Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Winklemann, 33178 Borchen
- Age at study initiation: Approximatley 6-7 weeks (males) and 8-10 weeks (females)
- Weight at study initiation: Approximately 160-200 g
- Housing: in fully airconditioned rooms in Makrolon cages (type 4) on soft wood granulate in groups of 5 animals
- Diet (e.g. ad libitum): Ssniff R/M-H (V1534)
- Water (e.g. ad libitum): tap water in plastic bottles, ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 C
- Humidity (%): 50 +/- 20 % (target)
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12 hours


IN-LIFE DATES: From: 13 Oct 1998 (males) To: 09 November 1998 (males)
IN-LIFE DATES: From: 14 Oct 1998 (females) To: 10 November 1998 (females)
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
other: unchanged (no vehicle)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Monitoring of the test atmosphere via IR analysis yielded for the following results:
Group 75 ppm 350 ppm 2000 ppm
Mean (ppm) 75 353 2033
Range (ppm) 55-88 330-381 1855-2116

Chemical analyses of the test atmosphere yielded the following results:
Group 75 ppm 350 ppm 2000 ppm
Mean (ppm) 79 335 2018
Range (ppm) 76.7-83.4 321.4-367.3 1829.9 - 2208.8
Duration of treatment / exposure:
28 days/ 6 hours
Frequency of treatment:
5 days a week
Dose / conc.:
0 ppm
Remarks:
nominal
Dose / conc.:
75 ppm
Remarks:
nominal
Dose / conc.:
350 ppm
Remarks:
nominal
Dose / conc.:
2 000 ppm
Remarks:
nominal
No. of animals per sex per dose:
10 male / 10 female
Control animals:
yes, concurrent no treatment
Details on study design:
Post-exposure period: 1 day
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: several times a day (once a day on weekend and holidays)
- Cage side observations checked in table were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: twice weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- 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

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: weekly
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes (Intraperitoneal injection of 67 mg Ketamine hydrochloride + 6.7 mg Xylazine / kg bw)
- Animals fasted: No
- How many animals: all animals
- Parameters checked in table yes were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination
- Animals fasted: No
- How many animals: all
- Parameters checked in table were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: few days before termination of treatment
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table in Section 5.6.4 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: weekly
- Dose groups that were examined: all
- Battery of functions tested: compound related signs of neurological disturbances
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes (see table)
Statistics:
Evaluation was performed by I/S Research and Preclinial Development, Hoechst Marion Roussel Deutschland GmbH with the aid of a program package for the evaluation of toxicological studies. The calculation methods used are referred to on the computer printouts.
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs (including neurological disturbances) attributable to the exposure methyl acetate were observed in the animals.
Mortality:
no mortality observed
Description (incidence):
No animal died during the course of the experiment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At the high concentration groups, the body weights were decreased in both sexes, particularly in males.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At the high concentration groups, food consumptions were decreased in both sexes (males>females) without significant differences in the mean relative food consumption /kg bw/d,
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
The food efficiency was similar in animals from all groups, therefore the reduction of body weights were considered to be related to the reduced food consumption.
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At the high concentration groups, the mean values of erythrocyte counts, hemoglobin and hematocrit were increased, while the total counts of leukocytes and lymphocytes were decreased for these groups.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Clinical chemistry examinations revealed a dose-related decrease of cholesterol levels gaining significance in both sexes at the high concentrations and in females at all dose groups.
Serum calcium concentrations were increased in both sexes at the high concentration.
The ALAT activities were slightly, but significantly increased in high concentration females (41 U/L vs. 34 U/L in controls). The increase of ALAT activity in rats is indicative for hepatocellular damage. In absence of any morphological lesion or any other corresponding change this was considered indicative for a minimal dysfunction of liver cells.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Females from the high concentration group showed significantly increases in urine volume and decreases in specific weight.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
The behavior and general health condition of the animals were observed several times daily (on weekends and public holidays once daily).
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Due to the effect on body weights several absolute organ weights were decreased and relative organ weights were increased in high concentration males.
Animals from the high concentrations groups had increased adrenal weights in both sexes and decreased thymus weights in females.
Slight significant changes of adrenal and thymus weights were also observed in females of the intermediate concentration group.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The toxicological significance of altered adrenal weight and reduced cholesterol levels was considered to be equivocal as no morphological abnormality of the adrenal was observed. It can be interpreted to indicate nonspecific toxic response e.g. due to stress.
However a specific response of the adrenal cortex cannot be excluded; data on the serum levels of steroid hormone concentrations were not generated. The red blood changes may indicate hemoconcentration due to treatment-related increased diuresis observed in high concentration females and/or reduced water consumption. This remains uncertain, because of missing data on the water consumption.
The increase of ALAT activity in rats is indicative for hepatocellular damage. In absence of any morphological lesion or any other corresponding change this was considered indicative for a minimal dysfunction of liver cells. The weight reduction of thymus and leucocytopenia/lymphopenia should be discussed to give hint on a possible immunosuppressive effect. This assumption seemed to be uncertain in view of the absence of morphological changes in the thymus or any other immune organ. Overall, there is a concern that diureses, minimal liver cell dysfunction, adrenal weight increase, and reduced serum cholesterol concentrations represented minimal adverse effects due to the methyl acetate treatment. Therefore the NOAEL for systemic effects was also derived to be 350 ppm (1.057 mg/l).
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At necropsy, no compound-related macroscopic findings were observed. Only sporadic findings could be observed in one animal of group 1, 2, and 4, each.
The main histopathological findings were topical irritations of the olfactory epithelium in the conchae nasales (degeneration and necrosis of epithelium).
Histopathologic examinations revealed slight to moderate degeneration and necrosis of the olfactory epithelium (at level 3 out of 4 sections) of mainly all males and females exposed to the high concentration of methyl acetate.
Any other treatment-related abnormality was observed in any other organs and in any other dose group. As degeneration of the olfactory mucosa occurred at 2000 ppm, the NOAEL for local effect was estimated to be 350 ppm (1.057 mg/l).
No changes attributable to the exposure of the test compound were detected in the animals from the other groups.
Histopathological findings: neoplastic:
not specified
Key result
Dose descriptor:
NOAEC
Effect level:
350 ppm
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
LOAEC
Effect level:
2 000 ppm
Sex:
male/female
Basis for effect level:
other: Impaired body weight gain, decreased food consumption and pathological changes in the olfactory epithelium.
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
2 000 ppm
System:
respiratory system: upper respiratory tract
Organ:
adrenal glands
blood
erythrocyte development
leucocyte development
thymus
other: degeneration and necrosis of the olfactory epithelium
Treatment related:
yes
Dose response relationship:
yes
Conclusions:
The subacute inhalational treatment of Sprague Dawley Rats with Methylacetat caused dose dependent alterations of the olfactory epithelium of the conchae nasales. The degeneration of the highly specified cells in the high dose (2000 ppm) revealed this organ as the target organ for Methylacetat. The epithelium of the mid dose (350 ppm) and low dose (75 ppm) animals is not affected.
Therefore, the No Obsered Adverse Effect Level (NOAEL) is 350 ppm methyl acetate for male and female Sprague-Dawley rats, equivalent to a concentration of 1057 mg/m3 air.
Executive summary:

Groups of 10 male and 10 female Sprague Dawley rats received methyl acetate by nose-only inhalation exposure at concentrations of 0, 75, 350 and 2000 ppm for a period of 28 days (6 hours per day, 5 days per week). One day after the last administration all animals were killed and necropsied.

Concentrations of the test compound in the respiratory aid were monitored several times daily via IR analysis. Additionally, chemical analyses were carried out at weekly intervals.

Behavior and state of health were observed daily in all groups. Body weights and food consumption were recorded twice weekly.

Hematological and clinical chemistry examinations were carried out at the termination of the study. Furthermore, urine analysis was performed at the termination of the study.

Blood levels of the test compound were determined at different times after cessation of the last exposure in order to characterize elimination kinetics.

During the necropsy the animals were examined for macroscopically visible abnormalities, the main organs weighed and the organ to body ratios calculated. many organs and tissues were processed for histopathological examination and checked for microscopically visible changes.

Body weights, hematological and clinical chemistry data, urine data, absolute and relative organ weights were analyzed with the aid of a statistical program to show differences compared with the controls.

Mean daily analytical concentrations of methyl acetate ranged from 55 -88 ppm (low concentration), 330 -381 ppm (intermediate concentration) and 1855 -2116 ppm (high concentration group) and thus were close to the target concentrations. Chemical analyses revealed mean concentrations of 79, 335, 2018 ppm, respectively.

No clinical signs attributable to the exposure methyl acetate were observed in the animals.

Toxicokinetic examinations revealed that the blood levels of methyl acetate were below the detection limit of 5 ppm immediately after the end of the exposure and thereafter.

Body weights were decreased in both sexes from the high concentration group, particularly in males. These groups showed also decreased food consumption. Body weights and food consumption remained unaffected by the exposure to the test compound in other groups.

Heamtological examinations revealed increases in erythrocyte counts, hemoglobin and hematocrit values in both sexes from the high concentration group. Leukocyte counts were decreased in these groups.

Clinical chemistry examinations revealed decreased cholesterol levels and increased calcium concentrations in both sexes from the high concentration group.

Due to the effect on the body weights several absolute organ weights were decreased and relative organ weights were increased in males from the high concentration group. Additionally, adrenal weights were increased in both sexes from the high dose group, thymus weights were decreased in females from the high concentration group. Slight changes in thymus and adrenal weights were still observed in females from the intermediate concentration group.

No compound-related macroscopic findings were observed at necropsy.

Histopathological examinations revealed degeneration and necrosis of the olfactory epithelium of mainly all male and female animals from the high concentration group. No changes attributable to the exposure of the test compound were detected in the animals from the other groups.

In conclusion, repeated exposure of Sprague Dawley rats to methyl acetate at the concentration of 2000 ppm cause impaired body weight gain, decreased food consumption and pathological changes of the olfactory epithelium. The toxicological relevance of the clinical chemistry findings remain unclear. The same applies to the hematological findings, for which an explanation could be hemoconcentration or oxygen deficit. Adrenal weights were increased in both sexes, possible reflecting stress of the animals caused by exposure to an irritant concentration of the test compound. Thymus weights were decreased in females. However, there were no changes observed by histopatholgical examination of adrenals and thymus.

At the concentration of 350 ppm, slight increases in adrenal weights and slight decreases in thymus weights were observed in females. As there were no histopathological changes in these organs even at the much higher concentration of 2000 ppm, these findings are considered not to be of toxicological relevance.

With regard to the present study, the No Obsered Adverse Effect Level (NOAEL) is 350 ppm methyl acetate for male and female Sprague-Dawley rats, equivalent to a concentration of 1057 mg/m3 air.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report date:
1999

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test) and EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Methyl acetate
EC Number:
201-185-2
EC Name:
Methyl acetate
Cas Number:
79-20-9
Molecular formula:
C3H6O2
IUPAC Name:
methyl acetate
Details on test material:
Formula C3H6O2
Molecular Weight 74.08 g/mol
Description: Clear, colorless liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Winklemann GmbH
- Age at study initiation: 6-7 weeks (males); 8-10 weeks (females)
- Weight at study initiation: 160-200 g
- Assigned to test groups randomly: yes
- Housing: makrolon cages type 4 (5 animals per cage) on soft wood granulate
- Diet: ad libitum, rat/mice small diet ssniff(r) R/M-H (V 1534)
- Water: ad libitum, tap water inplastic bottles
- Acclimation period: 5 days under study conditions


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/_ 3 C
- Humidity (%): 50 +/- 20%
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: October 13, 1998 To: December 15, 1998

Administration / exposure

Route of administration:
inhalation
Vehicle:
None
Details on exposure:
nose -only
Duration of treatment / exposure:
28 days, 20 exposures
Frequency of treatment:
6 hours/day 5 days a week
Post exposure period:
24 h
Doses / concentrationsopen allclose all
Dose / conc.:
0.227 mg/L air
Dose / conc.:
1.057 mg/L air
Dose / conc.:
6.04 mg/L air
No. of animals per sex per dose:
5 male / 5 female
Control animals:
yes, concurrent no treatment
Positive control(s):
Endoxan (r)
cyclophosphamide
C7H15Cl2N2P ~ H2O
50-18-0
ASTA Medica AG, Weismullerstr.45, D-60314 Frankfurt Germany
bathc Number: 603575B
Administered once orally by gavage at a dose of 25 mg/kg body weight 24 hours before killing.

Examinations

Tissues and cell types examined:
Erythrocytes from femora bone marrow
Details of tissue and slide preparation:
Animals of the negative and postive control groups were killed by dissection of the vena cava cranialis in deep narcosis and exsanguinated. the animals of the positive control group were killed by carbon dioxide asphyxiation. For each animal, about 6 ml fetal bovine serum was poured into a centrifuge tube. one femora was removed and the bones freed of muscle tissue. The proximal ends of the femora were opened and the bone marrow flushed into the centrifuge tube. A suspension was formed. the mixture was then centifuged for 5 minutes at approx. 1200 rpm, after which almost all of the supernatant was discarded. One drop of the thoroughly mixed sediment was smeared onto a cleaned slide, identified by project code and animal number and air-dried for about 12 hours. Staining was performed as follows:
-5 min in methanol
- 5 min in May-Grunwald's solution
- brief rinsing twice in distilled water
- 10 minutes staining in 1 part Giemsa solution to 6 parts buffer solution, pH 7.2
-rinsing in distilled water
-drying
-coating with Entellan(r)
Evaluation criteria:
1000 polychromatic and 1000 normochromatic erythrocytes were counted for each animal. The number of cells with the micronuclei was recorded, no the number of individual micronuclei. in addition, the ratio of polychromatic erythrocytes to 1000 normochromatic erythrocytes were determined. Main parameter for the the statistical anaylsis, i.e. validity assessment of the study and mutagenicity of the test substance, was the proportion of polychromatic erythrocytes with micronuclei out of the 1000 counted erythyrocytes. All bone marrow smears for evaluation were coded to ensure that the group from which they were taken remained unknown to the investigator.
The test substance is considered as positive if there is a significant dose-related increase in the number of micronucleated polychromatic erythrocytes compared with the concurrent negative control gruop. A test substance producing no significant dose -related increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in the test system.
Statistics:
A one-sided Wilcoxon-Test was evaluated to check the validity of the study. The study was considered as valid in case the proportion of polychromatic erythrocytes with micronuclei in the positive control was significantly higher than in the negative control (p=0.05).
Based on a monotone-dose-relationship one-sided Wilcoxon test were performed starting with the highest dose group. These tests were performed with a multiple level of significance of 5%. Tests on lower dose groups were only performed if all higher dose groups were significantly different from the control.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: systemic, probably secondary effects on a very low level.
Additional information on results:
Due to the rapid cleavage of the ester by hydrolases it was not possible to detect methyl acetat in the blood of rats (detection limit 5ppm)

Any other information on results incl. tables

Sex

Dose

Sample

Time

Number of Animals

Poly mean

Normo

mean

Erythrocytes

Eyrthrocytes with Micronuclei

Mean

P/N SD

Poly

mean

Mut.I

Normo

Mean

Mut. I.

No

%

SD

No

%

SD

Pooled

(-) Ctrl

24H

10

1000

1000

0.7

0.18

2.3

0.2

0.13

1.0

0.7

0.1

0.07

1.0

 

75

24H

10

1000

1000

0.6

0.18

1.8

0.2

0.1

1.8

0.2

0.1

0.04

0.3

 

350

24H

10

1000

1000

0.7

0.16

2.4

0.2

0.11

1.0

1.2

0.1

0.06

1.7

 

2000

24H

10

1000

1000

0.6

0.13

1.6

0.2

0.1

0.7

0.8

0.1

0.08

1.1

 

(+) ctrl

24H

10

1000

1000

0.7

0.16

17.9

1.8

0.45*

7.8

0.7

0.1

0.05

1.0

Sex

Dose

Sample

Time

Number of Animals

Poly mean

Normo

mean

Erythrocytes

Eyrthrocytes with Micronuclei

Mean

P/N SD

Poly

mean

Mut.I

Normo

Mean

Mut. I.

No

%

SD

No

%

SD

Male

(-) Ctrl

24H

5

1000

1000

0.5

0.13

2.2

0.2

0.15

1.0

0.8

0.1

0.08

1.0

 

75

24H

5

1000

1000

0.4

0.06

1.8

0.2

0.08

0.8

0.4

0.0

0.05

0.5

 

350

24H

5

1000

1000

0.6

0.11

1.8

0.2

0.08

0.8

1.4

0.1

0.05

1.8

 

2000

24H

5

1000

1000

0.6

0.11

1.6

0.2

0.05

0.7

1.2

0.1

0.08

1.5

 

(+) ctrl

24H

5

1000

1000

0.7

0.20

19.2

1.9

0.61

8.7

0.8

0.1

0.04

1.0

Female

(-) Ctrl

24H

5

1000

1000

0.8

0.09

2.4

0.2

0.11

1.0

0.6

0.1

0.05

1.0

 

75

24H

5

1000

1000

0.7

0.17

1.8

0.2

0.13

0.8

0.0

0.0

0.0

0.0

 

350

24H

5

1000

1000

0.8

0.16

3.0

0.3

0.10

1.3

1.0

0.1

0.07

1.7

 

2000

24H

5

1000

1000

0.7

0.13

1.6

0.2

0.13

0.7

0.4

0.0

0.05

0.7

 

(+) ctrl

24H

5

1000

1000

0.6

0.10

16.6

1.7

0.23

6.9

0.6

0.1

0.05

1.0

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The results lead to the conclusion that methyl acetate did not lead to a substantial increase of micronucleated polychromatic erythyrocytes and is not mutagenic in the micronucleus test.
Executive summary:

The micronucleus test was carried out with methyl acetate to evaluate potential for cytogenetic damage. The animals were administered with the test compound in a rat subacute inhalation toxicity study (20 exposures within 28 days, 6 hours/day, report no. 99.0011) at exposure concentrations of 75, 350 and 2000 ppm (226.5, 1057.0 and 6040.0 mg/m3). According to the test procedure the animals were killed 24 hours after the last administration.

Endoxan(r) (cyclophosphamide) was used as positive control substance and was administered once orally at a dose of 25 mg per kg body weight.

The number of polychromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic erythrocytes to normochromatic erythrocytes in both and female animals remained unaffected by the treatment with Methyl acetate adn was not less than 20% of the control value.

Endoxan(r) induced a marked statistically significant increase in the number of polychromatic erythrocytes to total erythrocytes was not changed to a significant extent.

Under the conditions of the present study the results indicate that Methyl acetate is not genotixic in the micronucleus test.