Reaction mass of diiron carbide, diiron phosphide and triiron phosphide

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:

11 August 2011 - 08 March 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was performed according to OECD test guidelines, and in compliance with GLP, so the data is considered reliable without restriction.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of diiron carbide, diiron phosphide and triiron phosphide

EC Number:

701-438-1

Cas Number:

not applicable

Molecular formula:

not applicable (multi constituent substance)

IUPAC Name:

Reaction mass of diiron carbide, diiron phosphide and triiron phosphide

Details on test material:

- Name of test material (as cited in study report): Fe3P, Ferrophosphorous
- Physical state: Solid (fine powder)
- Analytical purity:Phosphorous 16.0%, Carbon 0.61%, Iron as base.
- Purity test date: 24 Jan 2011
- Lot/batch No.: 1192691
- Expiration date of the lot/batch: 31 December 2011
- Storage condition of test material: Ambient temperature

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

The rat was chosen as the test species because of its acceptance as a predictor of toxic and reproductive change in man and the requirement for a rodent species by regulatory agencies. The Crl:CD(SD) strain was used because of the historical control data available in this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Approx. 72 days
- Weight at study initiation: 348 - 399 g (males), 230 - 287 g (females)
- Housing: Polycarbonate cages with either stainless steel grid floors during mating, and solid poly carbonate floors during other phases of testing.
- Diet (e.g. ad libitum): ad libitum / free access, except overnight before routine blood sampling
- Water (e.g. ad libitum): ad libitum / free access
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23°C
- Humidity (%): 40 - 70%
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 26 October 2011 (Treament commenced) To: 12 December 2011 (Last date of necropsy for main phase females)

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral route of administration was chosen to simulate the conditions of potential human exposure.

Vehicle:

other: 1% (w/v) aqueous methylcellulose

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
DIET PREPARATION
- Rate of preparation of diet (frequency): Formulations were prepared weekly, up to seven days in advance of the first day of dosing and were stored refrigerated (2-8 °C).

VEHICLE
- Concentration in vehicle: 10 - 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulation samples were analysed by Atomic Absorption Spectrometry, to determine Iron in the samples.

Prior to the start of treatment, the analytical method was validated with respect to specificity, limit of detection, linearity of detector response over the calibration range, precision of measurement at the lowest and highest calibration standards, and the accuracy and precision of the method, by the determination of six procedural recoveries at 1 mg/mL and 100 mg/mL. A stability tiral was also performed; formulations were found to be stable and homogenous for up to 2 hours at ambient temperature with paddle stirring, and following 15 days' refrigerated storage.

Samples from all formulations prepared in the first and last weeks of the study were analysed; the test material concentrations were found to be within acceptable limits, confirming accurate preparation.

Duration of treatment / exposure:

Main phase males and toxicity phase females were dosed for five consecutive weeks.
Main phase females were treated daily for two weeks before pairing, throughout mating, gestation and until day 6 of lactation.

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Main phase - 10 animals per sex per dose
Toxicity phase - 5 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were decided in a 7-day preliminary study in rats, conducted at the same laboratory - refer to "7 Day rangefinding_Huntingdon Life Sciences, 2011 (FGE0026)".
- Post-exposure recovery period in satellite groups: Not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:Prior to the start of treatment, and at least weekly for males and toxicity phase females. Main phase females were observed weekly before pairing, and on days 0, 6, 13 and 20 after mating, and days 1 and 7 of lactation.

BODY WEIGHT: Yes
- Time schedule for examinations: Main phase males and toxicity phase females were weighed on day 0 of treatment, then weekly and beofre necropsy. Main phase females were weighed on day 0 of treatment and weekly until mating was detected, and days 0, 6, 13 and 20 after mating, and on days 1, 4 and 7 of lactation.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/week: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 5 of treatment for 5 of the main phase males and for the toxicity phase females.
- Anaesthetic used for blood collection: Yes - isoflurane
- Animals fasted: Yes
- How many animals: As above
- Parameters checked: Haematocrit (Hct), Haemoglobin concentration (Hb), Erythrocyte count (RBC), Reticulocyte count (Retic), Mean cell haemoglobin (MCH), Mean cell haemoglobin concentration (MCHC), Mean cell volume (MCV), Total leucocyte count (WBC), Differential Leucocyte count (Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC)), Platelet count (Plt).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: conducted at the same time and using the same animals as Haematology, above.
- Parameters checked: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Total bilirubin (Bili), Bile acids (BIAC), Urea, Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Triglycerides (Trig), Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus (Phos), Total protein (Total Prot), Albumin (Alb).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: Sensory reactivity and grip strength measured in the first five males for the main phase, and all five females of the toxicity phase during week 5 of treatment.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Organ weights recorded at necropsy (F0 animals; L&R – Bilateral organs weighed individually): Adrenals, Prostate, Brain, Seminal vesicles with coagulating gland, Epididymides (L&R), Spleen, Heart, Testes (L&R), Kidneys, Thymus, Liver, Uterus (including cervix and oviducts), Ovaries (L&R)
- Gross necropsy, fixation (F0 animals): Adrenals, Pituitary, Brain, Prostate, Caecum, Rectum, Colon, Sciatic nerves (only one per adult animal processed for examination), Duodenum, Seminal vesicles with coagulation glands, Epididymides, Skeletal muscle (only one per adult animal processed for examination), Eyes, Spinal cord, Heart, Spleen, Ileum, Sternum with marrow, Jejunum, Stomach, Kidneys, Testes, Liver, Thymus, Lungs, Thyroid with parathyroids, Lymph nodes (axillary, mesenteric), Trachea, Urinary bladder, Oesophagus, Uterus (including cervix and oviducts), Ovaries, Vagina, Peyer’s patch

HISTOPATHOLOGY: Yes
- Tissues: Adrenal (cortex and medulla), Brain (cerebellum, cerebrum and pons), Heart (included auricular and ventricular regions), Kidneys (included cortex, medulla and papilla regions), Liver (section from two main lobes), Lungs (section from two major lobes, to include bronchi), Ovaries (qualitative evaluation of one section from each ovary), Seminal vesicles (included coagulating glands in section), Spinal cord (transverse and longitudinal section at the cervical, lumbar and thoracic levels), Sternum (included bone marrow), Stomach (included keratinised, glandular and antrum in sections), Thyroid (included parathyroids in section where possible), Uterus (uterine body with cervix section and oviducts)

Statistics:

The following sequence of statistical tests was used for grip strength, motor activity, body weight, food consumption, organ weight, litter size, survival indices and clinical pathology data:

A parametric analysis was performed if Bartlett's test for variance homogeneity was not significant at the 1% level. The F1 approximate test was applied. This test is designed to detect significant departure from monotonicity of means when the main test for the comparison of the means is a parametric monotonic trend test, such as Williams’ test. If the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test was performed instead.

A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. The H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied. This test is designed to be used when the main test for comparison of the means is a non-parametric monotonic trend test, such as Shirley's test. If the H1 approximate test for monotonicity of dose-response was not significant at the 1% level, Shirley's test for a monotonic trend was applied. If the H1 approximate test was significant, suggesting that the dose-response was not monotone, Steel's test was performed instead. For grip strength, motor activity survival indices and clinical pathology, if 75% of the data (across all groups) were the same value, for example c, Fisher’s Exact tests were performed. Treatment groups were compared using pairwise comparisons of each dose group against the control both for i) values c, as applicable.

For organ weight data, analysis of covariance was performed using terminal bodyweight as covariate.

Results and discussion

Results of examinations

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

Endocrine findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

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:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
During the study there were no signs that were considered to be related to the administration of Fe3P and no unscheduled deaths.

BODY WEIGHT AND WEIGHT GAIN
Bodyweight gain for males during the five weeks of treatment was unaffected by the administration of Fe3P.
Bodyweight gain for females during the first two weeks of treatment was considered to be unaffected by the administration of Fe3P. Toxicity phase females had lower bodyweight gain between weeks three and five of treatment (max 47% of Control) along with a lower overall bodyweight gain over the five weeks of treatment (max 60% of Control). However, there was no dose response and a relationship to treatment is uncertain.
Bodyweight gain for gestating females was considered the be unaffected by the administration of Fe3P, however lactating females given Fe3P at 300 and 1000 mg/kg/day had marginally lower bodyweight gain between days one and seven of lactation (max 82% of Control)

FOOD CONSUMPTION
Food consumption for males was considered to be unaffected by the administration of Fe3P.
During the first two weeks of treatment female food consumption was considered to be unaffected by the administration of Fe3P. Toxicity phase females showed some intergroup variation in food consumption during week 3 of treatment. During Weeks four and five food consumption was marginally lower in those animals given Fe3P (94% and 90% of Controls respectively). However, there was no dose response and a relationship to treatment is uncertain.
Food consumption for gestating and lactating females, with the exception of females given 1000 mg/kg/day, was considered to be unaffected by the administration of Fe3P. Females given 1000 mg/kg/day had marginally lower food consumption over the seven days of lactation when compared to Controls.

HAEMATOLOGY
Haematological parameters in both males and females in Week 5 of treatment were considered to be unaffected by the administration of Fe3P. Males given 300 or 1000 mg/kg/day had slightly low haematocrit and haemoglobin concentrations which attained statistical significance when compared to control, but there were no similar effects in females.

CLINICAL CHEMISTRY
Blood chemistry parameters in males and females in Week 5 of treatment were considered to be unaffected by the administration of Fe3P.

ORGAN WEIGHTS
There was no effect of treatment on organ weights for males receiving Fe3P.
Intergroup variability in uterine weight did not appear to follow any dose related pattern and may relate to differences in the stage of the oestrous cycle at necropsy.

GROSS PATHOLOGY
The macroscopic examination performed after 5 weeks of treatment revealed no test substance related lesions.
The nature and incidence of all findings were consistent with the commonly seen background of macroscopic changes.

HISTOPATHOLOGY
Microscopic examination was performed on the first five Main phase males killed at scheduled termination and Toxicity phase females in the Control and high dose group (1000 mg/kg/day).
In all the tissues examined, there was no evidence of changes considered related to the treatment with the test compound.
The seminiferous tubules of the testes were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage abnormalities were noted.

Effect levels

Dose descriptor:

NOAEL

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Oral administration of Fe3P to Sprague-Dawley rats for at least 5 weeks at doses of 100, 300 and 1000 mg/kg/day was generally well tolerated with no toxicologically significant systemic effects.

Based on the results from this study, for general toxicity the no-observed-adverse-effect level (NOAEL) was considered to be 1000 mg/kg/day.