Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 22, 2001 - November 9, 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
mammalian germ cell cytogenetic assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2-(3-oxazolidinyl)ethyl methacrylate
EC Number:
256-260-2
EC Name:
2-(3-oxazolidinyl)ethyl methacrylate
Cas Number:
46235-93-2
Molecular formula:
C9H15NO3
IUPAC Name:
2-(1,3-oxazolidin-3-yl)ethyl 2-methylprop-2-enoate
Test material form:
liquid
Remarks:
yellow

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
Male and female CD-1 mice, approximately 8 weeks old (weighmg approximately 22-30 g) were used for this study. The animals were obtained from Charles River Laboratories (Kingston, N.Y.) and acclimatized in animal facilities at Rohm and Haas Co. for a 7 day quarantine period. Animals were ear tagged prior to the initiation of the study. Purina Certified Rodent Chow 5002 C and water filtered through a reverse osmosis system were available & libitum. The animals were housed in an environmentally controlled room with temperature, relative humidity, and light cycle set according to Rohm and Haas Toxicology Department Standard Operating Procedures (SOP) (section 10). Daily temperatures averaged 23 degrees C with relative humidity averages between 38-49%, which were within the acceptable range as defined in the SOP. Animals were assigned to treatment groups through the use of a computerized randomization procedure according to sex and body weight prior to administration of the test articles. Animals were within the acceptable weight range of plus or minus 20% from the mean. Animals were fasted for approximately 3 hours prior to dose administration.

Administration / exposure

Route of administration:
other: The test article and the negative control were administered by gavage, in a single oral dose. The positive control was administered in a single dose by intraperitoneal (i.p.) injection since this is the accepted route for this substance.
Vehicle:
distilled water
Frequency of treatment:
single dose:
The test article and the negative control were administered by gavage, in a single oral dose. The positive control was administered in a single dose by intraperitoneal (i.p.) injection since this is the accepted route for this substance.
Post exposure period:
Animals from test article and vehicle control groups were euthanized by cervical dislocation at approximately 24'and 48 hours after dosing. Animals from the positive control groups were euthanized 24 hours after dosing. Animals were observed for the presence of clinical signs during the treatment period and prior to sacrifice.
Doses / concentrationsopen allclose all
Dose / conc.:
820 mg/kg bw (total dose)
Dose / conc.:
410 mg/kg bw (total dose)
Dose / conc.:
82 mg/kg bw (total dose)
No. of animals per sex per dose:
For each treatment group and vehicle and positive control group, 5 male and 5 female animals were dosed per time point, with a volume of 10 ml/kg. In the high dose group, 4 additional animals per time point were dosed to account for the possibility of unexpected deaths.
Control animals:
yes, concurrent vehicle
Positive control(s):
The positive control substance was Mitomycin-C (MMC) (Aldrich Chemical Co., Lot No. 10587MI), TD No. 01-111, dissolved in distilled water.

Examinations

Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
Animals were prepared for micronucleus evaluation as follows: The groin area was wetted with 70% ethanol in water. Both femurs were removed by making incisions at the hip joint and below the knee cap, and the knee cap was removed. The bone marrow was flushed into a 15-ml centrifuge tube, containing approximately 1 ml of Fetal Bovine Serum (FBS) using a 1-cc syringe fitted with a 25-gauge needle. The tubes were centrifuged at 120 x g for 5 minutes, and the supernatant was removed, leaving approximately 0.1 ml above the cell pellet. The cell pellet was re-suspended in the remaining serum until a homogeneous suspension was observed. A small drop of the cell suspension (approximately 10 u1) was placed on the unfrosted end of a clean microscope slide and spread along the length of the slide. The slides were air dried for at least 1 hour, and then fixed in methanol for 15 minutes and allowed to dry. The slides were stained with Acridine Orange staining solution.

Scoring:
Slides from at least five animals per seddose group were observed when possible. Three slides were prepared per animal. Slides were coded and read blind in order to avoid bias on the part of the scorer. The slides were read using an epifluorescence microscope to illuminate the acridine orange stain (Hayashi et al., 1983).
Slides were scanned for regions of suitable technical quality, where the cells were well spread, undamaged and well stained. These regions were normally located in a zone close to the middle of the smear. Staining was tan to faint grey in normochromatic erythrocytes (NCE) and bright orange in polychromatic erythrocytes (PCE). Micronuclei appeared bright green against an orange background in PCE and generally were round, although almond and ring-shaped micronuclei occasionally occur. Micronuclei have sharp borders and were usually between 1/20 and 1/5 the size of the PCE. The end point to be scored was the number of cells containing micronuclei (not the number of micronuclei per cell).
For each animal, a total of at least 1000 erythrocytes (polychromatic, referred to as PCE 1 and normochromatic) were recorded to calculate the PCE/NCE ratio. For each animal, the remaining number of polychromatic erythrocytes were recorded to total at least 2000 (referred to as PCE 2) and were scored for the presence or absence of micronuclei. The frequency of micronucleated polychromatic erythrocytes (MN-PCE) and the PCE/NCE ratio were calculated on the basis of these data.
Cell counting was accomplished using the Xybion Path/Tox computer software system, G Module (GICELL program version 4.2.2) which captures data from the cell counter keyboard and provides an audit trail for quality assurance.
Evaluation criteria:
Evaluation Criteria:
The test article is considered positive in this assay if it elicits a dose-response or a statistically significant increase in the number of micronucleated cells over that of the concurrent vehicle control at one or more dose levels. In the event that the test article elicits a significant increase in the number of MN-PCE due to an unusually low number of MN-PCE in the concurrent vehicle control, the data from that dose may be compared to historical vehicle control data.
A test article is considered negative in this assay if No indication of a dose-response is observed and the treatment groups do not show a statistically significant increase in the number of MN-PCE when compared to the vehicle control.
The above criteria are to be used as a guideline for evaluating the assay results. The study director may take other appropriate factors into consideration for evaluating the test results.
Statistics:
Data were analyzed separately for male and female animals using a Statistical Analysis System (SAS), version 6.09 enhanced. An arcsine square root transformation was applied to the percent of micronucleated PCE's; all subsequent analyses for this parameter were conducted on transformed data. Initially, a three-way analysis of variance model was applied to the data to determine the significance of each main effect (sex, group, and day) and all two-way and three-way interaction effects. If significant interaction effects were identified, then the data were analyzed separately for each sex and/or day. Three independent single degree of freedom contrasts of the group means were used to test for trends in the group means and included an assessment of 1) an overall effect of OXEMA treatment relative to control, and 2) a linear dose-response trend among the OXEMA, and 3) a quadratic dose-response trend among the OXEMA. Additionally, pair-wise comparisons between each of the three OXEMA groups and the control group were made using Dunnett's t-test (Kirkland, 1989).

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Clinical Signs:
Clinical signs included scant feces, passiveness, ataxia, and unkempt. Death occurred in two of the female mice treated with 820 mg/kg of the test article. No clinical signs of toxicity were observed in any of mice treated with 410 or 82 mg/kg of the test article or control articles.

Micronucleus Evaluation:
The test article did not induce an increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow cells of male or female mice when compared to the vehicle control values. This was true for both the 24 and 48 hour time points. There was no statistically significant change in the polychromatic/normochromatic ratio at either 24 or 48 hours, which is indicative of the absence of cytotoxicity. An increase in the frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow cells of male and female mice treated with 2.0 mg/kg of the positive control, mitomycin-C. When compared to the vehicle controls, the increase was greater than two-fold, indicating that the assay was sufficiently sensitive to detect induced cytogenetic damage.

Any other information on results incl. tables

Table 1. Clinical Signs

Dose Group

No. of Animals Exhibiting Signs

Day 0

Day 1

Day 2

0 mg/kg – Distilled Water

Male

Normal

10/10

10/10

5/5

Female

Normal

10/10

10/10

5/5

82 mg/kg OXEMA

Male

Normal

10/10

10/10

5/5

Female

Normal

10/10

10/10

5/5

410 mg/kg OXEMA

Male

Normal

10/10

10/10

5/5

Female

Normal

10/10

10/10

5/5

820 mg/kg OXEMA

Male

Normal

16/18

9/18

4/9

 

Passive

2/18

2/18

1/9

 

Unkempt

1/10

1/18

1/9

 

Ataxia

0/18

1/18

0/9

 

Scant Feces

0/18

9/18

5/9

Female

Normal

16/18

7/18

4/9

 

Passive

2/18

2/18

0/9

 

Ataxia

1/18

2/18

0/9

 

Scant Feces

0/18

8/18

5/9

 

Found Dead

0/18

2/18

0/9

2.0 mg/kg Mitomycin-C

Male

Normal

5/5

5/5

--

Female

Normal

5/5

5/5

--

Table 2a. Mean Summary Data: Male Animals

Group (number)

Day

Dose (mg/kg)

NCE

(Mean±SD)

PCE1 (Mean±SD)

MNP (Mean±SD)

PCE2 (Mean±SD)

PCE Total (Mean±SD)

PNR Ratio (Mean±SD)

MNC % (Mean±SD)

Control (n=5)

1

0

512±114

514±124

3±1

1532±107

2046±28

1.08±0.42

0.14±0.06

OXEMA (n=5)

1

82

527±135

532±140

2±1

1538±134

2070±20

1.11±0.48

0.08±0.06

OXEMA (n=5)

1

410

498±160

561±152

2±2

1518±172

2079±22

1.29±0.66

0.10±0.07

OXEMA (n=9)

1

820

539±192

547±198

3±1

1547±212

2093±49

1.26±0.84

0.16±0.07

Control (n=5)

2

0

496±105

578±95

3±2

1524±77

2102±42

1.24±0.41

0.13±0.08

OXEMA (n=5)

2

82

499±121

596±126

1±1

1505±75

2100±79

1.29±0.51

0.06±0.05

OXEMA (n=5)

2

410

450±119

616±105

3±1

1475±108

2091±26

1.51±0.70

0.13±0.07

OXEMA (n=9)

2

820

569±153

530±138

2±2

1564±138

2094±39

1.05±0.52

0.09±0.08

Mitomycin-C (n=5)

1

2

593±247

568±178

99±16

1504±151

2072±41

1.22±0.86

4.78±0.81#

Table 2b. Mean Summary Data: Female Animals

Group (number)

Day

Dose (mg/kg)

NCE

(Mean±SD)

PCE1 (Mean±SD)

MNP (Mean±SD)

PCE2 (Mean±SD)

PCE Total (Mean±SD)

PNR Ratio (Mean±SD)

MNC % (Mean±SD)

Control (n=5)

1

0

485±99

607±109

3±2

1518±114

2125±73

1.33±0.49

0.14±0.09

OXEMA (n=5)

1

82

517±158

571±164

3±2

1519±169

2090±45

1.29±0.79

0.16±0.08

OXEMA (n=5)

1

410

480±106

611±92

3±1

1488±149

2099±66

1.36±0.50

0.16±0.04

OXEMA (n=9)

1

820

526±97

575±106

2±1

1516±89

2091±47

1.16±0.39

0.09±0.05

Control (n=5)

2

0

507±127

583±116

2±1

1502±142

2086±38

1.25±0.53

0.09±0.05

OXEMA (n=5)

2

82

486±108

601±103

2±1

1484±120

2085±64

1.33±0.52

0.09±0.06

OXEMA (n=5)

2

410

473±58

598±96

1±1

1507±46

2105±90

1.30±0.35

0.06±0.06

OXEMA (n=7)

2

820

585±177

481±174

3±2

1605±206

2087±68

0.99±0.64

0.13±0.07

Mitomycin-C (n=5)

1

2

723±148

490±128

108±14

1585±111

2076±43

0.72±0.30

5.20±0.62#

NCE=Normochromatic Erythrocytes

PCE=Polychromatic Erythrocytes

PCE 1=Polychromatic Erythrocytes used in combination with Normochromatic

Erythrocytes to total at least 1000 cells and used to calculate the PCE/NCE ratio

PCE 2=The remaining number of Polychromatic Erythrocytes recorded and added to

PCE1to total at least 2000 Polychromatic Erythrocytes

MNP=Micronucleated Polychromatic Erythrocytes

CALCULATIONS:

PCE TOTAL=PCE 1 +PCE2

PNR RATIO=PCE/NCE RATIO=PCE l/NCE

MNC%=MICRONUCLEATED POLYCHROMATIC ERYTHROCYTE PERCENT=

MNP/(PCE 1 +PCE2) X 100

Statistical Methods: Analysis of Variance Followed by Dunnett's T-Test on Least Square Means. For All

Statistical Methods:'*'Indicates a Statistically Significant Difference from Control (p<0.05).

'#'Indicates a Greater Than 2 Fold Increase Over Control Values.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, OXEMA was not mutagenic in the micronucleus assay in CD-1 mouse bone marrow cells.
Executive summary:

OXEMA was evaluated for its potential to induce chromosomal damage in vivo, as assessed by the micronucleus assay with mouse bone marrow cells. Adult CD-1 male and female mice (5 male and 5 female animals per group, except for the high dose group, which had 4 additional animals per time point) received a single oral dose of the test article at concentrations of 82, 410 or 820 mg/kg. Control animals received a single oral dose of distilled water (vehicle control), or an intraperitoneal injection of 2.0 mg/kg mitomycin-C (positive control) (MMC). Animals from test article and vehicle control groups were euthanized at 24 or 48 hours after treatment. Animals from the positive control group were euthanized 24 hours after treatment. Bone marrow slides were prepared and the frequency of micronucleated polychromatic erythrocytes was measured as an indicator of cytogenetic damage. For each animal, a total of at least 2000 polychromatic erythrocytes were scored for the presence or absence of micronuclei. In addition, the polychromatic erythrocyte/normochromatic erythrocyte (PCE/NCE) ratio was measured to evaluate the cytotoxicity of the test agent.

The test article did not induce an increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow cells of male or female mice when compared to the vehicle controls.

An increase in the frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow cells of male and female mice treated with 2.0 mg/kg of the positive control, MMC. When compared to the vehicle controls, the increase was greater than two-fold, indicating that the assay was sufficiently sensitive to detect induced cytogenetic damage.

Under the conditions of this study, OXEMA was not mutagenic in the micronucleus assay in CD-1 mouse bone marrow cells.