Registration Dossier

Toxicological information

Genetic toxicity: in vivo

Currently viewing:

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 Jun - 2 Aug 1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
1000 immature erythrocytes/animal were scored for micronucleated immature erythrocytes which is fewer than the 4000 required, the highest dose level was more than four times higher than the recommended limit dose, the purity of the test substance was not specified.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994
Reference Type:
secondary source
Title:
Unnamed
Year:
2001

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted Sep 2014
Deviations:
yes
Remarks:
fewer than 4000 immature erythrocytes/animal were scored for micronucleated immature erythrocytes
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage, USA
- Age at study initiation: 49 days
- Weight at study initiation: 27 - 31 g (males), 20 - 26 g (females)
- Assigned to test groups randomly: yes, under following basis: according to weight
- Fasting period before study: no
- Housing: three (range-finding study) or five (main study) animals per cage, with hardwood chip bedding changed at least twice per week
- Diet: Purina certified rodent chow (Code No. 5002, lot No. Apr 13 94 1A74), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 24
- Humidity (%): 57 - 69
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: 0.5 mL of the test substance was miscible in 0.5 mL acetone and 0.5 mL corn oil. Corn oil was selected as the vehicle.
- Lot/batch no. (if required): D09B (CPC International Inc.)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was dissolved in the appropriate volume of corn oil just before dosing. The dosing volume was 10 mL/kg bw.
Duration of treatment / exposure:
not applicable
Frequency of treatment:
single treatment
Post exposure period:
24, 48 and 72 h
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
2.0, 5.0 and 10 mL/kg bw
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
1720, 4300 and 8600 mg/kg bw
Basis:
other: calculated based on a density of 860 mg/mL
No. of animals per sex per dose:
5/treatment period
Control animals:
yes, concurrent vehicle
Positive control(s):
triethylenemelamine
- Justification for choice of positive control(s): triethylenemelamine causes micronuclei
- Route of administration: intraperitoneal injection
- Doses / concentrations: 1.0 mg/kg bw

Examinations

Tissues and cell types examined:
Tissue: bone marrow
Cell type: bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: A range-finding study was performed to find the maximum tolerated dose. Dose groups, each comprising 3 males and 3 females, received a single dose of the test substance. The animals were observed for 3 days, during which mortality and physical condition were recorded daily and body wegiht was recorded on Day 1 prior to administration and on the day of sacrifice.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The animals were dosed once and sacrificed by cervical dislocation at 24, 48 or 72 h (test substance and vehicle control groups) or 24 h only (positive control group).

DETAILS OF SLIDE PREPARATION:
The femur bone was removed, and the bone marrow canal exposed and flushed with a small volume of foetal calf serum. The cell suspension was collected and centrifuged at 800 rpm for 5 min. The supernatant was removed, leaving approximately 0.1 mL above the pellet. The cells in the sediment were resuspended by flicking the tube until a homogenous suspension was observed. A drop of the cell suspension was placed on the end of a precleaned slide and the drop was spread along the length of the slide. The preparations were then air-dried, fixed for 15 min in methanol and air-dried again. The slides were stained using Wright-Giemsa stain for 3 minutes, rinsed in distilled water and allowed to air-dry. The dry slides were mounted in permount with a coverslip, the backsides were cleaned with methanol.

METHOD OF ANALYSIS:
The number of micronucleated polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE) was determined in 1000 erythrocytes.
The number of micronuleated PCE per 1000 PCE were determined (4000 should be counted according to the relevant guideline)
Evaluation criteria:
The criteria for a valid test were: (1) in the vehicle control, the average number of MPCE per 1000 PCE should not exceed 5; (2) in the positive control, the increase in the average number of MPCE per 1000 PCE over the average number of MPCE for the vehicle control should be statistically significant; (3) at least three animals from each sex must be alive at the time of sacrifice for each dose level.
The test substance was considered to have caused a positive response in this assay if: (1) the test article shows a positive dose-response trend and a statistically significant increase over that of the concurrent vehicle control in the number of MPCE at one or more dose levels. In the event that the test substance caused a statistically significant increase in the number of MPCE due to an unusually low number of MPCE (less than 0.05%) in the concurrent vehicle control, the data from that dose may be compared to historical vehicle control data; (2) in the event there is no positive dose-response trend, at least 2 consecutive test doses show a statistically significant increase in the number of MPCE.
The test substance was considered to have caused a negative response if no indication of a positive dose-response trend was observed and none of the test doses had a statistically significant increase in the number of MPCE when compared to the vehicle control.
Statistics:
Data were analysed separately for male and female animals. Unless otherwise indicated, the frequency of micronucleated PCE in each dose group was compared to that in the respective vehicle control using a 2-tailed Student’s t-test. Results were considered significant if the p-value is ≤ 0.05.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 0.01, 0.05, 0.1, 0.5, 1.0, 2.0 and 10 mL/kg bw
- Solubility: 0.5 mL of the test substance was only miscible in 0.5 mL of acetone and corn oil, showing a minimum miscible concentration of 1 mL/mL for each vehicle.
- Clinical signs of toxicity in test animals: one male administered 0.1 mL/kg bw died by accident on Day 1 due to being squeezed between the wires on top of the cage. No clinical signs were observed during the study period. the body weight increase was as expected for this species and strain under the current study conditions.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes from the bone marrow of test substance treated animals (see Table 1 and 2 under 'Any other results incl. tables). The positive control substance (cyclophosphamide) induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes in both sexes, showing that the positive control was valid.
- Ratio of PCE/NCE (for Micronucleus assay): The animals of the groups that were treated with the test substance did not show a decrease in the ratio of polychromatic to normochromatic erythrocytes compared with the vehicle controls. The groups that were treated with cyclophosphamide showed a decrease in the ratio of polychromatic to normochromatic erythrocytes compared with the vehicle controls.

Any other information on results incl. tables

Table 1: Results of the in vivo micronucleus assay in male animals

 

Mean

PCEs / 1000 NCEs

at sampling time

Total micronuclei

per 1000

PCEs at

sampling time

Exposure group

Number

of animals

Dose [mL/kg]

24 h

48 h

72 h

24 h

48 h

72 h

Vehicle control

(corn oil)

5

10

1.38

1.36

1.71

0.6

0.4

0.4

Positive control

(triethylenemelamine)

5

1.0 mg/kg bw

0.38

n.d.

n.d.

58.8*

n.d.

n.d.

Test substance

5

2.0

1.79

1.51

1.46

0.6

0.0

0.6

Test substance

5

5.0

1.30

1.56

1.88

0.4

0.4

0.4

Test substance

5

10

1.22

1.26

1.55

0.8

0.4

0.4

n.d. = not determined; *statistically significant (p<0.001);

 

 

Table 2: Results of the in vivo micronucleus assay in female animals

 

Mean

PCEs / 1000 NCEs

at sampling time

Total micronuclei

per 1000

PCEs at

sampling time

Exposure group

Number

of animals

Dose [mL/kg]

24 h

48 h

72 h

24 h

48 h

72 h

Vehicle control

(corn oil)

5

10

1.37

1.84

1.71

0.6

0.8

0.4

Positive control

(triethylenemelamine)

5

1.0 mg/kg bw

0.43

n.d.

n.d.

65.2*

n.d.

n.d.

Test substance

5

2.0

1.44

1.70

1.46

0.2

0.6

0.6

Test substance

5

5.0

1.03

1.38

1.88

0.4

1.0

0.4

Test substance

5

10

1.20

1.37

1.55

1.0

0.4

0.4

n.d. = not determined; * statistically significant (p<0.001);

Applicant's summary and conclusion

Conclusions:
CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.