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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 Aug - 4 Dec 2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted Jul 2016
Deviations:
yes
Remarks:
2000 immature erythrocytes were counted per animal, should be 4000
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
other: Japanese METI/MHLW/MAFF guidelines for testing of new chemical substances
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: in vivo micronucleus test in the mouse

Test material

Constituent 1
Chemical structure
Reference substance name:
2-{2-chloro-4-(methylsulfonyl)-3-[(tetrahydrofuran-2-ylmethoxy)methyl]benzoyl}cyclohexane-1,3-dione
Cas Number:
473278-76-1
Molecular formula:
C20 H23 Cl O7 S
IUPAC Name:
2-{2-chloro-4-(methylsulfonyl)-3-[(tetrahydrofuran-2-ylmethoxy)methyl]benzoyl}cyclohexane-1,3-dione
Test material form:
solid: particulate/powder

Test animals

Species:
mouse
Strain:
other: Crl:CD-1(ICR)BR
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, UK
- Age at study initiation: 7 - 10 weeks
- Weight at study initiation: 22 - 30 g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: the animals were housed in groups of up to 7 in solid-floor polypropylene cages with wood-flake bedding
- Diet: Certified Rat and Mouse Diet (Code 5LF2, IPS Limited, London, UK), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: arachis oil
- Concentration of test material in vehicle: 25, 50, 100 mg/mL
- Lot/batch no. (if required): M112

Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
A suspension of test substance in arachis oil was freshly prepared as required..

Duration of treatment / exposure:
Not applicable
Frequency of treatment:
Single treatment
Post exposure period:
24 and 48 h after last treatment
Doses / concentrationsopen allclose all
Dose / conc.:
250 mg/kg bw (total dose)
Remarks:
24 h post-exposure period
Dose / conc.:
500 mg/kg bw (total dose)
Remarks:
24 h post-exposure period
Dose / conc.:
1 000 mg/kg bw (total dose)
Remarks:
24 and 48 h post-exposure period
No. of animals per sex per dose:
7 males
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide;
- Justification for choice of positive control(s): cyclophosphamide is known to produce micronuclei under the conditions of this test
- Route of administration: oral
- Doses / concentrations: 50 mg/kg bw (5 mg/mL), 24 h post-exposure period

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 toxicity test was performed to determine a suitable dose level and route of administration for the main study, and whether a differences in effects is observed between males and females. The dose level of the main study should be the maximum tolerated dose level. 1 male and 1 female per group were administered 1000, 1500 and 2000 mg/kg bw per oral, and 1000 mg/kg bw intraperitoneal. the animals were observed for mortality and evidence of overt toxicity 1 h after dosing and once daily for 2 days thereafter.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The animals were dosed once only with 250, 500 and 1000 mg/kg bw test substance via the intraperitoneal route. One group of mice from each dose level was sacrificed by cervical dislocation 24 h after test substance administration, and a second group administered 1000 mg/kg bw were sacrificed 48 h after administration.
Both femurs were aspirated with fetal calf serum and bone marrow slides were prepared following centrifugation and re-suspension of the cells.

DETAILS OF SLIDE PREPARATION:
The samples were air-dried, fixed in absolute methanol, stained in May-Gruenwald/Giemsa solution, allowed to air-dry and cover-slipped using mounting medium.

METHOD OF ANALYSIS:
The slides were coded and examined blindly using light microscopy at x1000 magnification. The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE) per animal was scored. The number of normochromatic erythrocytes (NCE) associated with 1000 erythrocytes was counted and scored for incidence of micronuclei.
Evaluation criteria:
A positive mutagenic response was demonstrated when a statistically significant, dose-responsive, toxicologically relevant increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24- or 48 h groups when compared to the corresponding control group. If these criteria were not fulfilled, then the test substance was considered to be non-genotoxic under the conditions of the test.. A positive response for bone marrow toxicity was demonstrated when the dose group mean polychromatic to normochromatic ratio shown to be significantly lower than the concurrent vehicle control group.
Statistics:
All data was statistically analysed using appropriate statistical methods as recommended by the UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part III (1989). The data was analysed following a √(x + 1) transformation using Student's t-test (two-tailed) and any significant results were confirmed using the one-way analysis of variance.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
clinical signs (hunched posture, ptosis, lethargy, ataxia) were observed in an unspecified number of animals in all treatment groups, at the 24 and 48 h post-exposure periods
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 1000, 1500 and 2000 mg/kg bw via the oral route; 1000 mg/kg bw via the intraperitoneal route
- Clinical signs of toxicity in test animals: the animals in the 1000 mg/kg bw intraperitoneal group showed hunched posture, ptosis, lethargy and ataxia. There was no difference between males and females. No clinical signs were observed in the animals exposed via the oral route. Therefore the animals in the main study were administered 250, 500 and 1000 mg/kg bw test substance via the intraperitoneal route.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): There were no significant increases in the frequency of micronuclei in any of the treatment groups, compared with their respective control groups (see Table 1 under 'Any other information on results incl. tables').
- Ratio of PCE/NCE (for Micronucleus assay): There were no significant decreases in the PCE/NCE ratio of the treatment groups compared with their respective control groups. A small, though non-significant, reduction in the PCE/NCE ratio was observed in the 48-h high-dose group compared with the control.
- Appropriateness of dose levels and route: Clinical signs (hunched posture, ptosis, lethargy, ataxia) were observed in an unspecified number of animals in all treatment groups, at the 24 and 48 h post-exposure periods. No data on clinical signs was given for individual animals. Based on the systemic effects observed, the dose levels are considered to be appropriate. In the range-finding study, clinical signs were observed in animals exposed via the intraperitoneal route, while no clinical signs were observed in the animals exposed via the oral route. This indicates that little or no absorption may have occurred via the oral route, and that the intraperitoneal route is more suitable exposure route.
- Other: the results of the vehicle control fell within the range of the historical vehicle control data (see Table 2-4 under 'Any other information on results incl. tables')

Any other information on results incl. tables

Table 1: Results of the in vivo micronucleus assay

 

Mean PCE /NCE ratio

at sampling time

Total micronuclei per 2000 PCEs at sampling time

Exp group

Number of animals

Dose [mg/kg bw]

24 h

48 h

24 h

48 h

Vehicle control (arachis oil)

7

-

0.73 ± 0.10

0.91 ± 0.25

2.1 ± 1.9

0.9 ± 0.9

Positive control (cyclophosphamide)

5

 50

0.98 ± 0.43

n.d.

55.6 ± 17.6**

n.d.

Test substance

7

250

0.79 ± 0.31

n.d.

1.7 ± 1.4

n.d.

Test substance

7

500

0.86 ± 0.28

n.d.

3.9 ± 2.5

n.d.

Test substance

7

1000

0.70 ± 0.23

0.70 ± 0.18

3.7 ± 2.9

2.4 ± 1.8

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

Table 2: Historical vehicle control data,

relative group frequency categories of micronuclei per 1000 PCEs, 48 h group (60 groups)

Frequency categories

Groups [number and %]

0.0 – 0.4

21 (35%)

0.5 – 0.9

18 (30%)

1.0 – 1.4

14 (23%)

1.5 – 2.0

7 (12%)

2.1 – 2.5

0 (0%)

 

Table 3: Historical vehicle control data,

relative group frequency categories of micronuclei per 1000 PCEs, 24 h group (60 groups)

Frequency categories

Groups [number and %]

0.0 – 0.4

15 (25%)

0.5 – 0.9

25 (42%)

1.0 – 1.4

14 (23%)

1.5 – 2.0

3 (5%)

2.1 – 2.5

3 (5%)

 

Table 4: Historical vehicle control data, relative group frequency categories of

micronuclei per 1000 PCEs, combined 24 and 48 h (120 groups)

Frequency categories

Groups [number and %]

0.0 – 0.4

36 (30%)

0.5 – 0.9

43 (36%)

1.0 – 1.4

28 (23%)

1.5 – 2.0

10 (8%)

2.1 – 2.5

3 (3%)

 

 

 

 

Applicant's summary and conclusion