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

Endpoint summary

Description of key information

In a 28-day oral (gavage) key repeated dose toxicity study in rats conducted with the nearest analogue Fe(3K)EDDHSA, the NOEL was established at 1000 mg/kg bw/day, the highest dose level tested. No treatment- related effects were observed in animals in any parameter tested. In a key subacute 28-day dermal toxicity study conducted with another closely related substance Fe(Na)EDDHA, the NOEL was established at 100 mg/kg bw/day based on slight effects on the liver and skin and due to increased adrenal weight noted at the high dose level of 1000 mg/kg bw/day. No data on repeated inhalation exposure are required. Exposure by the inhalation route is considered to be negligible. The target substance Fe(3Na)EDDHSA does not need to be classified for systemic organ toxicity after repeated exposures.   
   

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

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

Adequacy of study:

key study

Study period:

2000-08-29 to 2000-10-06

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EEC, directive No. 96/54, B7, 30th September 1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 424 (Neurotoxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA, guideline 799, 9620-62-128, 15th August 1997

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
44 Sprague-Dawley rats (22 males and 22 females) were received at test facility.
- Source: Iffa Credo, L'Arbresle, France
- Age at study initiation: approximately 6 weeks old
- Weight at study initiation: 196 g (range: 182 g to 209 g) for the males and 156 g (range: 137 g to 175 g) for the females
- Fasting period before study: no
- Housing: individually in suspended wire-mesh cages (43.0 cm x 21.5 cm x 18.0 cm). A metal tray containing autoclaved sawdust (SICSA, Alfortville, France) was placed under each cage.
- Diet (e.g. ad libitum): free access to A04 C pelleted maintenance diet, batch No. 00331 (UAR, Villemoisson, Epinay-sur-Orge, France. Prior blood sampling the animals were fasted overnight.
- Water (e.g. ad libitum): filtered tape water
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2,
- Humidity (%): 50 ± 20
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

purified water, obtained by reverse osmosis

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was dissolved in the required quantity of vehicle in order to achieve the concentrations of 30, 90 and 200 mg/mL and then homogenized using a magnetic stirrer.
The test substance dosage forms were made up to 4 days from days 1 to 3 and up to 9 days, from day 4 of treatment, and were stored at +4°C prior to use. The dosage forms were delivered each day to the animal room.

VEHICLE

- Concentration in vehicle: 30, 90 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical analysis of the dosage forms
Before the start of treatment, the suitability of the proposed dosage form preparation procedure was determined by the analysis of stability of dosage forms which were prepared using this procedure. During the treatment period, the concentration of the test material was checked in dosage forms prepared for use in the study.

Stability
Two dosage forms were prepared to evaluate the stability:
- a dosage form at low concentration (2 mg/mL),
- a dosage form at high concentration (200 mg/mL). Each dosage form was analysed immediately after preparation and was then stored at 4°C (protected from light) and sampled after 4 and 9 days storage. Samples taken on days 4 and 9 were analyzed as soon as possible after sampling.


Concentration
The concentration of samples taken from each dosage form (including the control) prepared for use in weeks 1 and 4 was determined.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0, 150, 450 and 1000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose-levels were selected on the basis of the results of a 7-day range-finding toxicity study by oral route performed in the same species (CIT/Study No. 20508 TSR) in which no clinical signs or macroscopic findings were noted in any treated groups. Consequently the same dose-levels were selected.

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice a day for mortality or signs of morbidity and at least once a day for clinical signs

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before the first day of treatment and then at the end of weeks 1, 2, 3 and 4 (at least 12 hours after the last treatment. All animals of each group were observed in the cage, in the hand and in the standard arena, by observers unaware of the animal's treatment. Detailed clinical observation in week 4 was performed before blood sampling.
The following parameters were assessed:
- in the hand: fur appearance, salivation, lachrymation, piloerection, exophthalmia, reactivity to handling, pupil size (presence of myosis or mydriasis),
- in the standard arena (2-minute recording): grooming, palpebral closure, defecation and urination counts, tremors, twitches, convulsions, gait, arousal (hypo- and hyper- activity), posture, stereotypic behaviour and breathing, ataxia, hypotonia.

BODY WEIGHT: Yes
- Time schedule for examinations: once before allocation of the animals into groups, on the first day of treatment, and then once a week until the end of the study.

FOOD CONSUMPTION (gavage study):
The quantity of food consumed by the animals of each cage was recorded once a week, over a 7-day period, until the end of the study (calculated as mean values in g/animal/day)

OPHTHALMOSCOPIC EXAMINATION: No (pupil reflex was examined: see Neurobehavioural Examination)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of week 4
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, prior to blood sampling, the animals were deprived of food for an overnight period of at least 14 hours
- How many animals: all animals
- Parameters were examined:
Erythrocytes (RBC)
Hemoglobin (HB)
Mean Cell Volume (MCV)
Packed Cell Volume (PCV)
Mean Cell Hemoglobin Concentration (MCHC)
Mean Cell Hemoglobin (MCH)
Thrombocytes (PLAT)
Leucocytes (WBC)
Differential White Cell count with cell morphology: neutrophils (N), eosinophils (E), basophils (B), lymphocytes (L), monocytes (M)
Prothrombin Time
Activated Partial Thromboplastin Time
Fibrinogen

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of week 4
- Animals fasted: Yes, prior to blood sampling, the animals were deprived of food for an overnight period of at least 14 hours
- How many animals: all animals
- Parameters were examined:
Sodium (Na+), Potassium (K+), Chloride (C1-), Calcium (Ca++)
Inorganic phosphorus (I.PHOS) Glucose (GLUC)
Urea (UREA)
Creatinine (CREAT)
Total Bilirubin (TOT.BIL)
Total Proteins (PROT)
Albumin (ALB)
Albumin/globulin ratio (A/G)
Cholesterol (CHOL)
Triglycerides (TRIG)
Alkaline phosphatase (ALP)
Aspartate aminotransferase (ASAT)
Alanine aminotransferase (ALAT)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: All animals were evaluated before the first day of treatment, and then at the end of week 4, at least 12 hours after the last treatment. The observer performing the evaluation was not aware of the treatment group of the animal.
Reactivity to manipulation or to different stimuli in week 4 was performed before blood sampling.
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity / grip strength / motor activity (was measured by automated infra-red sensor equipment recording individual animal activity over 30-min period, before the first day of treatment and then in week 4)/ other: pupil reflex, visual stimulus, auditory startle reflex, tail pinch response, righting reflex, landing foot splay, at the end of observation: rectal temperature

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
A complete macroscopic post-mortem examination was performed on all study animals. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues.

HISTOPATHOLOGY: Yes
A microscopic examination was performed on:
- all tissues listed in the tissue procedures table (please refer to table in "Any other information on materials and methods incl. tables" for animals of the control and high-dose groups (groups 1 and 4) killed at the end of the treatment period,
all macroscopic lesions of all the animals of the low- and intermediate- dose groups (groups 2 and 3) killed on completion of the treatment period.

Other examinations:

ORGAN WEIGHTS
The organs specified in the Tissue Procedures Table ((please refer to table in "Any other information on materials and methods incl. tables") were weighed wet as soon as possible after dissection. The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated.

Statistics:

Scheme of statistical analysis is attached (see below)

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

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):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No mortalities and no treatment-related clinical signs were noted.

BODY WEIGHT AND WEIGHT GAIN
The mean body weight gain of males and females given the test substance at 150, 450 and 1000 mg/kg/day was similar to that of respective control groups throughout the treatment period (males: +158 g, +139 g and +145 g vs. +146 g in controls; females: +66 g, +74 g and + 76 g vs. +70 g in controls

FOOD CONSUMPTION
The mean food consumption of males and females given the test substance was similar to that of respective control groups throughout the treatment period.

HAEMATOLOGY
No toxicologically significant changes were noted

CLINICAL CHEMISTRY
No treatment-related differences from controls were noted in the treated animals for all the parameters examined.

NEUROBEHAVIOUR
No specific signs of a neurotoxic action of the test substance were noted.

ORGAN WEIGHTS
No treatment-related effects were noted in the organ weights.

GROSS PATHOLOGY
The following necropsy findings were noted in the digestive tract:
blackish contents were noted in several parts of the digestive tract of treated animals as follows:
- stomach, in 1/5 males given 1000 mg/kg/day,
- ileum in 1/5 females given 450 mg/kg/day and in 1/5 males given 1000 mg/kg/day,
- cecum, in 1/5 females given 150 mg/kg/day, in 4/5 males and all the females given 450 mg/kg/day and in 4/5 males and all the females given 1000 mg/kg/day,
- colon, in 1/5 females given 150 mg/kg/day, in 3/5 males and 4/5 females given 450 mg/kg/day and in 3/5 males and 1/5 females given 1000 mg/kg/day,
- rectum, in 1/5 males given 450 mg/kg/day and in 1/5 males given 1000 mg/kg/day.
Microscopic examination revealed no changes that related to this blackish material which was consequently considered to be a remnant of the test substance and therefore of no toxicological importance.

HISTOPATHOLOGY: NON-NEOPLASTIC
No changes were seen that were related to treatment.

OTHER FINDINGS
No other findings

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment related effects were noted in any parameter tested

Critical effects observed:

not specified

Chemical Analyses of the Dosage Forms

Stability

The results of the analyses demonstrated a satisfactory stability of the two dosage forms over a 9-day period at +4°C (protected from light).

Concentration

Throughout the study, a satisfactory agreement was observed between the nominal and actual concentrations of the test material in the dosage forms administered since the deviations from nominal concentration were in the requested range of +10%.

Conclusions:

The daily administration of the test substance, EDDHAS Fe 3K (batch No. CED 0701), at the dose-levels of 150, 450 and 1000 mg/kg/day, by oral route (gavage) to rats for 28 days did not produce any signs of toxicity.
Consequently, under our experimental conditions, the No Observable Effect Level (NOEL) was established at 1000 mg/kg/day.

Executive summary:

The objective of this study was to evaluate the potential toxicity of the test substance, EDDHAS Fe 3K, following daily oral administration (gavage) to Spague-Dawley rats for 28 days.

Methods

Groups of five males and five females Sprague-Dawley rats received the test substance, EDDHAS Fe 3K, daily by gavage at the dose-levels of 150, 450 or 1000 mg/kg/day for 28 days. An additional group of five males and five females received the vehicle alone (purified water) under the same experimental conditions, and acted as a control group. The animals were checked daily for mortality and clinical signs. A functional observation battery was performed on all animals of each group, once in predose and then in week 4; in addition, a detailed clinical observation was performed on all animals of each group, once a week until the end of the treatment period. Motor activity was recorded on all animals once in predose and in week 4. Body weight and food consumption were recorded once a week. Hematological and blood biochemical investigations were performed for all animals at the end of the treatment period. On completion of the treatment period, all animals were killed and submitted to a complete macroscopic post-mortem examination. Designated organs were weighed and selected tissues were preserved. A microscopic examination was performed on designated tissues of the animals of the control and high dose-level group.

Results

Mortality

No unscheduled deaths occurred during the study.

Clinical signs

No clinical signs of toxicological significance were observed in any treated animals.

Functional observation battery and motor activity

No specific signs of a neurotoxic action of the test substance were noted.

Body weight and food consumption

Overall body weight gains were similar in control and treated groups and food consumption was considered to be unaffected by treatment.

Hematology and blood biochemistry

No changes of toxicological significance were noted in any parameters.

Organ weights

No notable differences in organ weights were noted between control and treated groups.

Macroscopic and microscopic examinations

No changes of toxicological importance were observed.

Conclusion

The daily administration of the test substance, EDDHAS Fe 3K, at the dose-levels of 150, 450 and 1000 mg/kg/day, by oral route (gavage) to rats for 28 days did not produce any signs of toxicity. Consequently, under our experimental conditions, the No Observable Effect Level (NOEL) was established at 1000 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

There is no deficiencies in the available data set. The data on structurally related substances is reliable and consisitent.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: inhalation

Data waiving:

exposure considerations

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: inhalation

Data waiving:

exposure considerations

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

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

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

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

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

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).

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.

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.

Critical effects observed:

not specified

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The dermal study available meets required adequacy criteria and herewith demonstrating a good quality of data base

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

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

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

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

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

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).

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.

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.

Critical effects observed:

not specified

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

Study duration:

subacute

Species:

rat

Quality of whole database:

The dermal study available meets required adequacy criteria and herewith demonstrating a good quality of data base

Additional information

No repeated dose toxicity studies are available for the target substance Fe(3Na)EDDHSA. The data on other structurally related substances have been used to assess toxicity potential of Fe(3Na)EDDHSA at repeated exposures.

Oral route

Fe(3K)EDDHSA (EC 462- 490 -6) was tested in a 28-day oral gavage study in Sprague Dawley rats (Centre International de Toxicologie, 2001, OECD Guideline 407). Groups of five males and five females received the test substance daily at the dose-levels of 150, 450 or 1000 mg/kg/day for 28 days. An additional group of five males and five females received the vehicle alone (purified water) under the same experimental conditions, and acted as a control group.
The animals were checked daily for mortality and clinical signs. A functional observation battery was performed on all animals of each group, once in pre-dose and then in week 4; in addition, a detailed clinical observation was performed on all animals of each group, once a week until the end of the treatment period. Motor activity was recorded on all animals once in pre-dose and in week 4. Body weight and food consumption were recorded once a week. Haematological and blood biochemical investigations were performed for all animals at the end of the treatment period. On completion of the treatment period, all animals were killed and submitted to a complete macroscopic post-mortem examination. Designated organs were weighed and selected tissues were preserved. A microscopic examination was performed on designated tissues of the animals of the control and high dose-level group.

No mortalities and no clinical signs of toxicological significance were observed in any treated animals. No specific signs of a neurotoxic action of the test substance were noted. Overall body weight gains were similar in control and treated groups and food consumption was considered to be unaffected by treatment. Haematology and biochemistry parameters were without of toxicological significance. No notable differences in organ weights were noted between control and treated groups. Macroscopic and microscopic examinations revealed no changes of toxicological importance. The No Observable Effect Level (NOEL) was established at 1000 mg/kg bw.

The subchronic toxicity of Fe(Na)EDDHA (CAS 84539 -55 -9) by oral route was investigated in rats (Novartis Crop Protection AG, 1998). The test item was administered to 10 Sprague-Dawley derived rats/sex/dose level by oral gavage at 5, 50 or 200 mg/kg bw/day for 90 days. A concurrent control group was 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 impaired body weight development at 200 mg/kg bw/day. 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 addition, changes of blood chemistry and urine parameters concerning the liver and kidneys were noted. 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 Fe(Na)EDDHA when administered daily by oral gavage for three months was 10 mg/kg bw/day (estimated from the LOAEL).

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 from 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 appropriate 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.

In a dose range-finding subacute oral toxicity study (CIBA-GEIGY Limited, 1996a), Fe(Na)EDDHA was administered to 5 Sprague-Dawley derived rats/sex/dose level by oral gavage at 50, 200 or 1000 mg/kg bw/day for 28 days. A concurrent control group was treated with the vehicle only. Treatment with the test item resulted in impaired body weight development at 200 and 1000 mg/kg bw/day and correspondend lower food intake. Anaemia without erythropoietic response was noted at 200 and 1000 mg/kg bw/day. At the same dose levels, the kidney was revealed as target organ by microscopical examination, by blood chemistry data evaluation and by organ weight evaluation. In addition, body weight relative organ weight changes were noted in the heart, adrenals and spleen. However, the relevance of these findings was considered as equivocal. This study was used as scientific basis for dose level selection for the above-mentioned 90 -day repeated dose oral toxicity study in the rat.

It seems that the toxicity of Fe(Na)EDDHA is higher that that of Fe(3K)EDDHSA.

Dermal route

In a repeated dose dermal toxicity study (CIBA-GEIGY Limited, 1996b), Fe(Na)EDDHA was administered to the skin of 5 Sprague-Dawley derived rats/sex/dose level at 10, 100 or 1000 mg/kg bw/day for 28 days (5 days/week). A concurrent control group was treated with the vehicle only. Dermal treatment with the test item resulted in no mortality, no relevant clinical signs, and 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, the NOEL was established at 100 mg/kg bw/day.

Inhalation route

In accordance with column 2 of REACH Annex IX, the test repeated dose toxicity after inhalation (required in section 8.6) does not need to be conducted as repeated dose toxicity studies for oral and dermal application are available. Inhalation exposure is regarded negligible as the particle size distribution for particles below 100 µm were found to be 2.7 % and no particles were found less than 10 µm. In addition, the substance showed only very low toxicity after acute inhalation exposure with an LD50 of greater than 4200 mg/m³ in rats.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Key study conducted with the nearest analogue Fe(3K)EDDHSA (EC 462-490-6)

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
Inhalation is not relevant route of exposure

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
Inhalation is not relevant route of exposure

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
Key study conducted with the related substance Fe(Na)EDDHA (CAS 8453955-9)

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
Key study conducted with the related substance Fe(Na)EDDHA (CAS 8453955-9)

Justification for classification or non-classification

Based on the results of the key repeated dose toxicity study conducted with the nearest analogue Fe(3K)EDDHSA and considering the NOEL of 1000 mg/kg bw/day established for the oral route, Fe(3Na)EDDHSA is not subject to classification and labelling according to the Regulation No 1272/2008 (CLP) since no treatment-related effects were noted in any parameter tested.