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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
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)
Version / remarks:
1996-03-22
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
Reaction mass of diiron carbide, diiron phosphide and triiron phosphide
EC Number:
701-438-1
Molecular formula:
not applicable (multi constituent substance)
IUPAC Name:
Reaction mass of diiron carbide, diiron phosphide and triiron phosphide
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): Fe3P, Ferrophosphorous
- Physical state: fine powder
- 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
Vehicle:
other: 1% (w/v) aqueous methylcellulose
Details on 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
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Up to two weeks, or until evidence of mating was found
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of gestation
- After successful mating each pregnant female was caged (how): Individually
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
Details on study schedule:
- Age at mating of the mated animals in the study: Approx. 12 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Main Phase - 10 males and 10 females 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)".

Examinations

Parental animals: Observations and examinations:
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.

Oestrous cyclicity (parental animals):
For 15 days before pairing, daily vaginal smears (dry) were taken from all Main phase females, using cotton swabs moistened with saline. The smears were subsequently examined to establish the duration and regularity of the oestrous cycle. After pairing with the Main phase male, smearing was continued using pipette lavage, until evidence of mating was observed.
Sperm parameters (parental animals):
Parameters examined in male parental generations: testis weight, epididymis weight.
Seminal vesicles were subject to histopathological examination. 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.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Clinical signs, litter size, sex ratio, and bodyweight

GROSS EXAMINATION OF DEAD PUPS:
For offspring surviving to scheduled termination, a careful external examination for gross abnormalities was performed. Offspring that appeared normal externally were discarded without internal examination. Offspring which were externally abnormal were subjected to a full internal microscopic examination. Missing offspring and those grossly autolysed or cannibalised could not be examined; all other offspring which died before day 7 of age were examined as detailed. The necropsy also included an assessment for the presence of milk in the stomach, where possible.
Postmortem examinations (parental animals):
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)
Postmortem examinations (offspring):
Microscopic examination, as described in "Litter Observations", above.
Statistics:
please refer to "Any other information on material and methods incl. tables", below.
Reproductive indices:
- Percentage mating: Number animals mating / Animals paired x 100
- Conception rate (%): Number animals achieving pregnancy / Animals mated x 100
- Fertility index (%): Number animals achieving pregnancy / Animals paired x 100
- Gestation index (%): Number of live litters born / Number pregnant x 100
- Post - implantation survival index (%): Total number offspring born / Total number uterine implantation sites x 100
Offspring viability indices:
- Live birth index (%): Number live offspring on Day 1 after littering / Total number of offspring born x 100
- Viability index (%): Number live offspring on Day 4 after littering / Number live offspring on Day 1 after littering x 100
- Lactation index (%): Number live offspring on Day 7 after littering / Number live offspring on Day 1 after littering x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
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
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

Refer to detailed analysis of toxicological parameters in section 7.5.1.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Assessment of oestrous cycles during the two week pre-pairing period showed that nearly all of the main phase females had regular 4 or 4/5 day oestrous cycles and it was considered that this parameter was not affected by treatment with Fe3P.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
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.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
The pre-coital interval was unaffected by treatment, with animals mating at the earliest opportunity, when the female came into oestrus.
Mating performance and fertility as assessed by percentage mating, conception rate and fertility index, were unaffected by treatment.
The gestation length was within the normal range of 22 to 23 days for all animals and the gestation index was unaffected by treatment.

Effect levels (P0)

Dose descriptor:
NOAEL
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
The mean number of implantations and the live litter size on Days 1, 4 and 7 were similar to control values and unaffected by treatment.
Offspring survival up to Day 7 of age was unaffected by parental treatment.

CLINICAL SIGNS (OFFSPRING)
Refer to table in "Any other information on results".

BODY WEIGHT (OFFSPRING)
Mean bodyweights of male and female offspring of all groups of parents treated with Fe3P were marginally lower than in Controls on Day 1 of age but differences did not attain statistical significance and there was considered to be no effect of treatment on mean bodyweight gains of the offspring between Days 1 and 4 of age. However, between Day 4 and Day 7 of age, mean bodyweight gains of male and female offspring of all groups of parents treated with Fe3P were lower than in Controls and the differences attained statistical significance in the 1000 or 300 mg/kg/day groups (77 - 80% of Controls) and they showed a dose response. Overall bodyweight gain, between Days 1-7, was statistically lower in all male offspring of all groups of parents treated with Fe3P.

GROSS PATHOLOGY (OFFSPRING)
Macroscopic examination of offspring dying before scheduled termination or killed at scheduled termination on Day 7 of age did not reveal any findings that were attributed to parental treatment.

OTHER FINDINGS (OFFSPRING)
Sex ratio, as expressed in terms of % males, was not affected by parental treatment.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

Summary of offspring clinical observations

Observation

Group (Dose mg/kg/day)

1 (0)

2 (100)

3 (300)

4 (1000)

Number of offspring (litters) affected

Head: bruising and swelling

1(1)

 

 

 

Head: bruising

 

1(1)

1(1)

1(1)

Umbilical cord attached

1(1)

 

 

 

Missing

2(2)

 

1(1)

2(2)

Tail absent

1(1)

 

 

 

Dorsal surface: Scab and reddening

 

1(1)

 

 

Found dead

 

1(1)

 

 

Forepaw: Scab

 

1(1)

 

 

Colour: Pale

 

 

1(1)

 

Applicant's summary and conclusion

Conclusions:
The No Observed Adverse Effect Level (NOAEL) for Fe3P for reproductive and developmental effects was considered to be 1000 mg/kg/day.