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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from peer reviewed publication

Data source

Reference
Reference Type:
publication
Title:
Collaborative work to evaluate toxicity on male reproductive organa by repeated dose studies two week and 4 week administration study of in rats Methyl Methansulfonate (MMS)
Author:
Shigenari Ozawa, Ryohei Yokoi, Tsuyoshi Kitamura, Kazuya Kuriyama, Kazuo Kobayashi and Nobuo Shibata
Year:
2000
Bibliographic source:
Journal of Toxicological Sciences, Vol. 25, special issue, 155 – 162, 2000

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: Refer below principle
Principles of method if other than guideline:
Subacute repeated dose oral toxicity study of Methyl Methansulfonate (MMS) was performed in Sprague – Dawley Rat.
GLP compliance:
not specified
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Methyl methanesulphonate
EC Number:
200-625-0
EC Name:
Methyl methanesulphonate
Cas Number:
66-27-3
Molecular formula:
C2H6O3S
IUPAC Name:
methyl methanesulphonate
Test material form:
other: Liquid
Details on test material:
- Name of test material: Methyl Methanesulphonate (MMS)
- Molecular formula: C2H6O3S
- Molecular weight: 110.1324 g/mol
- Substance type: Organic
- Physical state: Solid
- Purity: 99.9%
- Impurities (identity and concentrations): 0.01%
Specific details on test material used for the study:
- Name of test material: Methyl Methanesulphonate (MMS)
- Molecular formula: C2H6O3S
- Molecular weight: 110.1324 g/mol
- Substance type: Organic
- Physical state: Solid
- Purity: 99.9%
- Impurities (identity and concentrations): 0.01%

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
No data
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan, Inc. (Astugi, Japan)
- Age at study initiation: No data available
- Weight at study initiation: No data available
- Fasting period before study: No data available
- Housing: Animals were housed in barrier system.
- Diet (e.g. ad libitum): Laboratory animals diet ( gamma irradiated CE-2, CLEA Japan, Inc., Tokyo, Japan), ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: Yes, duration not mention.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.8 to 23.6˚C
- Humidity (%): 43.5 to 55.8%
- Air changes (per hr): 16.3 air changes / hour
- Photoperiod (hrs dark / hrs light): 12 hr light/dark cycle

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
No data
Vehicle:
water
Remarks:
Distilled water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Methyl Methansulfonate (MMS) was dissolved in distilled water to give a dose level of 0, 20 or 40 mg/Kg bw/day.

DIET PREPARATION
- Rate of preparation of diet (frequency): No data available
- Mixing appropriate amounts with (Type of food): No data available
- Storage temperature of food: No data available

VEHICLE
- Justification for use and choice of vehicle (if other than water): Distilled water
- Concentration in vehicle: 0, 20 or 40 mg/kg body weight /day
- Amount of vehicle (if gavage): 5 ml/kg body weight
- Lot/batch no. (if required): No data available
- Purity: No data available
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data available
Duration of treatment / exposure:
2 weeks
Frequency of treatment:
Once daily
Doses / concentrations
Remarks:
0, 20 or 40 mg/kg body weight /day


No. of animals per sex per dose:
Total: 30
0 mg/kg body weight /day: 10 male
20 mg/kg body weight /day: 10 male
40mg/kg body weight /day: 10 male
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were selected on the basis of results of the 4 week micronucleus study. In the preliminary study, a 4 week repeated administration of 30 mg/kg of MMS cause degeneration of the seminiferous tubules of the testis and sloughing of germ cells into the lumen in all rats. Because of overt toxicity 20 and 40 mg/kg dose were selected for present study.
- Rationale for animal assignment (if not random): No data available
- Rationale for selecting satellite groups: No data available
- Post-exposure recovery period in satellite groups: No data available
- Section schedule rationale (if not random): No data available
Positive control:
No data

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations checked in table [No.?] were included: Survival was observed.

DETAILED CLINICAL OBSERVATIONS: No data available
- Time schedule: No data available

BODY WEIGHT: Yes
- Time schedule for examinations: Once a week

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet 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 data

FOOD EFFICIENCY: No data available
- 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 available

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data available
- Time schedule for examinations: No data available

OPHTHALMOSCOPIC EXAMINATION: No data available
- Time schedule for examinations: No data available
- Dose groups that were examined: No data available

HAEMATOLOGY: No data available
- Time schedule for collection of blood: No data available
- Anaesthetic used for blood collection: No data available
- Animals fasted: No data available
- How many animals: No data available
- Parameters checked in table [No.?] were examined. No data available

CLINICAL CHEMISTRY: No data available
- Time schedule for collection of blood: No data available
- Animals fasted: No data available
- How many animals: No data available
- Parameters checked in table [No.?] were examined. No data available

URINALYSIS: No data available
- Time schedule for collection of urine: No data available
- Metabolism cages used for collection of urine: No data available
- Animals fasted: No data available
- Parameters checked in table [No.?] were examined. No data available

NEUROBEHAVIOURAL EXAMINATION: No data available
- Time schedule for examinations: No data available
- Dose groups that were examined: No data available
- Battery of functions tested: sensory activity / grip strength / motor activity / other: No data available

OTHER:
Organ weights were taken: Testes and epididymis were weighted
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, animals were sacrificed after inhalation with carbon dioxide and necropsied on the day of final administration. Main organ like brain, heart, lungs, liver, spleem and kidneys were fixed in 10 % buffered formalin and preserved. Testes and epididymis were weighed and fixed in formalin acetic acid for 5 days before final fixation in 10% buffered formalin. They were then embedded in paraffin, sectioned and stained with hematoxylin and eosin, periodic acid Schiff and examined.

HISTOPATHOLOGY: Yes
Brain, heart, lungs, liver, spleen, kidneys, testes and epididymis were examined microscopically
Other examinations:
Stags of spermatogenesis were examined. PAS stained sections of the left testes were used to identify the spermatogonic stages. For quatitative evaluation, 3 spermatogonic stages (II-III, VII and XII) were selected for stage analysis with 3 seminiferous tubules of each stage of each rats. The cell types were also recognized.
Statistics:
Statistical analysis was performed by using F- test for homogeneity of variance. With homogenous data student’s t- test were used. Where variance was not homogenous Aspin – Welch method was employed. Comparisons among 3 groups, homogeneity of variance was tested using Barlett’s method. With homogenous data one – way analysis of variance was used. Kruskal – Wallis procedures for non-parametric analysis were used. For significant inter-group difference Dunnett multiple comparison test or Dunnett’s rank test were applied. The level of statistical significance of difference was set at less than 0.05.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY:
Mortality: No effect on survival were observed in treated male rat as compared to control.
Clinical signs: No Clinical signs of toxicity were observed in treated male rat as compared to control.

BODY WEIGHT AND WEIGHT GAIN: When treated with 40 mg/kg body weight/day, significant decrease in body weight was observed from day 8 of treatment in treated male rat as compard to control.

No significant body weight change was observed in 20 mg/kg body weight/day treated male rat as compared to control.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No significant changes in food consumtion was observed in 20 mg/Kg bw/day dose group as compared to controls. There was a tendency of decrease on day 2 of treatment in the 40 mg/Kg group and significant differences between 40 mg/Kg bw/day and control group were observed from day 9 of treatment

FOOD EFFICIENCY: No data available

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data available

OPHTHALMOSCOPIC EXAMINATION: No data available

HAEMATOLOGY: No data available

CLINICAL CHEMISTRY: No data available

URINALYSIS: No data available

NEUROBEHAVIOUR: No data available

ORGAN WEIGHTS: When treated with 40 mg/kg body weight/day, significant decrease in absolute testes and epididymides weight was observed in treated male rat as compard to control. However, there was no significant differences in relative organ weight.

GROSS PATHOLOGY: Slight atrophy of testes was observed in 40 mg/kg body weight/day as compared to control and spermatic granuloma was observed in the right epididymis in one animal of control group. No other gross abnormalities were observed.

HISTOPATHOLOGY: NON-NEOPLASTIC: Slight atrophy of seminiferous tubules (1/10), decrease (8/10) and exfoliation (3/10) of germ cells, vacuolar degeneration of sertoli cells of testes in and moderate cell debris in caput, slight caudal epididymal ducts, decrease of permatids (elongated) and spermatic grnuloma of epididymis was observed in 40 mg/kg body weight/day dose group.

Atrophy of seminiferous tubules (1/10) and dilatation of the smeiniferous tubules (1/10), Slight vacuolar degeneration of sertoli cells of testes and cell debris in caput and caudal epididymal ducts of epididymis (4/10) was observed in 20 mg/kg body weight/day as compared to control.


HISTOPATHOLOGY: NEOPLASTIC (if applicable): No data available

HISTORICAL CONTROL DATA (if applicable): No data available

OTHER FINDINGS: Stags of spermatogenesis:
Significant decrease in spermatogonia was observed in stage XII seminiferous tubules of 40 mg/kg body weight/day dose group and significant decreased in pachytene spermatocytes at stage XII were observed in 20 mg/kg body weight/day as compared to control.

Effect levels

Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No effect on survival, body weight, food consumption, organ weight, gross pathology, histopathology and Stags of spermatogenesis.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table: Organ weight of male rats treated with MMS

Dose (mg/Kg bw/day)

No of animals

Final BW

Absolute organ weights

Relative organ weights

Testis

Epididymis

Testis

Epididymis

Control

10

389.0±31.2

3.31±0.24

0.93±0.13

8.59±1.13

2.39±0.33

20

10

376.7±30.7

3.32±0.19

0.82±0.04

8.86±0.80

2.18±0.22

40

10

332.5±27.5**

2.89±0.39

0.70±0.09**

8.76±1.47

2.12±0.26

 

Table: Histopathology of male rats treated with MMS

Organ

Administration period

2 weeks

 

Dose (mg/Kg)

0

20

40

 

Findings/ Grade

-

+

++

+++

-

+

++

+++

-

+

++

+++

Testis

Atrophy of seminiferous tubules

10

0

0

0

9

1

0

0

9

1

0

0

 

Dilatation of seminiferous tubules

10

0

0

0

9

1

0

0

10

0

0

0

 

Decrease of germ cells

10

0

0

0

10

0

0

0

2

8

0

0

 

Exfoliation of germ cells

10

0

0

0

10

0

0

0

7

3

0

0

 

Vacoular degeneration of sertoli cells

5

4

1

0

4

6

0

0

1

9

0

0

Epididymis

Cells debris in caput epididymal ducts

8

2

0

0

8

2

0

0

0

3

7

0

Cells debris in caudal epididymal ducts

9

1

0

0

8

2

0

0

4

6

0

0

Decrease of spermatids (elongated)

10

0

0

0

10

0

0

0

6

4

0

0

Spermatic granuloma

8

1

1

0

10

0

0

0

10

0

0

0

Applicant's summary and conclusion

Conclusions:
The No observed adverse effect level (NOAEL) was considered to be 20 mg/kg body weight /day when Sprague – Dawley male rats were exposed to Methyl Methansulfonate (MMS) for 2 weeks
Executive summary:

In Repeated dose subacute oral toxicity study, male Sprague – Dawley rats were treated with Methyl Methansulfonate (MMS) at a concentration of 0, 20 or 40 mg/kg body weight/day orally by gavage for 2 weeks. The animals were observed for clinical signs, mortality, body weight changes, food consumption, gross pathology and histopathology. No effects were observed on mortality, clinical sign, body weight and food consumption at 20 mg/kg body weight/day treated rat as compared to control. However, when treated with 40 mg/kg body weight/day, significant decrease in body weight was observed from day 8 of treatment in treated male rat as compared to control and there was a tendency of decrease on day 2 of treatment in the 40 mg/Kg group and significant differences between 40 mg/Kg bw/day and control group were observed from day 9 of treatment. When treated with 40 mg/kg body weight/day, significant decrease in absolute testes and epididymides weight was observed in treated male rat as compared to control. However, there were no significant differences in relative organ weight. Slight atrophy of testes was observed in 40 mg/kg body weight/day as compared to control and spermatic granuloma was observed in the right epididymis in one animal of control group. No other gross abnormalities were observed. Slight atrophy of seminiferous tubules (1/10), decrease (8/10) and exfoliation (3/10) of germ cells, vacuolar degeneration of sertoli cells of testes in and moderate cell debris in caput, slight caudal epididymal ducts, decrease of spermatids (elongated) and spermatic grnuloma of epididymis was observed in 40 mg/kg body weight/day dose group. Atrophy of seminiferous tubules (1/10) and dilatation of the smeiniferous tubules (1/10), Slight vacuolar degeneration of sertoli cells of testes and cell debris in caput and caudal epididymal ducts of epididymis (4/10) was observed in 20 mg/kg body weight/day as compared to control. Significant decreased in spermatogoni was observed in stage XII seminiferous tubules of 40 mg/kg body weight/day dose group. Significant decreased in pachytene spermatocytes at stage XII were observed in 20 mg/kg body weight/day as compared to control. Observation made suggest the No observed adverse effect level (NOAEL) was considered to be 20 mg/kg body weight /day when Sprague – Dawley male rats were exposed to Methyl Methansulfonate (MMS) for 2 weeks.

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