Tricarbonyl(methylcyclopentadienyl)manganese

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

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

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

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

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.

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

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.

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: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

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.

Key result

Dose descriptor:

NOEC

Effect level:

8 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Key result

Critical effects observed:

no

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: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Key result

Dose descriptor:

other: not examined

Key result

Critical effects observed:

not specified

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

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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).

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.

Critical effects observed:

not specified

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

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOEC

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

8 mg/kg bw/day

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Species:

rat

Additional information

No reproductive or developmental effects were seen in the key study, performed according with OECD guidelines 421 following GLP guidance. Therefore in accordanc with REACH Annex X, the performance of a multi-generation reprotoxicity study is scientifically unjustified.

Short description of key information:
In a K1 reproductive toxicity study, mmt was administered to female and male rats by gavage at dose levels of 0, 0.5, 2 and 8 mg/kg body weight/day. There were no toxicologically relevant treatment related changes for mortality, clinical signs, body weights, food consumption, macroscopic examination, organ weights, reproduction , breeding data and pup development up to 8 mg/kg. Since no adverse effects were observed, the NOAEL derived from this study was determined to be the highest dose tested, 8 mg/kg body weight/day.

Effects on developmental toxicity

Description of key information

MMT was administered to rats by gavage at the dose levels of 2.0, 4.5, 6.5, and 9.0 mg/kg/day. MMT is not teratogenic at the tested doses, therefore the NOAEL is established as 9.0 mg/kg/day while the NOAEL for maternal toxicity is established at 6.5 mg/kg/day.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 1979-August 1979

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

The purity of the substance is not mentioned

GLP compliance:

yes

Remarks:

The report indicates compliance with GLP guidelines.

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Portage, Michigan)
- Age at study initiation: 15 weeks
- Weight at study initiation: 220 to 280 g
- Fasting period before study: No
- Housing: Individually housed except during mating in suspended wire mesh cages
- Diet (e.g. ad libitum): Purina Rodent Laboratory Chow #5001 ad. libitum
- Water (e.g. ad libitum): ad. libitum
- Acclimation period: 5 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 to 24
- Humidity (%): 45 +/- 10
- Air changes (per hr): -
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light


IN-LIFE DATES: The administration of the test compound was until the day 15 of gestation, and the rats were sacrificed at gestation day 20.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Dosage volumes administrated were 7.14 ml/kg, 7.50 ml/kg, 7.56 ml/kg and 6.72 ml/kg. Individual dosages were determined from individual body weight.



VEHICLE
The vehicle was prepared, assayed and shipped to the research laboratory.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

The solutions of the test compound in the vehicle were prepared by the sponsor. No verification of the test concentrations was performed.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Further matings after two unsuccessful attempts: N.A.
- Verification of same strain and source of both sexes: N.A.
- Proof of pregnancy: copulatory plug

Duration of treatment / exposure:

From gestation day 6 to gestation day 15 (total of 9 days of treatment)

Frequency of treatment:

Single daily dose.

Duration of test:

20 days.

No. of animals per sex per dose:

Vehicle control-25 Female Rats
2.0 mg/kg/day-25 Female Rats
4.5 mg/kg/day-25 Female Rats
6.5 mg/kg/day-25 Female Rats
9.0 mg/kg/day-25 Female Rats

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: A pilot study was conducted preceeding this study to determine the appropriate high-dose levels for a teratology study.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: No specified
- Cage side observations checked in table [No.?] were included. No

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: No

BODY WEIGHT: Yes
- Time schedule for examinations: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: Uterus and liver

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: 1/3 per litter
- Skeletal examinations: Yes: 2/3 per litter

Statistics:

Incidence of fetuses and litters with malformations were compared by the Wilcoxon test, as modified by Haseman and Hoel, to judge statiscal significance of differences.
Incidence on maternal deaths, pregnant dams and early resorptions and postimplatation loss were compared using the Chi-square test criterion with Yates correction for 2x2 contingency tables and/or Fisher's exact probability test as described by Siegel to judge significance of differences.
Maternal body weights, maternal liver weights, fetal body weights abd fetal crown-rump lengths were compared by analyses of variance (1-way classification) and t-test described by Steel and Torrie using Dunnets multiple comparison table.

Indices:

All statistical analyses compared the treatment groups to the control group with the level of significance at p<0.05

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Maternal toxicity due to treatment with mmt was evident at the 9.0 mg/kg/day dosage level as an increase in matting and staining of the anogenital haircoat and a mean maternal weight loss early in the treatment period which was statistically significant in gestation day 9.

Key result

Dose descriptor:

NOAEL

Effect level:

6.5 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Key result

Dose descriptor:

NOAEL

Effect level:

9 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Key result

Abnormalities:

effects observed, non-treatment-related

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
A slight reduction in mean fetal body weights were seen. An increase in the incidence of malformations was noted at all of the mmt dosage levels, exclusively due to the presence of bent ribs when compared with the control group. Bent ribs are not uncommon finding in this strain of rats based on historical data, therefore this effect was not considered a malformation and when present in the exclusion of other malformations is not considered a teratogenic response.

Key result

Dose descriptor:

NOAEL

Effect level:

6.5 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Lowest effective dose / conc.:

9 mg/kg bw/day (nominal)

Treatment related:

no

Maternal observations

1. Appearance and Behavior

There were no biologically meaningful differences in appearance and behaviour attributable to treatment with mmt in the 2.0, 4.5 or 6.5 mg/kg/day dosage groups when compared to the control group. Survival in these three dosage groups and the control group was 100%. A subcutaneous mass in thoracic area was observed in one rat in the 4.5 mg/kg/day dosage group at cesarean section. Upon histological examination, this mass was found to be a mammary adenocarcinoma. This mass was not considered to be a treatment-related effect, as it was located only 14-days after the initiation of test article administration.

A slight increase in matting and staining of the anogenital haircoat was noted in the 9.0 mg/kg/day dosage group when compared to the control group. One rat in this dosage group died on gestation day 11. At necropsy, death was attributed to pneumonia.

There was no statistically significant differences in the incidence of maternal deaths or pregnant dams in any of the mmt treated groups when compared to the control groups.

 

2. Body Weights

A very slight reduction in mean maternal body weight gain was noted from days 12 through 20 of gestation in the 2.0 mg/kg/day dosage groups and from days 6 though 20 of gestation in the 4.5 mg/kg/day dosage group when compared to the control group. This resulted in a slight reduction in mean weight gain over the entire gestation period in these two treatment groups when compared to the control group. A moderate reduction in mean weight gain from days 6 through 9 of gestation in the 6.5 mg/kg/day dosage group and a mean weight loss during this interval, accompanied by a statistically significant reduction in mean maternal body weight on gestation day 9, in the 9.0 mg/kg/day dosage group were noted when compared to the control group. This resulted in moderate reduction in mean maternal body weight gain over the entire gestation period in both of these treatment groups when compared to the control group. With the exception of gestation day 9 in the 9.0 mg/kg/day dosage group, all other mean maternal body weights for the treatment groups were not statistically significant when compared to the control group.

There were no statistically significant or biologically meaningful differences in mean maternal liver weights in any of the mmt treated groups when compared to the control group.

 

3. Caesarean Section Observations

No statistically significant differences in the mean number of early resorptions, post-implantation loss or mean fetal crown-rump lengths were noted in any of the mmt treated groups when compared to the control group. There were no biologically meaningful differences in the mean number of viable fetuses, early resorptions, post-implantation loss, total implantations, corpora lutea or the fetal sex distribution, mean fetal crown-rump length or mean maternal liver weights in any of the treatment groups when compared to the control group.

A slight increase in the mean number of early resorptions and post-implantation loss, a corresponding slight decrease in the mean number of viable foetuses and also a slight decrease in the mean number of total implantations were noted in all of the mmt treated groups when compared to the control group. However, these differences were not considered to be treatment-related, as no dose-related patterns were evident. A slight reduction in mean fetal crown-rump length and a statistically significant decrease in mean fetal body weight were noted in the 6.5 mg/kg/day dosage group. The mean fetal crown-rump length in the 9.0 mg/kg/day dosage group, however, was equal to that in the control group. A slight decrease in mean fetal body weight was noted in all of the mmt treated groups when compared to the control group.

 

Fetal morphological observations

A slight, though not statistically significant, increase in the incidence of fetuses and litters with malformations in the 2.0, 4.5 and 6.5 mg/kg/day dosage groups, and a statistically significant increase in the 9.0 mg/kg/day dosage group were noted when compared to the control group. These increases were deemed to be due to the presence of bent ribs in the mmt treatement groups, which were not dose-dependent increases. No bent ribs were observed in the control group.

Conclusions:

mmt is not teratogenic at the tested doses, therefore the developmental NOAEL is established as 9.0 mg/kg/day while the NOAEL for maternal toxicity is established at 6.5 mg/kg/day.   

Executive summary:

In a developmental toxicity study performed similar to OECD 422, mmt was administered by oral gavage to 25 females Sprague-Dawley rats at doses of 2.0, 4.5, 6.5 and 9/0 mg/kg/day in corn oil from day 6 through data 15 of gestation.

Maternal toxicity due to treatment with mmt was evident in the 9.0 mg/kg/day dosage level as an increase in matting and staining of the anogenital haircoat and a mean maternal weight loss early in the treatment period which was statistically significant on gestation day 0. A slight reduction in mean fetal body weights and a slight to moderate reduction in mean maternal body weight over the entire gestation period were noted in all of the mmt treatment levels.

An increase in the incidence of malformations was noted at all of the mmt dosage levels. This was exclusively due to the presence of bent ribs at all dosage levels when compared to the control group, however no dose-dependent increase were observed. Bent ribs are not an uncommon finding in this strain of rats based on the historic control data in this laboratory. Therefore bent ribs were not thought to be a malformation in the usual sense and when present with the exclusion of other malformations, as demonstrated in this study, was not considered a teratogenic response. mmt is not teratogenic at the tested doses, therefore the developmental NOAEL is established as 9.0 mg/kg/day while the NOAEL for maternal toxicity is esablished at 6.5 mg/kg/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

9 mg/kg bw/day

Additional information

The study conducted by International Research Development Corporation (IRDC) derived a NOAEL of 6.5 for maternal toxicity and a NOAEL of 9.0 mg/kg bw/day for developmental toxicity, while the study conducted by Biodynamics defined a NOAEL of 5 mg/kg bw/day due to severe effects seen in the higher concentrations. The group dosed with 40 mg/kg had to be terminated in the beginning of the test, and the following concentration, 20 mg/kg, still induce severe maternal toxicity. The results regarding the dose level of 10 mg/kg were inconclusive due to the lack of statistical significance. These deviations made complex the evaluation of the results.Hence, the study of IRDC was used as a key study due to the presence of less deviations from the original protocol. 

Justification for classification or non-classification

No classification is necessary since no effects were seen in reproduction or on development of rats in the three tests conducted.