potassium ferrite

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study methodology followed was basically according to OECD TG 415 and in accordance with the Principles of GLP. However, some deviations from the guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

yes

Remarks:

on a few occations, data recording was carried out a day late. On a number of occations, the room was not signed off and mortalities were not checked. On a number of occations there were omissions in data recording.

GLP compliance:

yes (incl. QA statement)

Remarks:

Safepharm Laboratories Limited Shardlow Business Park Shardlow Derbyshire UK

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

430-010-4

EC Name:

-

Cas Number:

12160-44-0

Molecular formula:

K2Fe22O34

IUPAC Name:

hydrate iron potassium hydride

Details on test material:

- Name of test material (as cited in study report): Potassium ferrite
- Substance type: Red/brown solid
- Lot/batch No.: LR 23388-17
- Storage condition of test material: Ambient conditions over silica gel

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 (UK) Limited, Margate, Kent
- Age at study initiation: approximately 6 weeks (male); 10 weeks (female)
- Weight at study initiation: Males: 181-253 g; Females: 211-297 g
- Housing: Initially, all animals were housed in groups of four in polypropylene cages with stainless steel grid floors and tops, suspended over polypropylene trays lined with absorbent paper. During the mating phase, animals were transferred to similar cages on a one male: one female basis. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation, in solid-floor polypropylene cages with stainless steel Iids furnished with softwood flakes (Datesand Ltd., Cheshire, UK). On isolated occasions, males were housed singly due to poor health.
- Diet: pelleted diet (Rodent PMI 5002, BCM IPS Limited, London, UK), ad libitum.
- Water: mains drinking water, ad libitum
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Formulations were prepared daily and dosed soon after due to instability of the test material in the vehicle. Males and females were treated at the appropriate dose level by oral gavage using 5 mL of vehicle (cornoil).

Details on mating procedure:

On Day 75, all animals were paired on a 1 male: 1 female basis within each dose group, for a maximum of twenty one days. Following evidence of mating, the males were returned to their original cages and females were transferred to individual cages. Pregnant females were allowed to give birth and maintain their offspring until Day 21 post partum.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Due to the complex nature of the test material and its limited solubility in organic and aqueous media, a substance specific quantitative method of analysis could not be developed. The active ingredient concentration of Potassium Ferrite in the test material formulations was determined using a gravimetric technique. The test material formulations were weighed into tared glass sintered crucibles and then rinsed with hexane to leave a test material residue. The samples were then dried in an oven at approximately 105°C before allowing to cool over silica gel in a desiccator and re-weighed. Homogencity Determinations: The test material formulations were mixed thoroughly and samples were taken from the top, middle and bottom of the container, shaking between sampling. Sampling was performed in triplicate.
- Stability Determinations: The test material formulations were sampled and analysed initially and then after storage at approximately +4°C in the dark for fourteen days. Verification of Test Material Formulation Concentrations: The test material formulations were sampled and analysed within three days of preparation.
- Test material formulation concentrations over the dosing period: Nominal concentration (mg/mL): 3, 30 and 100. Mean concentration found (mg/mL): 3.37, 31.8 and 112. Expressed as % of nominal: 112, 106 and 112.

Duration of treatment / exposure:

Males: up to 126 days; females: up to 77 days

Frequency of treatment:

daily

Details on study schedule:

Males and females were treated at the appropriate dose level for ten weeks and two weeks, respectively prior to pairing. On Day 75, all animals were paired on a 1 male: 1 female basis within each dose group, for a maximum of twenty one days. Following evidence of mating, the males were returned to their original cages and females were transferred to individual cages. Pregnant females were allowed to give birth and maintain their offspring until Day 21 post partum. Evaluation of each litter size, litter weight, clinical observations and landmark developmental signs were performed during this period. At Day 21 post partum, all females and offspring were killed and examined macroscopically. Subject to confirmation of successful mating, males were killed and examined macroscopically.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 15, 150 and 500 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

28

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

- Clinical Observations: All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before, immediately after and approximately one hour after dosing. All observations were recorded.
- Body weight: Individual bodyweights were recorded on Day 0 (the day before the start of treatment) and then weekly for males until termination. Females were weighed weekly until mating was evident. Bodyweights were then recorded on Day 1, 4, 7, 14 and 21 for both post coitum, and post partum periods.
- Food consumption: During the maturation period, food consumption was recorded for each cage of adults weekly. For females showing evidence of mating, food consumption was recorded for the periods covering Days 1-7, 7-14 and 14-21. For females with live litters, food consumption was recorded for the period covering Days 1 to 7 and 7 to 14 post partum.
- Water consumption: Water intake was observed daily by visual inspection of water bottles for any overt changes.
- Mating: Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to twenty one days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating and the males were subsequently returned to their original holding cages. Mated females were housed individually during the period of gestation and lactation. Where positive evidence of mating was not observed but pregnancy was later confirmed, the date of mating is assumed by the presence of a copulation plug on the cage trayliner.
-Pregnancy and Parturition: Each pregnant female was observed at approximately 08:30, 12:30 and 16:30h, at around the period of expected parturition. Observations were carried out at approximately 08:30h and 12:30h at weekends.

Litter observations:

Litter Observations: On completion of parturition, the number of live and dead offspring was recorded. The date and time of Day 1 post partum litter observations were standardised. For each litter the following was recorded; Number of offspring born. Number of offspring alive recorded daily and reported on Day 1, 4, 7, 14 and 21 post partum.The sex of offspring were recorded on Day 1 and 21 post partum. The sex of offspring were also recorded following interim deaths. Clinical condition of offspring from birth to weaning. Individual offspring and litter weights on Day 1, 4,7, 14 and 21 post partum. Necropsy findings of dead offspring. Physical development of offspring as assessed by intra-litter onset and duration of pinna unfolding, incisor eruption and eyelid separation. Surface righting reflex on Day 1, post partum and air righting reflex on Day 17 post partum. Pupillary reflex and auditory startle response on Day 21 post partum.

Postmortem examinations (parental animals):

-Pathology: Adult females were killed by carbon dioxide asphyxiation, followed by cervical dislocation on Day 21 post partum. Following the termination of all adult females and offspring, all males were killed by carbon dioxide asphyxiation, followed by cervical dislocation. All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. In addition, the corpora lutea of ovaries from pregnant females were counted at necropsy. The uterine implantation sites were counted.
-Organ Weights: The following organs, removed from adult animals that were killed at the end of the study, were dissected free from fat and weighed before fixation: Ovaries, Uterus (with cervix), Testes, Epididymides, Seminal vesicles (with coagulating gland), Prostate and Pituitary gland.
- Histopathology: Samples of the following tissues were removed from all adult animals and preserved in buffered 10% formalin with the exception of the testes with epididymides which were preserved in Bouins fluid and replaced by 70% industrial methylated spirits after 48 hours. Ovaries, Uterus, Cervix, Vagina, Testes, Epididymides, Seminal vesicles, Prostate, Coagulating gland, Pituitary gland and Lesion (target organs). All tissues were despatched to Propath UK Ltd, Rotherwas, Hereford, UK (Principal Investigator: N Candy). All tissues were embedded in paraffin wax BP (mp 56°C), sectioned at nominal thickness of 5µm and stained with haematoxylin and eosin for subsequent microscopic examination. Microscopic examination was conducted by the Study Pathologist. All findings were entered into the ROELEE Pathology computerisation system for tabulation and report production.

Postmortem examinations (offspring):

- Sacrifice: The F1 offspring were sacrificed at day 21 post partum.
- Pathology: These animals were subjected to postmortem examinations (macroscopic examination).

Statistics:

Data were processed to give group mean values and standard deviations where appropriate. Organ weight (absolute and relative to terminal bodyweight), weekly bodyweight gain, litter weights and offspring bodyweights data were assessed for dose response relationships using the following. Levene test for homogeneity of variance followed by either one way analysis of variance and pairwise comparison using a choice of versions of multiple comparison tests; or Kruskal-Wallis non parametric one way analysis of variance and Mann Whitney 'U' test/Wilcoxon signed ranktest. Reproductive and viability indices by Fisher's exact or chi-squared probability test, where applicable.

Reproductive indices:

The following parameters were calculated from the individual data during the mating period of the parental generation:
- Pre-Coital Interval : Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
- Fertility Indices: For each group the following were calculated: Mating Index (%) = (Number of animals mated)/(Number of animals paired) x 100, Pregnancy index (%) = (Number of pregnant females)/(Number of animals paired) x 100
-Gestation and Parturition Data: The following parameters were calculated for individual animals during the gestation and parturition period of the parental generation. Gestation Length : Calculated as the number of days of gestation including the day for observation of mating and the start of parturition. Where the start of parturition occurred overnight, the total was adjusted by subtracting half a day. Parturition Index: Parturition Index(%) = Number of females delivering live offspring divided by the number of pregnant females x 100

Offspring viability indices:

- Live Birth and Viability Indices: The following indices were calculated for each group from individual data; live birth index (%)= Number of offspring alive on Day 1/Number of offspring born x 100.
- Viability index 1 (%): Number of offspring alive on Day 4/Number of offspring alive on Day 1 x 100
- Viability index 2 (%): Number of offspring alive on Day 7/ Number of offspring alive on Day 4 x 100
- Viability index 3 (%): Number of offspring alive on Day 14 /Number of offspring alive on Day 7 x 100
- Viability index 4 (%): Number of offspring alive on Day 21/Number of offspring alive on Day 14 x 100
- Viability index 5 (%): Number of offspring alive at weaning/Number of offspring alive on Day 1 x 100
- Sex Ratio (% males): Group mean values calculated from each litter value on Day 1 and 21.
- Implantation Losses (%): Group mean percentile pre-implantation and post implantation loss were calculated as follows: % pre- implantation loss = Number of Corpora Lutea- Number of implantation sites/Number of corpora lutea x 100; % post-implantation losses (%) = Group number of implantation sites- number of offspring/Number of implantation sites x 100
- Offspring Physical Development: A continuity correction of half a day was subtracted from the age of appearance of physical landmarks of development for those litters born overnight

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

MORTALITY: There were eleven mortalities throughout the course of the study. One male and seven females dosed at 500 mg/kg/day, two females dosed at 150 mg/kg/day and one control female. Two females dosed at 500 mg/kg/day were killed in extremis due to excessive distress around the expected time of parturition. During pathological examinations, Myometritis was observed in both animals. One of both females additionally suffered from Pyometria. These findings most likely account for the difficulties during parturition. Another female was killed in extremis following two days of pairing due to a wound on her flank. Two further females were found partially cannibalized so the cause of death could not be determined. The remaining animals which were killed in extremis showed signs of respiratory distress following dosing. These animals plus the animals found dead showed fluid in the thoracic cavity and/or congested lungs at post mortem examination. These deaths are considered to be a consequence of the method of administration. Among the treated animals dying prematurely, there were no indications that early death was a consequence of test material toxicity. Certain changes, specifically necrosis associated with thoracic organs and the presence of foreign material in the thorax are indicative of mal-dosing.
CLINICAL SIGNS: Animals of either sex treated with 500 mg/kg/day showed isolated incidences of increased salivation at the pre-dose observations and up to ten minutes after dosing. Two females showed signs of respiratory distress. In addition to this, one displayed pilo-erection and emaciation. The other female showed pilo-erection, hunched posture, staining around the eyes and pallor of the extremities on the same day. One male vocalised on isolated occasions, this was a single isolated incidence and was therefore considered to be unrelated to treatment. On the second day of pairing, one female had a wound on the flank exposing underlying tissue. This injury was a severe result of fighting, which often occurs initially following pairing and was considered not to be related to treatment. One male and two females treated with 150 mg/kg/day showed isolated incidences of respiratory distress. In addition, these animals showed hunched posture, lethargy, tiptoe gait, staining around the eyes and pilo-erection. These observations were considered attributable to the misadministration of the test material and were not considered toxicologically significant. Males treated with 15 mg/kg/day showed isolated incidences of generalized fur loss, these were considered to be incidental and of no toxicological importance.
BODY WEIGHT: Maturation: No treatment related effects were seen on group mean bodyweight or bodyweight gain throughout maturation. Gestation: Bodyweight gain was comparable to controls for all dose levels during gestation. Lactation: Bodyweight gain was comparable to controls for all dose levels during lactation.
FOOD CONSUMPTION: Maturation: At 500 mg/kg/day there was a slight decrease in food consumed for females during week ten of the study. This was considered to be incidental as no such effects were seen in males and there were no intergroup differences in food efficiency. Gestation: There were no treatment related differences in food consumption or food efficiency during gestation at any dose level. Lactation: There were no treatment related differences in food consumption or food efficiency during lactation at any dose level.
WATER CONSUMPTION: Daily visual inspection of water bottles revealed no intergroup differences.
PATHOLOGY
-Organ weights: There were no intergroup differences in absolute or relative organ weights.
-Necropsy: Interim deaths: At 500 mg/kg/day one interim death male and two interim death females showed macroscopic changes consistent with dosing trauma including white fibrous adhesions around the heart and lungs and/or fluid in the thoracic cavity at post mortem examination. One female also exhibited sloughing and thickening of the glandular gastric epithelium. Two females dosed at 150 mg/kg/day and one control female showed similar findings at post mortem examination. Another female dosed at 500 mg/kg/day had a wound on the flank exposing underlying tissue. Two further females dosed at 500 mg/kg/day were partially cannibalized. Two female animals dosed at 500 mg/kg/day which were killed in extremis around the expected time of parturition had dead fetuses in utero. In addition, one female showed red/brown fluid in the uterus and a large swelling around the ano-genital region. The other female displayed red/brown staining around the eyes and red staining around vagina, large adrenals and a mottled appearance to the spleen. Terminal Necropsy: At 500 mg/kg/day one female displayed a damaged nipple and swollen mammary tissue filled with white fluid. One male showed a small, blue, flaccid testis and small, pale seminal vesicles. A solid mass, filled with thick, pale yellow/green fluid, was observed in the intestine. In addition to this the spleen was large. Another male showed patchy pallor of the liver. One further male showed a mottled appearance on one kidney. In isolation and in the absence of any histopathological findings these findings were considered to be incidental and of no toxicological significance. At 150 mg/kg/day one male showed sloughing of the glandular region of the stomach. Another male showed findings consistent with dosing trauma including fibrous adhesions around the lungs and a yellow/green substance around the lungs. The spleen was also enlarged. One control male had a small testis. The occurrence of a small testis in a control animal confirms the opinion that the small testis and seminal vesicles observed in a male treated with 500 mg/kg/day was not a consequence of administration of the test material.
HISTOPATHOLOGY:
- Mortalities: Among the treated animals dying prematurely, there were no indications that early death was a consequence of test material toxicity. Certain changes, specifically necrosis associated with thoracic organs and the presence of foreign material in the thorax are indicative of mal-dosing.
- Treatment Related Histopathology: No treatment-related changes were observed.
- Other Histopathology: Several conditions warrant specific mention: Pituitary: Developmental cysts in the pars anterior or pars intermedia are common findings in laboratory maintained rats and were considered to have no toxicological significance. Prostate: Interstitial chronic inflammatory cell infiltrates are a commonly observed background finding in laboratory maintained rats. Uterus: Areas of fibrosis and haemosiderin pigment accumulation were seen in the peripheral myometrium of the uterus in the majority of female animals examined from control and high dose groups. These conditions are consistent with normal post partum uterine changes in the rat and were considered to have no toxicological significance. All remaining morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed and, since there were no differences in incidence or severity between control and treatment groups, all were considered to be without toxicological significance.
REPRODUCTIVE SCREENING
-Mating: There were no intergroup differences in pre coital intervals or gestation lengths. Two females dosed at 500 mg/kg/day were killed in extremis around the expected time of parturition. Due to the fact that these animals both suffered from myometritis (together with pyouretria in one of these animals), and due to the fact that difficulties in parturition are common on this type of study, these deaths were considered to be incidental and not a consequence of treatment.

Effect levels (P0)

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

500 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Results: F1 generation

Details on results (F1)

-Offspring Viability and Growth: There were no intergroup differences in litter size or offspring viability throughout lactation. Group mean litter weights and mean offspring weights were comparable to controls for all dose levels. There were no intergroup differences in the time of completion of physical landmarks of development or the percentage of offspring passing reflexological response assessments.
- Litter observations: The type and incidence of clinical observations and post mortem findings were those commonly seen on this type of study. There were no intergroup differences in the time of completion of physical landmarks of development or the percentage of offspring passing reflexological response assessments. No treatment-related macroscopic abnormalities were detected at terminal kill. The findings observed for interim and terminal kill offspring throughout the dose groups were consistent with normally expected low incidence findings in rats of the age examined, and of no toxicological importance.
-Uterine Examination: There were no treatment related differences in the number of corpora lutea or implantation sites observed at necropsy. There were also no treatment related differences in pre or post implantation losses.

Overall reproductive toxicity

Reproductive effects observed:

not specified