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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:
17 February 1989 - 21 March 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read across to similar substance. Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Justification for type of information:
See read-across justification in Section 13.
Cross-reference
Reason / purpose:
other: Target substance
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted on read across material
Justification for type of information:
See read-across justification in Section 13.
Reason / purpose:
read-across source
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not examined
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: in vivo mammalian cell micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Description: White powder

Test animals

Species:
mouse
Strain:
other: Albino BKW
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 5 – 8 weeks.
- Weight at study initiation: Males 22 – 28g; Females 20 – 26g
- Housing: The animals were housed in groups of up to five by sex in solid-floor polypropylene cages with sawdust bedding.
- Diet (e.g. ad libitum): ad libitum with the exception of a 3-4 hour fast immediately before dosing and for approximately two hours after dosing; free access to food (Rat and Mouse Expanded Diet No. 1, Special Diet Services Limited, Witham, Essex, U.K.) was allowed throughout the study.
- Water (e.g. ad libitum): ad libitum with the exception of a 3-4 hour fast immediately before dosing and for approximately two hours after dosing; free access to mains drinking water was allowed throughout the study.
- Acclimation period: minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 – 21 °C
- Humidity (%): 52 – 60% relative
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours darkness.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Distilled water
Details on exposure:
A confirmatory range-finding study was performed to determine a suitable dose level for the micronucleus study. The dose level selected was a maximum practical dose of 5000 mg/kg.
The study was performed using one dose level (the maximum practical dose of 5000 mg/kg) at three kill times of 24, 48 and 72 hours after dosing.
Duration of treatment / exposure:
One dose administered by gavage.
Frequency of treatment:
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 fasted bodyweight at the time of dosing.
Post exposure period:
Animals were observed 1 and 4 hours after dosing and subsequently once daily for 3 days.
Doses / concentrations
Remarks:
Doses / Concentrations:
5000 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
Three groups, each of ten mice (five males and five females) were dosed once only with test material at the "maximum practical dose level".
Control animals:
yes
Positive control(s):
Four further groups of ten mice (five males and five females) were included in the study; three groups were treated with the vehicle alone (distilled water) and the fourth group was treated with cyclophosphamide, a positive control material.

Examinations

Details of tissue and slide preparation:
PROCEDURE
i) Range-finding Toxicity Study
A confirmatory range-finding 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 practical dose of 5000 mg/kg.
Groups of mice were dosed as follows:
DOSE LEVEL CONCENTRATION DOSE VOLUME NUMBER OF MICE
mg/kg mg/mL mL/kg MALE FEMALE

5000 500 10 7 7

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 fasted bodyweight at the time of dosing.

Animals were observed 1 and 4 hours after dosing and subsequently once daily for 3 days. Deaths and evidence of overt toxicity were recorded at each observation. No necropsies were performed. At 72 hours after dosing, up to 2 mice of each sex were sampled for bone-marrow cells to evaluate any specific bone-marrow toxicity in the absence of deaths.
ii) Micronucleus Study
The study was performed using one dose level (the maximum practical dose) at three kill times of 24, 48 and 72 hours after dosing.
Three groups, each of ten mice (five males and five females) were dosed once only with test material at the "maximum practical dose level". One group of mice was killed by cervical dislocation 24 hours following treatment, a second at 48 hours and a third at 72 hours. In addition, four further groups of ten mice (five males and five females) were included in the study; three groups were treated with the vehicle alone (distilled water) and the fourth group was treated with cyclophosphamide, a positive control material known to produce micronuclei under the conditions of the test. The vehicle control groups were killed 24, 48 and 72 hours following treatment and positive control group animals were killed 24 hours following treatment.
All animals were observed for signs of overt toxicity and death 1 and 4 hours after dosing and then once daily as applicable.
iii) Slide Preparation
Immediately following sacrifice (i.e. 24, 48 or 72 hours following dosing), one femur was dissected from each animal, aspirated with foetal calf serum and bone marrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol, and stained in May-Grünwald/Giemsa.
iv) Evaluation of Slides
Stained bone marrow smears were examined at random using light microscopy at x 1000 magnification. The incidence of micronucleated cells per 1000 polychromatic erythrocytes PCE (blue stained immature cells) per animal was scored. In addition, the number of normochromatic erythrocytes NCE (pink stained mature cells) associated with 1000 polychromatic erythrocytes were counted; these cells were also scored for incidence of micronuclei.
The ratio of normochromatic to polychromatic erythrocytes was calculated together with appropriate group mean values for males and females separately and combined.
Evaluation criteria:
Interpretation of Results
A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the three test material groups and the number occurring in the corresponding vehicle control groups.
A positive mutagenic response is demonstrated when a statistically significant increase in the number of micronucleated polychromatic erythrocytes is observed for either the 24, 48 or 72 hour kill times.
If the above criteria are not demonstrated, then the test material is considered to be non-mutagenic under the conditions of the test.
A positive response for bone marrow toxicity is demonstrated when the treatment group mean normochromatic to polychromatic ratio is twice the vehicle control value or when a treatment related increase is shown to be statistically significant.
Statistics:
If necessary, and where possible, all data were statistically analysed using the Kruskal-Wallis one-way analysis of variance by ranks (Kruskal W.H. and Wallis W.A. 1952 J. Am. Statist. Soc. 47 583).

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not examined
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING TOXICITY STUDY
The mortality data are summarised as follows:

Dose Level Sex Number of Deaths on day TOTAL DEATHS
mg/kg animals treated 0 1 2 3

5000 Male 7 0 0 0 0 0/14
Female 7 0 0 0 0

No adverse clinical symptoms were observed in any of the animals treated with Substance 1658/5 and there were no premature mortalities.
MICRONUCLEUS STUDY
i) Mortality Data and Clinical Observations
No adverse clinical symptoms were observed in any of the animals treated with Substance 1658/5 in the micronucleus study and there were no premature mortalities.
ii) Evaluation of Bone Marrow Slides
A summary of the results of the micronucleus study is given in Table 1.
There were no significant increases in the frequency of micronucleated PCE's in any of the groups of animals treated with Substance 1658/5 when compared to the corresponding vehicle control group. Furthermore, there was no increase in the frequency of micronucleated NCE's or in the NCE/PCE ratio after treatment with Substance 1658/5.

The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the known mutagenic activity of cyclophosphamide under the conditions of the test.

The test material, Substance 1658/5, was found not to produce micronuclei in polychromatic erythrocytes of mice under the conditions of the test.

Any other information on results incl. tables

Summary of Group Mean Data (Males and Females Combined)

Table 1

Treatment group

Number of PCE with Micronuclei per 1000 PCE

Number of NCE with Micronuclei per 1000 PCE

NCE PCE Ratio

Group Mean

SD

Group Mean

SD

Group Mean

SD

Vehicle Control

72 hour sampling time

0.8

0.9

0.4

0.6

1.00

1.07

Vehicle Control

48 hour sampling time

0.6

0.7

0.3

1.0

0.59

0.30

Vehicle Control

24 hour sampling time

0.9

1.3

0.4

0.7

0.79

0.22

PositiveControl

24 hour sampling time

41.5

9.1

0.7

0.5

1.22

0.36

Substance 1658/5 5000mg/kg

72 hour sampling time

0.9

1.1

0.3

1.1

0.92

0.28

Substance 1658/5 5000mg/kg

48 hour sampling time

1.1

1.3

0.2

0.5

0.81

0.37

Substance 1658/5 5000mg/kg

24 hour sampling time

0.7

0.8

0.3

0.7

0.75

0.17

PCE = polychromatic erythrocytes

NCE = normochromatic erythrocytes

SD = standard deviation

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test material was considered to be non-mutagenic under the conditions of the study.
Executive summary:

The test material was considered to be non-mutagenic under the conditions of the study. The test followed the OECD Guidelines for Testing of Chemicals No. 474 "Genetic Toxicology: Micronucleus Test" and Annex V method B12 of EEC Commission Directive 84/449/EEC.