Tricarbonyl(methylcyclopentadienyl)manganese

Endpoint summary

Description of key information

The most relevant route of exposure to mmt is the inhalation route.  A reliable subchronic study on mice, rats, and monkeys, performed similar to OECD 413, was determined to be the key study for this endpoint as it addresses the requirements for repeated-dose toxicity.   From this study, it was determined that the NOAEC was 30 mg/m3 of mmt in air for the monkey based on a lack of toxicologically significant effects observed at this dose in the monkey. 

Key value for chemical safety assessment

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1977-03-03-1978-08-10

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted similar to OECD 413 with limited restrictions. Sufficient information of methods and results was available for an assessment.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

yes

Remarks:

The rats and mice were not medically examined prior to the beggining of the study. Animal husbandry is not specified, but the investigators do state they are accordantly to the 'Inhalation Toxicology Department'.

GLP compliance:

no

Remarks:

Study conducted prior to GLP guidelines.

Limit test:

no

Species:

other: Rat (Female and Male), Mice (Female and Male) and Monkey (Male)

Strain:

other: Sprague-Dawley, Swiss Webster and cynomolgus monkeys (M. fasicularis)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS: Rat Sprague-Dawley

- Source: Charles River Breeding Laboratories
- Age at study initiation: 7 weeks old
- Weight at study initiation: Males - 377-604 grams and Females 230-317 grams
- Fasting period before study: No
- Housing: Housed in groups of 10
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Two weeks
ENVIRONMENTAL CONDITIONS
Photo period, ambient temperature, and humidity were according to Inhalation Toxicology Department's Standard Procedure
IN-LIFE DATES: From: 0 To: 12 weeks

TEST ANIMALS: Mice Swiss Webster
- Source: Charles River Breeding Laboratories
- Age at study initiation: 8 weeks old
- Weight at study initiation: Males - 39.7-53.3 gr
- Fasting period before study: No
- Housing: Housed in groups of 10
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Two weeks


ENVIRONMENTAL CONDITIONS
Photo period, ambient temperature, and humidity were according to Inhalation Toxicology Department's Standard Procedure


IN-LIFE DATES: From: 0 To: 12 weeks

TEST ANIMALS: cynomolgus monkeys (M. fasicularis)
- Source: Prime Labs
- Weight at study initiation: Males - 2711-5043 grams
- Fasting period before study: No
- Housing: Housed individually
- Diet (e.g. ad libitum): 8 Purina monkey biscuits (25% protein) twice per day and fruit
- Water (e.g. ad libitum): ad libitum



ENVIRONMENTAL CONDITIONS
Photo period, ambient temperature, and humidity were according to Inhalation Toxicology Department's Standard Procedure


IN-LIFE DATES: From: 0 To: 12 weeks

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: Not applicable, mmt does not creat particles.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 6m^3 stainless steel and glass exposure chambers
- Method of holding animals in test chamber: N.A.
- Source and rate of air: airflow of approximately 1000L/min
- Method of conditioning air: supplied and filtered air by a system separate from the general building system
- System of generating particulates/aerosols: Vapors were generated by two systems: the low level (0.3 ug/L) generator consisted of a 25 ml capacity midget bubbler with a 145-175 ml micron porosity glass frit. The generator was protected from light and supplied with an airflow of 0.3-0.6 L/min. The intermediate (3 ug/L) and high (30 ug/L) level generators were based on the following principle: mmt was delivered at a controlled rate by a Sage Syringe drive to a U-Style custom fabricated counter current vaporizer packed with 4 mm glass beads and fitted with a 300 watt quartz immersion heater. The vaporizer delivered mmt to the chamber air inlet through heated teflon delivery lines.
- Temperature, humidity, pressure in air chamber: No information
- Air flow rate: 0.3 ug/L: 0.3-0.6 L/min; 3.0 ug/L and 30 ug/L: 24 L/min


TEST ATMOSPHERE
- Brief description of analytical method used:
- Samples taken from breathing zone: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chamber concentrations of mmt were monitored by a single four to six hour integrated sample. Each chamber was monitored daily by collecting samples with adsorption tubes packed with chromosorb 102. The adsorption tubes for chambers II and chambers III were packed with 1.3 g chromosorb. The adsorption tube for chamber IV was packed with 1.4 g chromosorb. Prior to first use, each adsorption tube was washed with 15-20 ml hexane. Following collection, samples were eluted from chromosorb 102 with hexane, and the eluates were analyzed for mmt using a Perkin-Elmer model 267 infrared spectrophotometer set for 1934 cm-1, using a Beckman RIIC F-05 cell with a 2 mm path length and a 22x41x7 mm NaCl windows. Concentrations of mmt were read using a standard curve established at the beginning of exposures.

Duration of treatment / exposure:

12 weeks.

Frequency of treatment:

Six hours per day and five days per week.

Remarks:

Doses / Concentrations:
Control group or group I (air ventilation), 0.3 (group II), 3.0 (group III) and 30 ug/L (group IV)
Basis:
nominal conc.

No. of animals per sex per dose:

Group I: control
-Rats: 10 (males) 10 (females)
-Mice: 10 (males) 10 (females)
-Primates: 6 (males)

Group II: 0.3 ug/L
-Rats: 10 (males) 10 (females)
-Mice: 10 (males) 10 (females)
-Primates: 6 (males)

Group III: 3.0 ug/L
-Rats: 10 (males) 10 (females)
-Mice: 10 (males) 10 (females)
-Primates: 6 (males)

Group IV: 30.0 ug/L
-Rats: 10 (males) 10 (females)
-Mice: 10 (males) 10 (females)
-Primates: 6 (males)

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: No dose-range finding study was performed, nevertheless a low, intermediate and high concentrations were chosen by the sponsor.
- Rationale for animal assignment (if not random): No information
- Rationale for selecting satellite groups: No information
- Post-exposure recovery period in satellite groups: 14 days post-exposure

Positive control:

No.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: no data

DETAILED CLINICAL OBSERVATIONS: yes
- Time schedule: no information

BODY WEIGHT: yes
- Time schedule for examinations: no information

FOOD CONSUMPTION: yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No

FOOD EFFICIENCY: no
- 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: yes
- Time schedule for examinations: no information

OPHTHALMOSCOPIC EXAMINATION: no

HAEMATOLOGY: yes
- Time schedule for collection of blood: no information
- Anaesthetic used for blood collection: no data
- Animals fasted: no
- How many animals: 5 female and 5 male rats and monkeys
- Parameters checked in table were examined: six week interval-male rats (Table 2), female rats (Table 4) and monkeys (Table 6) twelve week interva-male rats (Table 8), female rats (Table 10) and monkeys (Table 12);
Parameters: hematocrit, hemoglobin, erythrocyte count, leukocyte count and differential leukocyte count.

CLINICAL CHEMISTRY: yes
- Time schedule for collection of blood: no information
- Animals fasted: no
- How many animals: 5 female and 5 male rats and monkeys
- Parameters checked in table were examined. twelve week intervale-male rats (Table 14), female rats (Table 16) and monkeys (Table 18)
Parameters: blood urea nitrogen, blood glucose, serum glutamic oxaloacetic transminase, serum glutamic pyruvic transminase and serum alkaline phosphatase

URINALYSIS: yes
- Time schedule for collection of urine: no information
- Metabolism cages used for collection of urine: no
- Animals fasted: no
- Parameters checked in table were examined. twelve week interval-male rats , female rats and monkeys at twelve week interval and on three primates from each group at two, four and eight days post-exposure (Table 50)
Parameters: bilirubin, occult blood, pH, glucose, ketones, protein, specific gravity

NEUROBEHAVIOURAL EXAMINATION: no

Sacrifice and pathology:

SACRIFICE
Group IV mice were terminated at week 5 of exposure due to a high incidence of death and severe weight loss.
All the rat, remaining mice (Groups 1-III) and 3 monkeys were sacrified in week 12 immediatly after exposure. The remaining 3 monkeys were sacrificied 14 days post-exposure. All the animals were sacrificied by pentobarbital sodium anesthesia and exsanguination. Only the rats were fasted prior to necropsy.

GROSS PATHOLOGY: Yes
Lungs from all animals were removed in an inflated state, examined for macroscopic abnormalities, deflated and re-inflated.

The following organs were weighed at the time of necropsy:
-Heart, liver, kidney, teste, spleen, brain, lung and trachea.

The following organs were preserved in formalin:
-Abnormal tissue, adipose tissue, adrenals, bone, brain, epididymis, esophagus, eye and optic nerve, gall blader, heart and aorta, intestine (colon, duodenum, ileum and jejunum), kidneys, liver, lung and trachea, lymph nodes (mesenteric and peribronchial, mammary gland, ovaries and oviduct, pancreas, peripheral nerve (sciatic), pituitary gland, prostate gland, salivary gland, skeletal muscle, skin, spinal cord, spleen, stomach (cardiac, fundus and pylorus), testes, thymus, thyroid, urinary bladder and uterus.

The following organs from all primate as well as 5 male and 5 female rats, 5 male and 5 female mice from the control (group I) and high level (group IV) groups were imbedded in paraffin and stained with hematoxylin and eosin and evaluated by a pathologist:
-Adrenal, adipose tissue, bone marrow, brain (cerebellum and brain stem), epidedymis, esophagus, gall bladder, heart and aorta, intestine (ileum and colon), kidney, liver, ovary and oviduct, pancreas, peripheral nerve, pituitary, prostate, salivary gland, skeletal muscle, skin, spinal cord, spleen, stomach, thyroid and parathyroid, lungs and bronchi, lymph nodes (mesenteric), mammary tissue, teste and trachea.

Samples of the following tissues from all monkeys were collected at the time of necropsy and sent for manganese analysis:
-Adipose tissue, blood, brain, hair, heart, kidney, liver, lungs, muscle, pancreas, spleen, testes and thyroid.

Other examinations:

Body and organ weights

Statistics:

Hematology, clinical chemistry, organ weight and body weight data was evaluated using a two-way analysis of variance and where appropriate, a Dunnet's test. The significance for all the cases was p≤0.05

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

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:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY: -Group IV rodents presented the most severe manifestations of toxicity characterized by rough coat, lethargy, dyspnea and death. Group IV male and female mice were terminated at five weeks of exposure, due to the severity of toxic signs.

BODY WEIGHT AND WEIGHT GAIN: -Decreases in body weight were noted in group IV male and female rats beginning at approximately two weeks of exposure. Male and female mice in group IV showed decreases in body weight beginning at about 1 or 1/2 weeks of exposure. Female and male mice in group III began to show decreases in body weight at week 3 and 4, respectively. No body weight effects were seen in monkeys at any of the exposure levels.

HAEMATOLOGY: - Hematologic evaluations at six and twelve weeks of exposure to mmt vapors showed few effects in any of the experimental animals. Under the conditions of this experimental design, there were no hematologic effects in either rats or monkeys that could be attributed to exposure to mmt vapors for periods up to 12-weeks.

CLINICAL CHEMISTRY: At the 12-week interval, blood urea nitrogen (BUN) was elevated in both male and female rats. There are indications of an exposure related effect on SAP in male and female rats. There was effect on glucose in both male and female rats; however, there was insufficient evidence to consider it an exposure related effect. Variations in fasting time could account for the higher glucose levels in the control group. No effect was seen on serum calcium or phosphorous in male monkeys.

URINALYSIS: Rodents in all groups were comparable to controls. Monkeys, however, showed elevation in urinary ketones in group IV as compared to controls. Monkeys in group II and III were comparable to controls.

ORGAN WEIGHTS: Because of the effects on body and brain weight in some of the exposure groups, organ weights were sometimes difficult to interpret. Body weights at necropsy were significantly depressed in male and female rats and mice. Brain weights were significantly depressed, on an absolute basis, in female rats, and male and female mice. Therefore, a conservative method of interpretation was adopted; changes were not considered to be significant or compound related unless they occurred in the same direction on an absolute, percent and ratio basis.
Kidney weights were elevated in male and female mice of group III, and female mice of group II, at the termination of exposure. Liver weights were elevated in female mice of groups II and III. Male mice, group III, showed indications of increased liver weights on an absolute and percent basis. Female mice in group II showed a decreased heart weight, while female mice in group IV showed indications of an increased lung weight on a percent and ratio basis.

GROSS PATHOLOGY: Microscopic examination of tissues taken at necropsy revealed exposure related changes in the lungs of group IV rats and mice. The changes in the mice were more severe than the rat. An exposure related vacuolation of the white matter of the brain stem and cerebellar folia was noted in group IV monkeys. Monkeys did not show microscopic lung alterations, nor did they show any other manifestation attributable to exposure conditions with the exception of vacuolation of the white matter.

HISTOPATHOLOGY: NON-NEOPLASTIC

Rats: Male and females from group IV (30ug/L) revealed exposure-related histological changes: increase in the number of alveolar macrophages, varying degrees of focal pneumonitis, pleuritis and alveolar wall thickening were seen in some exposed rats. The alveolar macrophages were generally present in the lumen of the alveoli adjacent to the terminal airway and contained a finely granular brownish material in their cytoplasm. The pneumonitis was generally focal and involved the terminal bronchioles and proximal alveoli. Occasionally, a minimal non-suppurative pericholangitis was seen in the liver, and a few scattered instances of mineralization were seen in the kidney. Extramedullary hematopoiesis and pigment deposition were present from a minimal to slight degree in the spleens from both exposed and control rats. Bonne marrow activity, as judged by the degree if cellularity, was generally moderate in both the control and treated rats.

Monkeys: With the exception of the brain, no exposure related microscopic alterations were seen in the monkeys exposed to 0.3, 3.0 and 30 ug/L of mmt. In the brain, vacuolation was present in the white matter of the brain stem and cerebellar folia from a slight to moderate degree in five of the six monkeys exposed to 30 ug/L. Vacuolation in the same location was present to a minimal degree in three of six monkeys in the control group, two of six monkeys exposed to 0.3 ug/L and 3 of 6 monkeys exposed to 3.0 ug/L. Hepatocyte vacuolation was present in a few monkeys from the control and exposed animals and a a few scattered instances of focal gastrititis were seen in the stomach. Bone marrow activity, as judged by the degree of cellularity, was generally moderate in both the control and mmt exposed monkeys. In the lungs, scattered incidental microscopic alterations were seen in the majority of monkeys from both the control and exposed groups.

Mice: Exposure related microscopic alterations were seen in the lungs from the male and female mice exposed to 30.0 ug/L. These alterations were characterized by bronchial epithelial erosion and in several instances bronchial wall fibrosis. Extra-medullary hematopoiesis was consistently seen in the spleen and varied from minimal to moderate in severity. Bone marrow activity, as judged by the degree of cellularity, was generally moderate in to moderately high.

HISTOPATHOLOGY: NEOPLASTIC
Mice: Exposure related microscopic alterations were seen in the lungs from the male and female mice exposed to 30.0 ug/L of mmt. These microscopic alterations were characterized by varying degrees of bronchial epithelial hyperplasia and bronchial squamous metaplasia.

Dose descriptor:

NOAEC

Remarks:

Monkeys

Effect level:

30 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOAEC

Remarks:

Rat

Effect level:

3 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOAEC

Remarks:

Mice

Effect level:

3 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

Conclusions:

There appears to be a sex related difference in response, with female rodents being more sensitive than male rodents.
The results of the microscopic examination indicate that the inhalation exposure of rats and mice to 30.0 ug/L of mmt results in exposure related histological changes in the lung. These microscopic alterations were characterized by an increase number of alveolar macrophages. Different degrees of focal pneumonitis, pleuritis and alveolar wall thickening were also seen in a few of the exposed rats. The histological appearance of the microscopic changes was different between the mice and rats and somewhat greater in severity in the mice than in the rats.

Although vacuolation of the white matter was identified in the brain stem and cerebellar folia, the same effect was also seen to a lesser degree in the control monkeys. No significant exposure related microscopic alterations were seen in the lungs from monkeys exposed to mmt.

Based on study findings, a NOAEC of 3 mg/m^3 was derived for mice and rats while a NOAEC of 30.0 mg/m^3 was derived for monkeys.

Executive summary:

In a subchronic inhalation toxicity study similar to OECD 413, methylcyclopentadienyl manganese tricarbonyl (mmt) was administered to 10 male and 10 female Sprague-Dawley rats, 10 males and 10 females Swiss Webster mice and 6 male cynomolgus monkeys (M. fasicularis). The animals were exposed whole-body to the vapors of three concentrations of mmt, 0.3, 3.0 and 30.0 ug/L (group II, III and IV respectively) and one negative control group (group I). Hematological evaluations did not show dose related effects that could be attributed to mmt exposure. Clinical chemistry evaluations indicate an exposure related elevation in BUN and SAP in male and female rats. Monkeys were evaluated for calcium and phosphorous levels with no effect seen. Urinalysis did not show any differences from the controls for rats, of either sex, at any exposure level. Decreases in body weight were noted in group IV male and female rats at about 1 to ½ weeks of exposure. Male and female mice in group III began to show decreases in body weight at study weeks 3 and 4 respectively. No effects on body weights were seen in monkeys at any of the exposure levels. Kidney weights were elevated in group III male and female mice. Liver weights were elevated in group II and III female mice only. Decreases in spleen and gonad weights were seen in group IV. Monkeys showed no organ weight effects. Group IV rodents presented the most severe manifestations of toxicity; characterized by rough coat, lethargy, dyspnea and death. Group IV male and female mice were terminated at 5 weeks of exposure, due to severity of toxic signs. Microscopic examination of tissues taken at necropsy revealed exposure related changes in the lungs of group IV rats and mice. The changes in the mice were somewhat more severe than in the rat. An exposure related vacuolation of the white matter of the brain stem and cerebellar folia was noted in group IV monkeys. Monkeys did not show microscopic lung alterations, nor did they show any other toxicity manifestations other than the vacuolation of the white matter which was also seen to a lesser degree in the control monkeys.

There seems to be a sex related difference in response, with female rodents being more sensitive than male rodents. Based on study findings, a NOAEC of 3 mg/m^3 was derived for mice and rats while a NOAEC of 30.0 mg/m^3 for monkeys.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

30 mg/m³

Study duration:

subchronic

Species:

monkey

Additional information

Oral exposure

 

The data requirement for endpoint 7.5.1 (repeat dose oral) was waived as exposure to mmt via the oral route is expected to be negligible, the inhalation route is the most relevant, and a reliable repeat dose inhalation study is available for this route of exposure.

 

Dermal exposure

 

In a subchronic study on male rabbits and male and female rats exposed dermally to leaded and unleaded gasoline containing mmt, and a NOAEL of 16.0 g/l was derived for both species. The NOAEL was set to the highest concentration tested since all the adverse effects seen in the animals, were also observed in controls. The author concluded that the effects were due to the vehicle itself, rather than mmt. The study was disregarded based on insufficient documentation/reporting of essential information and the choice of an unsuitable vehicle. 

 

A reliable study for this endpoint 7.5.3 (repeat dose dermal), is not available; however, as exposure to mmt via the inhalation route is the most relevant and a reliable repeat dose inhalation study is available no further testing is required.

 

Inhalation exposure

 

The most relevant route of exposure to mmt is the inhalation route. A reliable subchronic study on mice, rats and monkeys, performed similar to OECD 413, was determined to be the key study for this endpoint as it addresses the requirements for repeated-dose toxicity.  From this study, it was determined that the NOAEC was 30 mg/m3 of mmt for the monkey based on a lack of toxicologically significant effects observed at this dose (30 mg/m3 was the highest dose evaluated in the study).  The monkey is generally considered a better model for human health toxicity than rodents. This is further substantiated based on the in vitro metabolism data on mmt (Biopharmaceutical Research Inc., 2014a & 2014b). Therefore, because the monkey represents a better model for human health toxicity, the monkey NOAEC of 30 mg/m3 was used for the evaluation of repeat dose toxicity.

Justification for classification or non-classification

The key study for the repeat dose toxicity endpoint is the subchronic inhalation toxicity study on mice, rats, and monkeys. As discussed, the monkey NOAEC of 30 mg/m3 (0.03 mg/L vapor) was selected for use in the CSA for mmt for repeat dose toxicity (30 mg/m3 was the highest dose tested, with no effects were observed in the monkey). 

Substance classification is based on the dose at which effects are observed (i.e., LOAEC); as no effects were observed at the highest dose tested, it is not possible to assess the level at which toxicologically significant effects may occur in the monkey. Therefore, a repeat dose STOT classification based on available test data is not possible based on inconclusive evidence from the 90 day inhalation study on monkeys with a NOAEC of 0.03 mg/L vapor.