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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:
20 March to 26 June 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study performed equivalent or similar to OECD test guideline No. 474 without deviation.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspected on 22 January 1996/ signed on 27 February 1996)
Type of assay:
other: micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Remarks:
Powder
Details on test material:
- Description: White powder
- Storage condition of test material: 4 °C under nitrogen

Test animals

Species:
mouse
Strain:
CD-1
Details on species / strain selection:
The test system was chosen because the mouse has been shown to be a suitable model for this type of study and is recommended in the test method.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent.
- Age at study initiation: ca.5-7 weeks
- Weight at study initiation: Males: 22-30 g; females: 20-26 g
- Assigned to test groups randomly: Yes; after a minimum acclimatisation period of five days the animals were selected at random.
- Housing: Animals were housed in groups of five by sex in solid-floor polypropylene cages furnished with woodflakes
- Diet: Food (Rat and Mouse Expanded Diet No.1, Special Diets Services Limited, Witham, Essex, UK), ad libitum
- Water: Mains drinking water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20-23 °C
- Humidity: 50-51 %
- Air changes: Rate of air exchange was ca.15 changes per hour
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: 20 March to 26 June 1996

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Distilled water
- Concentration of test material in vehicle: 18.75, 37.5, 75, 150 and 175 mg/mL
- Amount of vehicle (gavage): 10 mL/kg bw
- Lot/batch no.: Steripak, 501041
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Test item formulation: For the purpose of this study the test item was freshly prepared as required as a solution at the appropriate concentrations in distilled water. Initially the test item was formulated as a suspension in arachis oil. However, it was apparent that poor homogeneity of the formulations was giving inconsistent results in the initial range-finding investigation. Following discussion with the sponsor the vehicle was changed to distilled water for the final range-finding study and main study.

DOSE VOLUME: 10 mL/kg bw
Duration of treatment / exposure:
Single treatment (One day)
Frequency of treatment:
Aall animals were dosed once only at the appropriate dose level by gavage.
Post exposure period:
24 and 48 h after administration of test item
Doses / concentrationsopen allclose all
Dose / conc.:
187.5 mg/kg bw/day (nominal)
Dose / conc.:
375 mg/kg bw/day (nominal)
Dose / conc.:
750 mg/kg bw/day (nominal)
Dose / conc.:
1 500 mg/kg bw/day (nominal)
Dose / conc.:
1 750 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positive control: Cyclophosphamide
- Route of administration: Oral (gavage)
- Concentration: 5 mg/mL
- Dose volume: 10 mL/kg bw
- Dose: 50 mg/kg bw
- For the purpose of this study the positive control material was freshly prepared as required as a solution at the appropriate concentration in distilled water.

Examinations

Tissues and cell types examined:
- The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored.
- Number of normochromatic erythrocytes (NCE) associated with 1000 erythrocytes were counted; the ratio of polychromatic to normochromatic erythrocytes was calculated as a measure of bone marrow toxicity.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Range-finding Toxicity Study:
- A range-finding toxicity study was performed to determine a suitable dose level for the micronucleus study. The dose level selected should ideally be the maximum tolerated dose level or that which produces some evidence of cytotoxicity up to a maximum recommended dose of 2000 mg/kg bw.
- Dose levels for range-finding toxicity study: 1500, 1750 and 2000 mg/kg bw
- Animals were observed one hour after dosing and subsequently once daily for up to two days. Any deaths and evidence of overt toxicity were recorded at each observation. Animals were killed 24 or 48 hours after dosing, femurs were removed and slides prepared for cytotoxicity evaluation.
- Based on the results of the range-finding study the vehicle to be used in the main study was distilled water which gave a homogenous solution of the test item. Though no premature deaths were observed at 2000 mg/kg bw using distilled water as the vehicle, it was decided following discussion with the sponsor that the toxic effects at 2000 mg/kg bw were excessive and to limit the maximum dose level to 1750 mg/kg bw. The other dose levels to be used in the main study were 187.5, 375, 750 and 1500 mg/kg bw.

TREATMENT AND SAMPLING TIMES
- All animals were dosed once only at the appropriate dose level by gavage using a metal cannula attached to a graduated syringe. The volume administered to each animal was calculated according to its bodyweight at the time of dosing.
- One group of mice from each dose level was killed by cervical dislocation 24 h following treatment and a second group from each dose level at 48 h. The vehicle controls were killed 24 or 48 h following dosing and positive control group animals were killed 24 h following dosing.

DETAILS OF SLIDE PREPARATION:
- Immediately following sacrifice (i.e., 24 or 48 h following dosing), both femurs were dissected from each animal, aspirated with foetal calf serum and bone marrow smears prepared following centrifugation and resuspension. The smears were air-dried, fixed in absolute methanol and stained in May-Grunwald/Giemsa.

METHOD OF ANALYSIS:
- Stained bone marrow smears were coded and examined blind using light microscopy at x1000 magnification.
- The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored. Micronuclei are normally circular in shape, although occasionally they may be oval or half-moon shaped, and have a sharp contour with even staining.
- In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes were counted; these cells were also scored for incidence of micronuclei.
- The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values.

OTHER:
- All animals were observed for signs of overt toxicity and death one hour after dosing and then once daily as applicable and immediately prior to sacrifice.
Evaluation criteria:
- A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test item groups and the number occurring in the corresponding vehicle control group.
- A positive mutagenic response was demonstrated when a statistically significant increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24 or 48 h kill times when compared to their corresponding control group.
- If these criteria were not demonstrated, then the test item 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 was shown to be statistically significantly lower than the concurrent vehicle control group.
Statistics:
All data were 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 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/female
Genotoxicity:
positive
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 1500, 1750 and 2000 mg/kg bw
- Solubility: Test item was dissolved in distilled water.
- Clinical signs of toxicity in test animals: There were no premature deaths in animals dosed with the test item formulated in distilled water. Clinical signs were observed in the 48 h dose group animals as follows: hunched posture, lethargy, ataxia, splayed gait, decreased respiratory rate and ptosis. Animals used to obtain 24 h PCE/NCE ratios exhibited the following clinical signs: hunched posture, lethargy, ataxia, decreased respiratory rate, laboured respiration, splayed gait and ptosis.
- Harvest times: 24 and 48 h
- Evidence of cytotoxicity in tissue analyzed: A summary of the mean PCE/NCE ratio values for the 24 and 48 h dose groups were presented in Table 7.6.2/1.
- Other: As in the arachis oil formulated test item range-finding study some animals were observed to have an elevated frequency of micronucleated polychromatic erythrocytes.

RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity in test animals: There were no premature deaths seen in any of the dose groups. Clinical signs were observed in animals dosed with the test item at ≥ 750 mg/kg bw in the 24 h groups and at ≥1500 mg/kg bw in the 48 h groups, these were as follows: lethargy, ataxia, ptosis and hunched posture.
- Induction of micronuclei (for Micronucleus assay): There were dose-related statistically significant increases in the frequency of micronucleated PCEs in the 24 h test item dose groups at ≥ 1500 mg/kg bw when compared to their concurrent vehicle control group. In both the 1500 and 1750 mg/kg bw 24 h groups there were several animals which had micronucleated polychromatic erythrocyte values similar to those normally seen in vehicle control animals. However, the majority of animals had values far greater than those seen in control animals. A possible hypothesis for the low values is that inhibition of erythropoiesis due to the toxicity of the test material to the bone marrow cells, delayed erythrocyte production and any accompanying micronucleated erythrocytes. This theory is supported by the cytotoxic response observed in the 24 h test item groups. There was no evidence of a statistically significant increase in the frequency of micronucleated PCEs in the 48 h test item dose groups. There were statistically significant decreases in the PCE/NCE ratio in the 24 h test item groups when compared to their concurrent vehicle control group. This would indicate that a cytotoxic response in the target tissue, bone marrow, had been achieved.
- Ratio of PCE/NCE (for Micronucleus assay): Table 7.6.2/2

Any other information on results incl. tables

Table 7.6.2/1: Range finding study

Dose group (mg/kg bw)

48 h PCE/NCE ratio

24 h PCE/NCE ratio

Mean

SD

Mean

SD

1500

1.53

0.42

1.40

0.22

1750

1.59

0.69

1.37

0.40

2000

1.85

0.69

1.68

1.20

 

Table 7.6.2/2: Micronucleus study

Dose group (mg/kg bw)

Sampling

Number of PCE with micronuclei per 2000 PCE

PCE/NCE ratio

Group Mean

SD

Group Mean

SD

Vehicle control

48 h

2.4

2.2

1.29

0.34

Vehicle control

24 h

2.6

1.7

1.44

0.51

Positive control

24 h

50.1***

28.7

1.22

0.50

Test item 1750 mg/kg bw

48 h

2.7

2.8

1.09

0.37

Test item 1500 mg/kg bw

2.8

1.9

1.47

0.43

Test item 750 mg/kg bw

3.5

4.8

1.62

0.92

Test item 375 mg/kg bw

3.2

1.3

1.36

0.48

Test item 187.5 mg/kg bw

3.4

3.0

1.46

0.42

Test item 1750 mg/kg bw

24 h

18.4**

15.4

0.79**

0.34

Test item 1500 mg/kg bw

12.2***

8.9

0.92*

0.30

Test item 750 mg/kg bw

3.0

3.0

0.85**

0.27

Test item 375 mg/kg bw

4.0

3.6

1.36

0.58

Test item 187.5 mg/kg bw

2.2

1.4

1.21

0.36

 

PCE-Polychromatic erythrocytes 

NCE-Normochromatic erythrocytes 

SD-standard deviation

*** -p < 0.001

**-P < 0.01

*-P < 0.05

Applicant's summary and conclusion

Conclusions:
The test item was found to produce a dose related significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice at dose levels greater than 750 mg/kg bw 24 hours after dosing. Therefore, test item was considered to be genotoxic under the conditions of the test.
Executive summary:

In an in vivo bone marrow micronucleus test, groups of CD-1 mice (5/sex/dose) were exposed to test item in distilled water at doses of 187.5, 375, 750, 1500 and 1750 mg/kg bw by gavage. Further groups of mice were given a single oral dose of distilled water or cyclophosphamide, to serve as vehicle and positive controls respectively. Animals were killed 24 or 48 hours later, the bone marrow extracted and smear preparations made and stained. Polychromatic and normochromatic erythrocytes were scored for the presence of micronuclei.

 

There was evidence of a dose-related increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test item in the 24 h harvest group when compared to the concurrent vehicle control group but no statistically significant increases in the incidence of micronucleated cells at 750 mg/kg bw and below. There was no evidence of a statistically significant increase in the frequency of micronucleated PCEs in the 48 h test item dose groups. Statistically significant decreases in the PCE/NCE ratio were observed in the 24 h 750, 1500 and 1750 mg/kg bw test item dose groups when compared to their concurrent control group. This confirms that systemic absorption was achieved and that a cytotoxic response was achieved in the target tissue, bone marrow, even at the 750 mg/kg bw dose level.

 

The positive control item produced a marked increase in the frequency of micronucleated polychromatic erythrocytes.

 

Test item was found to produce a dose related significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice at dose levels greater than 750 mg/kg bw 24 hours after dosing. The dose response exhibited marked evidence of a clear threshold between 750 and 1500 mg/kg bw. The test item was considered to be genotoxic (clastogenic) to somatic cells under the conditions of the test.