarm water
- Time after start of exposure: 6 hours (on each day of treatment)

TEST MATERIAL/VEHICLE
- Amount applied: 4 mL/kg bw
- Concentrations: 2.5, 25 and 250 mg/mL
- Constant volume used: yes, adjusted weekly to individual animal body weight

USE OF RESTRAINERS FOR PREVENTING INGESTION: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Control analyses of the test item concentration in the vehicle was carried out at all dose levels on samples collected on experimental days 1, 7, 14 and 21. Samples were analysed by RCC Umweltchemie AG, 4452 Itingen, Switzerland (Project No. 329207). The test article concentrations in the vehicle were found to be in the range from 82.5 % to 105.3 % of the nominal concentrations.

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

6 hours per day, on 5 days per week

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 10, 100 and 1000 mg/kg bw/day
Basis:
nominal per unit body weight

No. of animals per sex per dose:

5 animals

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: no rationale is given in the report.

Positive control:

none

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
- Time schedule: twice daily on working days and once daily on weekend days for mortality, and once daily for general observations.
- Skin irritation: Following each application, approximately 17 hours after patch removal, local skin reactions at the application site were assessed according to Draize et al. (1944).

BODY WEIGHT:
- Time schedule: on study days -7, 1, 8,15, 22 and 28

FOOD CONSUMPTION:
- Food consumption for each animal was determined weekly and mean daily diet consumption calculated as g food/kg bw/day.

HAEMATOLOGY:
- Time schedule for collection of blood: once, at the end of the treatment period
- 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 and leukocyte, neutrophil, eosinophil, basophil, lymphocyte, monocyte, large unstained cells and thrombocyte counts

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: once, at the end of the treatment period
- 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 and alkaline phosphatase activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY:
The following organs or tissues were examined microscopically:
skin application site, skin remote site, liver, spleen, kidneys, thymus, thyroid with parathyroid gland, prostate, seminal vesicle, testis, epididymis, ovary

Other examinations:

ORGAN WEIGHT
The weights of the following organs were determined at necropsy:
brain, heart, liver, kidneys, adrenals, thymus, ovaries or testes, and spleen

Statistics:

Each treated group was compared to the control group by Wilcoxon's two-sample test (non-parametric) and tested for increasing or decreasing trends from control up to the respective dose group by Jonckheere's test for ordered alternatives (parametric).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Dermal irritation:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

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:

effects observed, treatment-related

Histopathological findings: neoplastic:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY
No treatment related clinical signs and changes in behavior were noted and all animals survived.

BODY WEIGHT AND WEIGHT GAIN
A slight body weight loss was noted in females at 1000 mg/kg bw/day during the first week of treatment. Otherwise the body weight was comparable in all groups: "During week 1 of treatment, female group 4 (1000 mg/kg) did not gain body weight. Afterwards, mean body weight gain was essentially comparable to that of the control group. At end of treatment the mean body weight was 4% lower than that of the control group".

FOOD CONSUMPTION
Food consumption was not influenced by treatment.

HAEMATOLOGY AND CLINICAL CHEMISTRY
No effects on haematology and clinical chemistry parameters were noted.

ORGAN WEIGHTS
There was an increased adrenal weight in males at 1000 mg/kg bw/day: "Mean absolute and relative adrenal weights of males in group 4 (1000 mg/kg) were 23 % and 22 % higher than those of the control group, respectively".

GROSS PATHOLOGY
No treatment related macroscopical findings were noted.

HISTOPATHOLOGY
The skin application site of females treated at 1000 mg/kg bw revealed epidermal hyperkeratosis and an increased severity of acanthosis. In one female, an additional epidermal parakeratosis and a chronic dermal inflammation were observed. In 2/5 males at 1000 mg/kg bw, centrilobular hypertrophy of hepatocytes was noted.

Effect levels

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on slight effects on the liver and skin and due to increased adrenal weight noted at 1000 mg/kg bw/day.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

SIGNS OF LOCAL IRRITATION

The skin application site of all animals of the high dose group (1000 mg/kg) was discolored after treatment start, due to staining properties of the test article. Therefore, evaluation of erythema was impeded in the high dose group. From treatment day 19 onwards, one female presented with slight crust formation at the site of application. No further local skin reactions were noted in this and all other groups.

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 102, 103 and 89.7 % of the nominal concentrations, respectively (RCC Project No. 392207). The test item was proved to be stable in the vehicle under actual conditions of administration over the period of dosing.

Applicant's summary and conclusion

Conclusions:

Under the conditions of this test, dermal treatment of rats with FeNaEDDHA resulted in depressed body weight gain of females during week 1 of treatment at 1000 mg/kg body weight with consequent marginal body weight reduction at study end as compared to controls. All animals survived to scheduled sacrifice, and no clinical signs of overt toxicity were noted.
Histopathological examination revealed local intolerance at the skin application site for the high dose females (1000 mg/kg body weight). In addition, hypertrophy of liver hepatocytes in individual males of the high dose group was found, most likely indicative of adaptive changes.
For both sexes the NOEL (No observable effect level) was 100 mg/kg body weight.

Executive summary:

In a repeat-dose dermal toxicity study (CIBA-GEIGY Limited, 1996b), FeNaEDDHA in distilled water was administered to the skin (clipped fur) of 5 Sprague-Dawley derived rats/sex/dose level at dose levels of 10, 100 or 1000 mg/kg bw/day for a period of 28 days (5 days per week basis). Male and female animals of the concurrent control group were treated with the vehicle only. Dermal treatment with the test item resulted in no mortality, no relevant clinical signs, no changes in food consumption, no effects on haematology and clinical chemistry parameters and no gross findings. A transient slight body weight loss was noted in females at 1000 mg/kg bw/day during the first week of treatment. There was an increase in adrenal weight in males at 1000 mg/kg bw/day. Microscopically, the skin application sites of females at 1000 mg/kg bw/day revealed epidermal hyperkeratosis associated with an increased severity of acanthosis. In 2/5 males at 1000 mg/kg bw/day centrilobular hypertrophy of hepatocytes was noted. Based on the slight effects on the liver and skin and due to the increased adrenal weight noted at 1000 mg/kg bw/day under the conditions of this study, the NOEL was established at 100 mg/kg bw/day.

This dermal toxicity study in the rat is acceptable and satisfies the requirement for test guideline OECD 410.