Ferrocene

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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:

yes

Remarks:

Body wt.range stated 180-200g. Actual-males(269-309g), females(193-221g). The target range for RH (50 ± 20%), achieved range was 39- 86%. Neither deviation from Study Plan was considered to have had no impact on the scientific integrity of the study

Principles of method if other than guideline:

DEVIATIONS FROM STUDY PLAN
The predicted body weight range stated in the Study Plan at the start of treatment was 180-200 g, but this range proved to be too low for the age of animals required. At the start of treatment, males weighed 269 to 309g and females weighed 193 to 221g; all were of the correct age and were considered acceptable for the study. The deviation from Study Plan for the weight ranges for these animals was considered to have had no impact on the scientific integrity of the study.
The Study Plan target range for relative humidity was 50 ± 20% respectively but this was exceeded on a number of occasions during the study (achieved range was 39- 86% RH). While the deviations observed for relative humidity were not ideal, it was considered that they had not impacted on the scientific integrity of the study.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Ferrocene

EC Number:

203-039-3

EC Name:

Ferrocene

Cas Number:

102-54-5

Molecular formula:

C10H10Fe

IUPAC Name:

iron(2+) dicyclopenta-2,4-dienide

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Description : Orange coloured crystalline
Chemical name : Dicyclopentadienyl Iron
CAS number : 102-54-5
Storage conditions : room temperature in the dark

Test animals

Species:

rat

Strain:

other: Wistar Han™:RccHan™:WIST

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Number: 80, (40 males and 40 females)
- Source: Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK.
- Age at study initiation: (P) 10 wks
- Weight at study initiation: (P) Males: 269-309 g; Females: 193-221 g (see Deviations from Study Plan)
- Housing: Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. 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 mesh lids and softwood flakes.
- Diet: ad libitu, Pelleted, Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK
- Water: ad libitum, mains drinking water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 2-+3
- Humidity (%): 39-86 - it was considered that this had not impacted on the scientific integrity of the study (see Deviation from Study Plan).
- Air changes (per hr): >15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

i) Groups of ten male and ten female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
ii) Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioural toxicity.
iii) On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.

Details on mating procedure:

Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen 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 oestrus 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 (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation.

Duration of treatment / exposure:

28days

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

5 mg/kg bw/day

Remarks:

Basis: actual ingested

Doses / concentrations 2

Dose / conc.:

10 mg/kg bw/day

Remarks:

Basis: actual ingested

Doses / concentrations 3

Dose / conc.:

15 mg/kg bw/day

Remarks:

Basis: actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

Several observations were made. Parameters included:
Clinical Observations
Functional Observations-Behavioural Assessments, Functional Performance Tests; Motor Activity.and Forelimb/Hindlimb Grip Strength., Sensory Reactivity
Body Weight
Food Consumption
Water Consumption
Reproduction Screening-Mating, Pregnancy and Parturition, Litter Data, Physical Development
Laboratory Investigations-Haematology, Blood Chemistry
Pathology-Organ Weights, Histopathology

Litter observations:

The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using the individual litter values. Group mean values generally included all litters reared to termination (Day 5 of age), however at 25 mg/kg bw/day group mean values were calculated for all pregnant animals (where appropriate) and also for those females that littered.

Statistics:

Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05.

Reproductive indices:

Mating Performance and Fertility
The following parameters were calculated from the individual data during the mating period of the parental generation:
i) Pre-coital Interval
ii) Fertility Indices
Gestation and Parturition Data
The following parameters were calculated for individual data during the gestation and parturition period of the parental generation.
i) Gestation Length
ii) Parturition Index
Litter Responses
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using the individual litter values. Group mean values generally included all litters reared to termination (Day 5 of age), however at 25 mg/kg bw/day group mean values were calculated for all pregnant animals (where appropriate) and also for those females that littered.
i) Implantation Losses (%)
ii) Live Birth and Viability Indices
iii) Sex Ratio (% males)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

The clinical signs observed on the study were unremarkable and did not indicate any adverse effect of treatment at 5, 10 or 25 mg/kg bw/day.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Both had effects see discussion

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Both had effects see discussion

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

There were no clear effects of treatment on fertility at 5,10 mg/kg bw/day. However, at 5, 10 mg/kg bw/day, 1 female had implantations but was not observed to litter; additionally at both dosages, 1 female showed total litter loss post partum

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating performance, as assessed by pre-coital interval and the evidence of mating observed (copulation plugs, sperm in the vagina), was unaffected by treatment at 5, 10 and 25 mg/kg bw/day.

Effect levels (P0)

1

Dose descriptor:

NOEL

Effect level:

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

Sex:

male/female

Basis for effect level:

other: reproduction and offspring survival, growth and development

2

Key result

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male/female

Basis for effect level:

other: Reproduction and systemic toxicity

Results: F1 generation

Effect levels (F1)

1

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Sex:

male/female

Basis for effect level:

other: systemic toxicity

2

Dose descriptor:

NOEL

Generation:

F1

Effect level:

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

Sex:

male/female

Basis for effect level:

other: offspring survival, growth and development

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Ferrocene was assessed to investigate the systemic toxicity and its potential adverse effects on reproduction (including offspring development). the study was performed according to OECD Guideline 422.

There were clear treatment-related effects on reproduction at 25 mg/kg bw/day. At 5 and 10 mg/kg bw/day, reproduction per se was not affected by treatment but at both dosage two females failed to maintain their litter to Day 4 of age due to either in utero or post partum offspring losses. Very high pre and post natal losses were evident at 25 mg/kg bw/day, therefore the litter deaths at lower dosages become problematic when establishing a NOEL effect level for offspring survival, which may become clearer in a study of larger dimensions. Within the context of this study, there was no evidence of effects of treatment on surviving offspring at 5 mg/kg bw/day, although there was a suggestion of marginally lower weight gain at 10 mg/kg bw/day.

As effects of treatment were apparent at dosages as low as 5 mg/kg bw/day it was not possible to establish a No Observed Effect Level (NOEL) for systemic toxicity. A dosage of 10 mg/kg bw/day was considered to be a No Observed Adverse Effect Level (NOAEL) for systemic toxicity. In view of this the NOEL for reproduction and offspring survival, growth and development was considered to be at 5 mg/kg bw/day.