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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
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 Nov 1992 - 22 Jan 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1993

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Lucullus 235/02
- Physical state / appearance: dark brown liquid
- Composition of Lucullus 235/02 'batch 235/04': water content: 24.4%, dry mass: 75.6%, ash: 23.44%, pH: 7.97
total nitrogen: 4.5%, NH4-N: 50 ppm, K: 0.13%, Na: 7.55%, SO4: 0.97%, Chloride: 2.06%, phosphorus: 0.03%, Ca: 110 ppm
organic material (e.g. amino acids and sugar)
- Purity (of the dry extract): 65 - 70 %
- Lot/batch No.: 235/04
- Storage condition of test material: in the dark at room temperature (protected from light)

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Ltd., Margate, UK
- Age at study initiation: 36 - 43 days (males), 36 - 43 days (females)
- Weight at study initiation: 24 - 31 g (males), 21 - 25 g (females)
- Assigned to test groups randomly: yes
- Housing: no more than 3 animals of the same sex in polypropylene cages with wire mesh lids and solid floors containing wood shavings to a depth of approx. 1 cm.
- Diet: ad libitum, Special Diets Services Ltd., RM1. (E). SQC. pellets
- Water: tap water ad libitum
- Acclimation period: 7

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23
- Humidity (%): 60 - 65
- Air changes (per hr): 20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: purified water
- Concentration of test material in vehicle: 100 mg/mL
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Prepared and administered on 2 consecutive days
Dosing preparations were made by dissolving Lucullus 235/02 in purified water to give 100 mg/mL. Dilutions were made using purified water and the test chemical preparations used within 1.5 hours of formulation.


Duration of treatment / exposure:
24 and 48 hours (5 males and 5 females of both groups [vehicle, Lucullus 235/02 2000 mg/kg] at each time point)
(positive control: only 24 hours)
Test substance was prepared and administered on 2 consecutive days, positive control CPA as a single adminsitration
Frequency of treatment:
given as a single administration per day on two consecutive days
Post exposure period:
mice were killed after 24 or 48 hours (after administration of test chemical or vehicle)
Doses / concentrations
Remarks:
Doses / Concentrations:
2x 2000 mg/kg
Basis:
other: administered by gavage
No. of animals per sex per dose:
15 (includes an additional 5 mice to be used in the event of deaths among similarly dosed animals)
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (CPA, Sigma Chemical Co, Poole, UK)
- freshly dissolved in purified water at 4 mg/mL- administered at 20 mL/kg
- Route of administration: orally
- Doses / concentrations:80 mg/kg

Examinations

Tissues and cell types examined:
bone marrow, erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: range-finding study

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): Mice were killed after 24 hours after administration; slides and reserve slides sampled at 24 hours.

DETAILS OF SLIDE PREPARATION: Both femurs from each animal were exposed, removed, cleaned of adherent tissue and the ends removed from the shanks. Using a syringe and needle bone marrows were flushed with 1 mL FBS into apprpriately labelled centrifuge tubes. The tubes were centrifuged (1250 g, 2-3 minutes) and the serum was aspirated to leave one or two drops and the cell pellet. The pellet was mixed into this small volume of serum in each tube using a Pasteur pipette and from each tube one drop of suspension was placed on the end of each of 2 labelled slides. Analysis could be conducted "blind". Slides were allowed to air-dry and were fixed in methanol before staining with giemsa solution according to the modification of Gollapudi and Kamra, 1979.

METHOD OF ANALYSIS: Initially the relative proportions of PCE and NCE were determined until a total of at least 1000 cells (PCE plus NCE) had been analysed. Counting continued (but of PCE only) until at least 2000 PCE had been observed. All PCE containing micronuclei observed during these 2 phases of counting were recorded. The vernier coordinates of all cells containing micronuclei were recorded for test chemical and vehicle control animals.

OTHER:
Evaluation criteria:
The test chemical was to be considered as clearly positive in this assay if:
- a statistically significant increase in the frequency of micronucleated PCE occurred for at least one kill time
- the frequency of micronucleated PCE at sucha point exceeded the historical vehicle control range
- corroborating evidence was obtained, for example, increased but statistically insignificant frequencies of micronucleated PCE at the other kill time.
Statistics:
Chi square test

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 357 - 2000 mg/kg (x2)
- Clinical signs of toxicity in test animals: no effects



RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): Lucullus 235/02 did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of mice treated up to 2000 mg/kg (x2) a dose considered to a suitable maximum dose level for micronucleus tests.
- Ratio of PCE/NCE (for Micronucleus assay): Similar to vehicle controls at both sampling times, also similar to those seen in controls.

Any other information on results incl. tables

Table 1: Summary of group mean data for test chemical, vehicle and positive controls (Lucullus 235/02)

Treatment group (mg/kg x2)

Kill time (hours)

Sex

Mean ratio PCE/NCE

Group mean frequency of micronucleated PCE (per 1000)

per sex

per treatment group

Vehicle control

24

M

0.83

0.1

0.25

F

0.86

0.4

48

M

1.03

0.9

0.6

F

1.09

0.3

2000

24

M

0.82

0.5

0.4

F

0.89

0.3

48

M

1.00

0.5

0.65

F

1.02

0.8

CPA, 80 +

24

M

0.77

22.8

23.05

F

0.58

22.3

+ administered as a single dose

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative