Iron tris(2-ethylhexanoate)

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From December 13, 2018 to February 21 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

July 29, 2016

Deviations:

yes

Remarks:

Female in the weight range of 190-204 g and not 175-200 g, while males were in the range of 220-234 g, instead of 200-225 g. This deviation was not considered to have compromised the purpose or conduct of the study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Iron tris(2-ethylhexanoate)

IUPAC Name:

Iron tris(2-ethylhexanoate)

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Germany, Sulzfeld, Germany
- Weight at study initiation: 220-234 g for males, 190-204 g for females
- Housing: from arrival to mating, animals were housed up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5×38×20cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Nesting material was provided inside suitable bedding bags and changed at least twice a week.
During mating, animals were housed one male to one female in clear polysulfone cages
measuring 42.5×26.6×18.5cm with a stainless steel mesh lid and floor (Tecniplast Gazzada
S.a.r.l., Buguggiate, Varese). Each cage tray held absorbent material which was inspected
and changed daily.
After mating, the males were re-caged as they were before mating. The females were
transferred to individual solid bottomed cages for the gestation period, birth and lactation
(measuring 42.5×26.6×18.5 cm). Nesting material was provided inside suitable bedding
bags.
- Diet (e.g. ad libitum): laboratory rodent diet (4 RF 21,Mucedola S.r.l., Via G. Galilei, 4,
20019 SettimoMilanese (MI), Italy) ad libitum.
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks before the start of treatment

DETAILS OF FOOD AND WATER QUALITY: Records of analyses of water and diet are kept on file at RTC.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C ± 2 °C
- Humidity (%): 55 % ± 15 %
- Air changes (per hr): approximately 15 to 20 air changes per hour
- Photoperiod (hrs dark / hrs light): artificial light for 12 hours each day

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS: The required amount of the test item was suspended in the vehicle.
The preparations were made daily (concentrations of 15.0, 30 and 60 mg/mL). Concentrations were calculated and expressed in terms of test item as supplied.
The test item was weighed directly into a clear glass container by using disposable smartSpatula ® (Sigma-Aldrich Code number: Z740505). The container was subsequently filled to approximately 75% of vehicle and put into a water bath (set at 50°C). Every approximately ten minutes, the flasks were shaken manually until homogeneous distribution of the test item was visually observed. The preparations were then brought to room temperature (approximately 25°C) filled with the remaining amount of vehicle and shaken by hand again.

- VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item is insoluble in water. The vehicle was cor oil as in it the test item is stable and forms an homogeneous solution.
- Concentration in vehicle: 15.0, 30 and 60 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before treatment, analysis was performed in a separate study (IES Study No. 20180064) in order to validate the analytical method and the preparation procedure in the test item concentration range of 0.8 to 250 mg/mL and to verify the stability of the preparation at 28 hours at room temperature (20 ± 5 °C) and ambient light conditions and for 8 days at room temperature (20 ± 5 °C) in the dark at the concentrations of 0.8 and 250 mg/mL.
Samples of the preparations made during the current study (Week 1 and Week 4) were analysed to check the homogeneity and concentration. Analysis of test item preparations were carried out at the Test Site under the Delegated phase (IES Study No. 20180065). Samples were packed and transferred for analysis at room temperature (20 ± 5 °C) by a dedicated courier to the Principal Investigator of the Test Site.
Six replicates of approximately 10 mL were drawn from Groups 2/6 and 4/8 preparation (2 from the top, 2 from the middle and 2 from the bottom of the preparation), while 2 replicates approximately 10 mL were drawn from Groups 1/5 and 3/7 preparations. Therefore, for each sampling date a total of 16 specimens were prepared for shipment.
For each specimen an additional back up sample of approximately 10 mL was retained at the test facility. Samples were labelled as follows: study number, sampling date and nominal concentration.
The 16 specimens were transported by a dedicated courier. Prior to shipment, the Principal Investigator was notified by e-mail. Back up samples were maintained at -20 ± 5°C until positive results of the analysis were available and approved.
The recoveries for test samples from treatment Week 1 were found to be within the range of 86 % to 113 %. The % nominals determined for test samples from treatment Week 4 were within the range of 91 % to 104 %. This confirms the correct preparation of dose formulation samples at the test facility and, since both sample sets included samples drawn from top, middle and bottom of dose group 2/6 (15 g Iron Tris(2 Ethylhexanoate)/L and 1.71 g Fe/L, respectively) and dose group 4/8 (60 g Iron Tris(2 Ethylhexanoate)/L and 6.84 g Fe/L, respectively) preparations, further confirms the homogeneous distribution of the test
item in the selected corn oil vehicle.
All control samples (dose group 1/5) were found to be free of residues of Iron Tris(2 Ethylhexanoate) and iron, respectively. Thus, the concentrations in these samples were below the limit of detection (LOD) of 14.5 µg Iron Tris(2-Ethylhexanoate)/L and 1.65 µg Fe/L.

Duration of treatment / exposure:

Males
Animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing, during pairing with females until the day before necropsy, for a total of 32/33 days. Dose volumes were adjusted once per week for each animal according to the last recorded body weight.
Females
Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 13 post partum, for a total of 42 to 63 days.
Dose volumes were adjusted once per week for each animal according to the last recorded body weight up to mating. During the gestation and lactation periods, dose volumes were calculated according to the last recorded body weight.
Recovery groups (Groups 5 to 8)
Male animals were dosed once a day, 7 days a week, for a total of 28 days. Female animals were dosed once a day, 7 days a week, for a total of 63 days. Dose volumes were adjusted once per week for each animal according to the last recorded body weight.
No treatment was given during the recovery period.

Frequency of treatment:

Each day for the periods described above (duration of treatment/exposure)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

75 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

150 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Main groups
10 males + 10 females for the control and for each tested dose

Recovery groups
5 males + 5 females for the control and for each tested dose

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: Based on the dose range finding test, where signs of toxicity were seen at 320 mg/kg bw, the highest dosage selected for the final test was 300 mg/kg bw.
- Rationale for selecting satellite groups: As toxicity was expected at 300 mg/kg bw, based on the range finding test, a recovery group was considered necessary in oreder to understand which sign should be considered as adverse.
- Post-exposure recovery period in satellite groups: 4 weeks

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Once before commencement of treatment and at least once daily during the study

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Once before commencement of treatment and at least once a week thereafter

BODY WEIGHT: Yes
- Time schedule for examinations
Main groups
Males were weighed weekly from allocation to termination. Females were weighed weekly from allocation to positive identification of mating and on Days 0, 7, 14 and 20 post coitum.
Dams were also weighed on Days 1, 4, 7, 13 post partum and just before necropsy.
Recovery groups
Each animal was weighed on the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter and just prior to necropsy.

FOOD Consumption
- Time schedule for examinations: the weight of food consumed by each cage of males and females were recorded weekly during the pre-mating period starting from Day 1 of dosing. Individual food consumption for the females were measured on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Day 7 and 13 post partum starting from Day 1 post partum.
Recovery groups
The weight of food consumed by each cage of rats were recorded at weekly intervals following allocation.


HAEMATOLOGY: Yes
- Time schedule for collection of blood:
As a part of the necropsy procedure, approximately 0.8mL of blood was taken from all parental males and females.
- Parameters checked in the "Any other information on materials...." field of the IUCLID dossier were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
see HAEMATOLOGY
- Parameters checked in the "Any other information on materials...." field of the IUCLID dossier were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
see DETAILD CLINICAL OBSERVATIONS
- Battery of functions tested: sensory activity / grip strength / motor activity.

IMMUNOLOGY: No

OTHER:
Thyroid hormone determination

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, see "Any other information on materials...."

HISTOPATHOLOGY: Yes, see "Any other information on materials...."

Statistics:

Standard deviations were calculated as appropriate. For variables such as body weight, food consumption, clinical pathology parameters and organ weight, the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test if n was more than 5.
The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters.
Intergroup differences between the control and treated groups were assessed by the nonparametric version of the Williams test. The criterion for statistical significance was p<0.05.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation was observed in main and recovery phase females dosed at 300mg/kg/day during treatment period.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Very slight reductions in body weight, only occasionally significant at statistical analysis, were observed in the high dose females of the recovery group (Group 8, dosed at 300 mg/kg/day) in the last 3 weeks of treatment period. Reductions gradually recovered during the recovery period.

Food efficiency:

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Slight decrease of erythrocytes was recorded in males dosed at 150 and 300 mg/kg/day. Due to the minimal severity, this change was considered to be not adverse.

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Subcutaneous masses were observed in one female dosed at 75mg/kg/day and in 2 females dosed at 300mg/kg/day. Two of these were recognised at histopathological examination as inflammation or adenocarcinoma of the mammary gland, respectively, while the third one
was unremarkable.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Subcutaneous masses were observed in one female dosed at 75mg/kg/day and in 2 females dosed at 300mg/kg/day. Two of these were recognised at histopathological examination as inflammation or adenocarcinoma of the mammary gland, respectively, while the third one was unremarkable.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid hormone determination
An increase of thyroid stimulating hormone was recorded in one animal receiving 75 mg/kg/day (no. X0990034), with no related changes of the other thyroid hormones. Due to the absence of dose-relation, this finding was considered to be incidental.

Effect levels

Dose descriptor:

NOAEL

Effect level:

>= 300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no toxicity recorded

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

1. Clinical signs

Main and Recovery groups

No treatment-related clinical signs were observed in the male from the main phase and in those from the recovery phase, during treatment and recovery periods.

Salivation was observed in 8/10 main phase females dosed at 300mg/kg/day during mating phase and post partum phases and in 4/5 recovery females, dosed at the same dose level, during treatment period. In addition, pioloerection, pallor and vaginal discharge were seen in a single female from the low dose group (no. X0990039, dosed at 75mg/kg/day) from Day 1 to Day 4 post partum.

No clinical signs were observed during recovery period.

A subcutaneous mass was observed in one female dosed at 75mg/kg/day (no. X0990045) in the right ventral area of the neck (data not tabulated). This was recognised at histopathological examination as acute, multifocal inflammation of the mammary gland.

A second subcutaneous mass was observed in one female dosed at 300mg/kg/day (no. X0990063) in the left dorsal area (data not tabulated). This was recognised at histopathological examination as an adenocarcinoma of the mammary gland.

A third subcutaneous mass was observed in a second female dosed at 300mg/kg/day (no. X0990079) in the right ventral area of the neck (data not tabulated). This was recognised at histopathological examination as unremarkable.

2. Clinical observations (Functional Observation Battery Tests)

Main and Recovery groups

Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item, compared to controls.

3.Motor activity, grip strength and sensory reactivity to stimuli

Main and Recovery groups

A slight statistically significant increase in motor activity was observed in the females of the mid- and high dose recovery groups (dosed at 150 and 300mg/kg/day) when compared to controls. No other changes in motor activity were observed during treatment of recovery periods in either males of females. Due to the episodical nature of this change and to the low magnitude of the measured values (below the mean historical control value), this increase was condidered to be of no toxicological significance.

No changes of neurotoxicological significance were observed in grip strength, landing foot splay or other sensory reactivity parameters evaluated.

4. Body weight

Main and Recovery groups

Very slight reductions in body weight (not greater than 10%), only occasionally significant at statistical analysis, were observed in the high dose females of the recovery group (Group 8, dosed at 300mg/kg/day) in the last 3 weeks of treatment period. Reductions gradually recovered during the recovery period.

No significant changes in body weight and body weight gain were observed in the treated male animals and in the main group females, when compared to controls during treatment or recovery periods.

5. Food consumption

Main and Recovery groups

No changes of toxicological significance were observed in food consumption during the study in the males and in the females from the main phase groups.

Slight reductions in food consumption (from 7% to 20%) were seen in the recovery low and high dose females (Groups 6 and 8, dosed at 75 and 300mg/kg/day) during the treatment period. These reductions were no longer present during the recovery period. Due to the absence of dose relation and to the low magnitude, this change was considered of no toxicological relevance and, therefore, not adverse.

6. Haematology

Dosing phase (main phase animals)

Slight decrease of erythrocytes was recorded in males dosed at 150 and 300mg/kg/day (7% and 6% below controls, respectively). Due to the minimal severity, this change was considered to be not adverse.

In addition, male no. X0990068 (300mg/kg/day) showed moderate neutrophilia. Compared with mean control data, the increment was 4.1 fold. Due to the minimal incidence, this finding was considered to be unrelated to treatment. The statistically significant difference of reticulocytes recorded between controls and females dosed at 150mg/kg/day (39% above controls) was not dose-related, therefore it was considered to be incidental.

Recovery phase (recovery phase animals)

No changes were recorded.

Coagulation

Dosing phase (main phase animals)

No changes were recorded.

Recovery phase (recovery phase animals)

The statistically significant difference of prothrombin time recorded between controls and females dosed at 75mg/kg/day (4% above controls) was not dose-related, therefore it was considered to be incidental.

7. Clinical chemistry

Dosing phase (main phase animals)

One male dosed at 150mg/kg/day (no. X0990050) showed an increase of aspartate aminotransferase (2.8 fold compared with controls). Due to the absence of dose-relation, this change was considered to be unrelated to treatment.

Recovery phase (recovery phase animals)

The statistically significant differences recorded between control and treated animals (protein, globulin, chloride and potassium in males, chloride and bile acids in females) were not observed during the Dosing Phase and/or not dose-related, therefore they were considered to be unrelated to treatment.

8. Thyroid hormone determination

Parental males

No changes were recorded. An increase of thyroid stimulating hormone was recorded in one animal receiving 75mg/kg/day (no. X0990034), with no related changes of the other thyroid hormones. Due to the absence of dose-relation, this finding was considered to be incidental.

9. Terminal body weight and organ weights

Final and recovery sacrifice

No relevant changes were observed on terminal body, absolute and relative organ weights of treated animals that completed the treatment or recovery period, when compared to controls.

10. Macroscopic observations

Final and recovery sacrifice

No remarkable changes were noted at post mortem examination in treated animals at the end of treatment and recovery period, when compared with controls.

The sporadic changes such as adhesions to lungs and sternum in one mid-dose male (no.X099048), subcutaneous mass observed in one mid- and high dose female (no. X0990045 and X0990063, respectively), pelvic dilatation of kidneys or dark and/or red areas in the stomach of control and/or treated animals of both sexes of treatment and/or recovery period, were considered spontaneous and incidental, having a comparable incidence in control and treated groups and/or are characteristically seen in untreated Sprague Dawley SD rats of the same age.

11. Microscopic observations

No treatment-related changes were noted in animals sacrificed at the end of the treatment period.

The sporadic lesions such as mucosal erosion of non glandular region of the stomach (sometimes associated in treated females with pigmentation - test item like) or pelvic dilatation of kidneys reported in control and/or treated animals of both sexes, chronic inflammation of prostate in one high dose male (no. X0990068) or M-Adenocarcinoma in the mammary glands of one high dose female (no. X0990063) correlated to the subcutaneous mass observed during necropsy were considered to be an expression of spontaneous and/or incidental pathology, commonly seen in this species and age. In particular, acute inflammation of lungs associated with necrosis, fibrosis and presence of foreign material (test item like), correlated to adhesions to lungs, sternum and thymus.

Applicant's summary and conclusion

Conclusions:

NOAEL for REPEATED DOSE TOXICITY >= 300 mg/kg bw

Executive summary:

Method

The toxic effects on Sprague Dawley rats after repeated dosing by oral route with the test item, as well as any effects of the test item on male and female reproductive performance, such as gonadal function, mating behaviour, conception, parturition

and early lactation of the offspring were investigated. The vehicle was corn oil. All doses (0, 75, 150 and 300 mg/kg/day) were administered at a constant volume of 5mL/kg body weight.

Males of the main groups were treated for 14 days prior to pairing and during pairing with females until the day before necropsy, for a total of 32/33 days. Males of the recovery group were treated for a total of 28 days. Females of the main groups were treated for 14 days prior to pairing, during pairing and throughout the gestation and lactation periods until Day 13 post partum, for a total of 42 to 63 days. Females of the recovery groups were treated for a total of 63 days.

The following investigations were performed in all groups (main and recovery): body weight, clinical signs (including neurotoxicity assessment, motor activity and sensory reactivity to stimuli), food consumption, clinical pathology investigations (haematology and clinical chemistry in five animals/sex/main group randomly selected and in all recovery

animals), post mortem macroscopic observations, organ weights. In addition, for main phase groups only (parental animals), oestrous cycle evaluation for parental females (1 week before dosing, during pre-mating and mating phases, prior to necropsy), mating performance, thyroid hormone determination (parental main phase males) and litter data were performed. Clinical signs, anogenital distance, external and internal examination and thyroid weight at necropsy were recorded for pups. Thyroid hormone levels were also determined in 1 pup/sex/group randomly selected at Day 14 post partum.

Routine histopathological examination was performed only in main control and high dose groups (five animals/sex/group randomly selected). It included identification of the stages of the spermatogenic cycle in five males.

Observations

Mortality and Clinical signs: no treatment-related clinical signs were observed in the males from the main phase and in

those from the recovery phase, during treatment and recovery periods.

Neurotoxicity assessment: no treatment-related alterations in motor activity, grip strength, landing footsplay and sensory reactivity to stimuli were observed in any treatment group at the examination performed at the end of treatment.

Body weight and body weight gain: very slight reductions in body weight were observed in the high dose females of the recovery group. Reductions gradually recovered during the recovery period.

No significant changes in body weight and body weight gain were observed in the treated male animals and in the main group females.

Food consumption: no changes of toxicological relevance, which could be considered adverse, were observed in food consumption during the study.

Haematology: slight decrease of erythrocytes was recorded in males dosed at 150 and 300mg/kg/day. Due to the minimal severity, this change was considered to be not adverse. No changes were recorded on the recovery phase.

Coagulation: no changes of toxicological relevance were observed.

Terminal body weight and organ weights: terminal body weight unaffected by treatment in both sexes. Absolute and relative organ weights were comparable in all groups.

Macroscopic observations: no remarkable differences were noted at post mortem examination in treated animals sacrificed at the end of treatment and recovery period, when compared with controls.

Microscopic observations: no treatment-related changes were noted in animals sacrificed at the end of the treatment period.

Conclusion

No adverse effects indicating systemic toxicity were observed in male or female animals from main and recovery groups at any of the dose levels investigated (75, 150 and 300 mg/kg/day).

Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for general toxicity was considered to be 300 mg/kg/day for males and females.