Tricarbonyl(methylcyclopentadienyl)manganese

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

other company data

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Statement of GLP compliance

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Tricarbonyl(methylcyclopentadienyl)manganese

EC Number:

235-166-5

EC Name:

Tricarbonyl(methylcyclopentadienyl)manganese

Cas Number:

12108-13-3

Molecular formula:

C9H7MnO3

IUPAC Name:

tricarbonyl(methyl-η5-cyclopentadienyl)manganese

Details on test material:

- Name of test material: X-16039 (methylcyclopentadienyl manganese tricarbonyl)
- Molecular Weight: 218.10 g/mol
- CAS: 12108-13-3
- Physical state: Orange-yellow liquid
- Analytical purity: 97.5%
- Purity test date:
- Lot/batch No.: #109206020B001
- Expiration date of the lot/batch: 2008-10-04
- Stability under test conditions: Avoid exposure to normal room and daylight, use yellow light (>510 nm) and protected pots.
- Storage condition of test material: At room temperature protected from light.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approximately 11 weeks; Pups yet not born.
- Weight at study initiation: not mentioned.
- Fasting period before study: no.
- Housing: housing in a controlled environment:
- Pre-mating - animals were housed in groups of 5 animals/sex/cage in Macrolon cages;
- Mating - females were caged together with males on a one-to-one basis in Macrolon cages.
- Post-mating - males were housed in a home cage with a maximum of 5 animals/sex/cage. Females were housed individually in Macrolon cages.
- Lactation - offspring was kept with the dam until termination.
- General - sterilised sawdust as bedding material and a paper as cage-enrichment was supplied.
- Use of restrainers for preventing ingestion (if dermal): Not Applied.
- Diet : ad. libitum
- Water : ad. libitum
- Acclimation period: 5 days.


ENVIRONMENTAL CONDITIONS
- Temperature: 20.4 - 22.9°C
- Humidity : 39-75 %
- Air changes : 15 air changes per hour
- Photoperiod: 12 hours day and 12 hours night

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared daily within 4 hours prior to dosing and were homogenised to a visually acceptable level. Adjustment was made for specific gravityof the vehicle and test substance.

DIET PREPARATION
Not applicable, not a feeding study.

VEHICLE
- Justification for use and choice of vehicle (if other than water): based on trial formulations prepared by NOTOX. And also due to the poor solubility of mmt in water
- Dose-volume: 5 ml/kg bw

Details on mating procedure:

- M/F ratio per cage: 5 animals/sex/cage. During mating, females were caged together with males in a one-to-one basis.
- Length of cohabitation: maximum of 14 days to enable mating.
- Proof of pregnancy: evidence of sperm in the vaginal lavage or by the appearence of an intravaginal copulatory plug referred to as day 0 post-coitum.
- Further matings after two unsuccessful attempts: no.
- After successful mating each pregnant female was caged: individually in Macrolon cages.
- Any deviations from standard protocol: none.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Quantitative analysis was based on the analytical method validated for the test substance.
Analytical conditions:
-Instrument: Varian Cary 50 (Varian, Mulgrave, Victoria, Australia)
-Wavelenght of detection: 330 nm
-Slit: 1.5 nm
-Type of probe: Ultra mini immersion probe with titanium tube stainless steel handle, quartz prism
-Path lenght 10 mm
-Accuracy of preparation: The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (between 101 and 108%)
-Homogeneity: The formulations of Group 2 and Group 4 were homogeneous (1.3 and 04% coefficient of variation, respectively)
-Stability: Analysis of Group 2 and Group 4 formulations after 5 hours of storage at room temperature yielded a relative difference of 0.5% and -1.1%, respectively. Therefore, the samples were considered stable at room temperature for at least at 5 hours.
-Mean recoveries of the procedural recoveries of the procedural recovery samples were 92 and 101%, the results for the test samples were accepted since they meet the criterion (should be between 70 and 110%).
For detailed information see table 1

Duration of treatment / exposure:

Males were exposed for 28 days, i.e, 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 43 to 51 days, i.e 2 weeks prior to mating, during mating, post-coitum, and during at least 3 days of lactation.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 4 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to scheduled necropsy.

Details on study schedule:

N.A.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 0.5, 2.0, 8.0 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

Group 1 (0 mg/kg bw) - 10 females and 10 males
Group 2 (0.5 mg/kg bw) - 10 females and 10 males
Group 3 (2.0 mg/kg bw) - 10 females and 10 males
Group 4 (8.0 mg/kg bw) - 10 females and 10 males

Control animals:

yes, concurrent no treatment

Positive control:

No

Examinations

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: First day of exposure and weekly thereafter. Mated females were weighted on days 0, 4, 7, 11, 14, 17 and 20 of gestation, and during lactation on days 1 and 4.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

Oestrous cyclicity (parental animals):

Not performed.

Sperm parameters (parental animals):

Parameters examined in [all/P/F1/F2] male parental generations:
Macroscopic examination of seminal vesicle, prostate gland and testes. Organ weights of epididymides and testes.

Litter observations:

STANDARDISATION OF LITTERS
Litters were not standardised.

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring: number and sex of pups, stillbirths, live births, postnatal mortality, clinical signs, body weights.


GROSS EXAMINATION OF DEAD PUPS: Yes, if possible, defects or cause of death were evaluated.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: males were sacrificied following completion of the mating period (28 days of dose administration).
- Maternal animals: females which delivered were sacrificied on lactation day 4. For the females which failed to deliver were sacrificied on post-cointum day 24-27. No deaths occured within all the female rats.

GROSS NECROPSY
- Gross necropsy consisted of macroscopic examinations of the cranial, thoracic and abdominal tissues and organs, with special atention paid to the reproductive organs.
Organ weights were recorded from all surviving F0 (Males and Females): Epididymides, Kidneys, Liver, Tungs and Testes.
Histopathology was performed on the following observed organs: Liver, lungs, kidneys, ovaries, testes and epididymides of animals of group 1 and 4. Testes of animals of group 1 and 4 were also examined for staging of spermatogenesis. All gross lesions of all animals. The reproductive organs of all animals that failed to conceive or sire (Males 4, 7, 37 and Females 44, 47, 77).

Postmortem examinations (offspring):

SACRIFICE
Pups (F1) were killed by decapitation on day 4 of lactation or shortly thereafter.
All offspring was sexed and externally examined if practically possible. The stomach was examinated for the presence of milk . Descriptions of all
external abnormalities were recorded. Where possible, defects or cause of death were recorded.

HISTOPATHOLOGY / ORGAN WEIGTHS
Not done.

Statistics:

-If the variables could be assumed to follow a normal distribution, the Dunnett-test (Dunnett, 1955) (many-to-one t-test) was applied based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
-The Steel-test (Miller, 1981) (many-to-one t-test) was applied if the data could not be assumed to follow a normal distribution.
-The Fish Exact-test (Fisher, 1950) was applied to frequency data.

Reproductive indices:

100 %. For detailed information see tables 2 and 3.

Offspring viability indices:

100 %. For detailed information see tables 2 and 3.

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:

no effects observed

Haematological findings:

no effects observed

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:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No toxicological relevant clinical signs were noted up to 8 mg/kg. Slight salivation was noted for all high dose animals (8 mg/kg). This observation
was considered related to the taste of the formulation, and was not considered toxicologically relevant. Incidental findings included rales, scabs and alopecia at several body parts, watery discharge from the eye, piloerection, hunched posture, and chromodacryorrhea. These signs were considered to be within the normal range of biological variation for rats of this age and strain.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Body weight and body weight gain were unaffected by treatment of X-16039 up to 8 mg/kg bw/day.
No toxicologically significant changes were observed for food consumption before or after allowance for body weight. In the 2 mg/kg dose group, absolute and relative food consumption was decreased for females on Days 1-15 of the pre-mating period. At 8 mg/kg absolute and relative food consumption was decreased for females on Days 1-8 of the pre-mating period and relative food consumption was decreased on Days 17-20 post-coitum. As these changes were very slightand were inconsistent over time, they were not considered toxicologically significant.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Not measured.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Not measured.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Not measured.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Reproduction parameters were unaffected by treatment up to 8 mg/kg bw/day . No treatment related findings were observed on mating performance, fertility parameters, duration of gestation, number of dead and living pups at first litter check, corpora lutea and implantation sites. One female of the control (Female 41) delivered only two pups (of which one was dead at first litter check). As this was observed at the control group, it was not treatment related.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Increased absolute and relative lungs weights were observed for males and females treated at 8 mg/kg. All other statistical significant changes (testes and liver of male rats) were considered unrelated to treatment as no dose response relationship was apparent.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Observations at necropsy did not reveal any alterations that were considered to be toxicologically relevant.
Incidental findings included a yellowish nodule on the tail of the epididymides, dark red discolouration of the kidney, fluid in the uterus, dark red discolouration of the papillary process of the liver, alopecia at several body parts, and dark red discolouration of the accesssory lung lobe. These findings are occasionally seen among rats used in these types of study and in the absence of a dose response relationship they were considered changes of no toxicological significance.

HISTOPATHOLOGY (PARENTAL ANIMALS)
There were no treatment-related observations. There were no significant changes in the reproductive organs of those animals that were suspected of infertility. Usual distribution of stages types of the testes stained with PAS. No variations were found in the subjectively judged stages of testicular spermatogenesis. Occasional observations of commonplace changes in the tissues of young laboratory rats, all of small degree and variable incidence. None were associated with treatment.

Effect levels (P0)

Key result

Dose descriptor:

NOEC

Effect level:

8 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

not examined

Mortality:

not examined

Body weight and weight changes:

not examined

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Effect levels (P1)

Key result

Dose descriptor:

other: not examined

Target system / organ toxicity (P1)

Key result

Critical effects observed:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

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:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

VIABILITY (OFFSPRING)
No treatment related findings were observed in the number of dead and living pups at first litter check. One female of the control group (Female 41) delivered only two pups (of which one was dead at first litter check). Viability index was similar for the control and treated groups.

CLINICAL SIGNS (OFFSPRING)
Development of pups was unaffected by treatment up to 8 mg/kg bw/day.
Incidental clinical symptoms consisted of blue and red discolouration of several body parts, scabs at several body parts, small appearance, wound at the right ear, absence of milk, purple spot on the snout, swelling of the head, pale appearance, white or missing tail apex, and broken tail. Macroscopic examination of the pups revealed scabs at the right foreleg or at the back, wound at the right ear, small appearance, missing tail apex, absence of milk, broken tail, and autolysis or cannabalism for dead pups. No relationship with treatment was established for these observation and they were considered to be within the normal biological variation for rats of this age and strain.

BODY WEIGHT (OFFSPRING)
(Mean) body weights were similar for the control and treated groups, i.e., all pups increased their body weight from day 0 to day 4 (termination).

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

8 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Highest dose tested. No toxicologically relevant effects were seen in any of the doses evaluated.

Target system / organ toxicity (F1)

Critical effects observed:

not specified

Results: F2 generation

General toxicity (F2)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not specified

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not specified

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:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not specified

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

Key result

Dose descriptor:

NOEC

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F2)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

Treatment with the test substance by oral gavage in male and female Wistar Han rats at dose levels of 0.5, 2 and 8 mg/kg bw/day revealed no parental, reproduction, breeding or developmental toxicity up to 8 mg/kg body weight/day. Based on these findings, the parental, reproduction, breeding and developmental No Observed Adverse Effect Level (NOAEL) was established to be greater than 8 mg/kg body weight/day.

Executive summary:

In a reproduction/developmental toxicity study performed according to OECD 421 following GLP guidelines, mmt was administered to 41 Females and 41 males Winstar Han rats by oral gavage at dose levels of 0, 0.5, 2 and 8 mg/kg body weight/day. Males were exposed for 28 days: 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 43 to 51 days: 2 weeks prior to mating, during mating, during post-coitum, and during at least 3 days of lactation. The following eindpoints were evaluated: clinical signs, body weight, food consumption, reproduction processes, the observations of offspring, macroscopy, organ weights, and histopathology. Formulation analysis conducted during the study showed that the test substance doses were prepared accurately and were homogeneous and stable for at least 5 hours when stored at room temperature. There were no toxicologically relevant treatment related changes for mortality, clinical signs, body weights, food consumption, macroscopic examination, organ weights, reproduction , breeding data or pup development up to 8 mg/kg. Since no adverse effects were observed, the NOAEL derived from this study was 8 mg/kg body weight/day.