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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:
2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
the racemate is characterized by CAS-number 7696-12-0
Specific details on test material used for the study:
Batch No.: TM1027

Test animals

Species:
mouse
Strain:
other: Swiss albino (HsdOla : MF1 strain)
Details on species / strain selection:
Source: Toxicology department
Advinus Therapeutics Private Limited
Bangalore —560 058
India
Sex:
male/female
Details on test animals and environmental conditions:
Age at the start of acclimatisation: 11 weeks
Acclimatisation: Five days under experimental conditions after veterinary examination.
Grouping: Mice were assigned to 3 study groups by body weight stratification during acclimatisation.
Body weight range after grouping: Males : 27.69 — 37.10 g Females : 24.60 — 29.10 g.
Identification: At the start of acclimatisation, mice were identified by serial numbers and crystal violet body marking. After grouping, they were identified by turmeric body marking, cage cards and permanent accession numbers.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Carboxymethyl cellulose (sodium salt)
Details: 0.5% aqueous carboxymethyl cellulose (CMC) with Tween 80 (1 mL/L)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

TEST ITEM PREPARATION FOR ORAL GAVAGING: Just prior to the treatment, a precisely weighed aliquot of 4.0 g of the finely ground test item (weighed using an electronic balance) was suspended in 0.5% aqueous CMC with Tween 80 (1mI/L) and the final volume was made up to 20 ml to obtain the nominal concentration of 200 mg of the test item per ml of the suspension. The homogeneity of the test item suspension was maintained by constant stirring using a magnetic stirrer.

PREPARATION OF POSITIVE CONTROL FOR ORAL GAVAGING: Before each treatment, 40 mg of cyclophosphamide (monohydrate) was dissolved in 10 ml of distilled water in a clean glass beaker to obtain a concentration of 4 mg/ml of the solution.
Duration of treatment / exposure:
48 h
Frequency of treatment:
The test item, the positive control and the vehicle control were administered orally as gavage twice at 24 hour intervals to each group of animals.
Post exposure period:
The animals were sacrificed 23 to 24 hours after the second treatment.
Doses / concentrations
Dose / conc.:
2 000 mg/kg bw/day
No. of animals per sex per dose:
10 animals (5 males + 5 females)/group
3 groups have been tested: 1 test item concentration, 1 positive control and 1 vehicle control
Control animals:
yes, concurrent vehicle
Positive control(s):
The positive control group has been exposed to 40 mg/kg of cyclophosphamide (monohydrate).

Examinations

Tissues and cell types examined:
Erythrocytes from bone marrow
Details of tissue and slide preparation:
Terminal Sacrifice
The treated mice were sacrificed 23 - 24 hours after the second treatment for sampling of femur bone marrow cells. All mice sacrificed at term were subjected to gross pathological observations. The mice were killed by cervical dislocation and the femora from both sides were removed after clearing the musculature. Femur heads were trimmed to expose the marrow canals, the bone marrow was flushed with 0.9% sodium chloride and collected in a centrifuge tube.
Bone Marrow Smear Preparation
The cell suspensions were centrifuged at 2500 - 2750 rpm for 10 minutes and supernatants discarded. Approximately 10 microlitre of the cell suspension was spread evenly on a glass slide and air-dried. The slide was etched with the study number, mouse accession number, slide number and sex immediately thereafter. The smears were fixed in methanol for about 30 minutes. 4 to 5 slides were prepared for each animal.
Staining
Slides were stained by a combination of May-Gruenwald and Giemsa stain in succession. The slides were blow-dried, immersed in xylene and cover slips mounted with DPX. The slides were then coded after which blind evaluation was carried out.




Evaluation criteria:
A minimum of 2000 polychromatic erythrocytes (PCE) was scored from each animal for the incidence of PCE with micronuclei. The proportion of immature erythrocytes among total RBC (number of PCE divided by number of total erythrocytes) was determined for each animal by counting 295 to 556 erythrocytes per animal.
From these observations, the following were derived for each animal:
·       Total RBC/erythrocytes scored
·       No. of PCE differentiated
·       No. and percentage of PCE with micronuclei
·       Mean and SD of PCE with micronuclei
·       Ratio of PCE : total RBC
After completing the evaluation of all the slides, the slides were verified or cross-checked by another in-house cytogeneticist. This included verification of all the micronucleated PCE in the vehicle control and the treatment group and 10% of the micronucleated PCE in the positive control group. The micronucleated PCE to be verified were chosen using the random numbers generation method as per the in-house developed and validated randomisation program.
Statistics:
For intragroup comparison, the body weights on day 2 and at sacrifice were compared with the initial body weight, and the body weight at sacrifice was also compared with the body weight on day 2 by paired 't' test. (Snedecor and Cochran, 1980).
For intergroup comparison, the net body weight change was analysed by Bartlett's test for homogeneity of intragroup variances followed by ANOVA and Dunnett's test. (Sokal and Rohlf, 1981).
The data of the treatment group and the positive control group was compared with the vehicle control by Bartlett's test followed by ANOVA and Dunnett's test (Gomez and Gomez, 1984) for the percentage of PCE with micronuclei and the proportion of PCE among total RBC.
The above statistical analyses were carried out using in-house developed and validated software.
All analyses and comparisons were evaluated at 5% (p <0.05) level

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Vehicle Control Group
There were no incidences of micronucleated PCE in males in the vehicle control group. The incidences of micronucleated PCE in females and PCE : total RBC ratio in males, females and combined sex in the concurrent vehicle control group was within the historical data range.

Treatment Group
The incidences of micronucleated PCE and the PCE : total RBC ratio in the limit dose group in males, females and combined sex were comparable to the vehicle control values.

Positive Control Group
Cyclophosphamide (monohydrate) significantly increased the percentage of micronucleated PCE in combined sex and significantly altered (reduced) the PCE : total RBC ratio in males, females and combined sex. The percentage of micronucleated PCE and PCE: Total RBC ratio was within the positive control historical data range in males, females and combined sex.


Any other information on results incl. tables

CLINICAL SIGNS, MORTALITY AND NECROPSY FINDINGS

There were no clinical signs, mortality or necropsy findings in the tested dose groups in males, females and combined sex.

BODY WEIGHT

In the limit dose group, the intra group comparison of the body weights showed a statistically significant increase:

a.       In sacrifice body weight as compared to the initial body weight in combined sex.

b.       In sacrifice body weight as compared to day 2 body weight in females and combined sex.

The intergroup comparison of the net body weight change showed:

a.       A statistically significant increase in combined sex in the limit dose group.

b.       A decrease in body weights in females and combined sex in the vehicle control group.

As the treatment increased the body weights of mice as compared to the concurrent control group, it was inferred that treatment did not affect the body weights of mice.

In the positive control group, the intragroup comparison of body weights as compared to the initial body weights showed a statistically significant decrease:

a.       In day 2 body weights in females and combined sex

b.       In sacrifice body weights in combined sex.

The intergroup comparison of net body weight change showed a decrease in body weights in males, females and combined sex as compared to the vehicle control group

Applicant's summary and conclusion

Conclusions:
The test item tetramethrin was not mutagenic in this micronucleus test in Swiss albino mice at the tested limit dose of 2000 mg/kg under the testing conditions adopted.


Executive summary:

Tetramethrin was tested for its potential to induce cytogenetic damage in bone marrow cells of Swiss albino mice by a micronucleus test. The test item was administered twice at an interval of 24 hours by oral gavage to a group of Swiss albino mice at the limit dose of 2000 mg/kg body weight at the dosage volume of 10 ml/kg. A concurrent vehicle control group {0.5% Carboxy methyl cellulose with Tween 80 1 mI/L} and a concurrent positive control group {cyclophosphamide (monohydrate)} were also included. The mice were sacrificed 23 - 24 hours after the second treatment. From each animal, a minimum of 2000 polychromatic erythrocytes (PCE) were scored for the incidence of micronucleated PCE. The ratio of PCE : total RBC was determined by counting 295 - 556 RBC per animal.

The results are summarized as follows:

Tetramethrin at the tested limit dose of 2000 mg/kg body weight did not affect the body weights of mice and did not cause clinical signs, mortality or gross lesions.

There were no incidences of micronucleated PCE in males in the vehicle control group. The incidences of micronucleated PCE in females and PCE : total RBC ratio in males, females and combined sex in the concurrent vehicle control group was within the historical data range.

The incidences of micronucleated PCE and the PCE : total RBC ratio in the limit dose group in males, females and combined sex was comparable to the vehicle control values.

Cyclophosphamide (monohydrate) significantly increased the percentage of micronucleated PCE in combined sex and significantly altered (reduced) the PCE : total RBC ratio in males, females and combined sex. The percentage of micronucleated PCE and PCE: Total RBC ratio was within the positive control historical data range in males, females and combined sex.

This result indicated the effectiveness of the methodology adopted and that the test system used was sensitive to a known mutagen.

The test item tetramethrin was not mutagenic in this micronucleus test in Swiss albino mice at the tested limit dose of 2000 mg/kg body weight under the conditions adopted.