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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:
From 10 September 1992 To 15 October 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

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

Materials and methods

Test guideline
Qualifier:
according 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)
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): ITC 288 (reaction mixture consisting mainly of the following components: n=1= phosphonoethane-1,2-dicarboxylic acid tetrasodium salt and n = 2= 1-phosphonobutane-1, 2, 3, 4-tetracarboxylic acid hexasodium salt)
- Physical state: white powder
- Storage condition of test material: room temperature under silica gel

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd., Manston, Kent.
- Age at study initiation: 5-8 weeks old
- Weight at study initiation: 23-28 g (males), 20-26 g (females)
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: up to 5 per cage par sex
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-21
- Humidity (%): 52-55
- Air changes (per hr): 15
- Photoperiod : 12 hrs dark / 12 hrs light


IN-LIFE DATES: From: 10 September 1992 To: 15 October 1992

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: sterile distilled water
- Concentration of test material in vehicle: 100 mg/ml

Details on exposure:
Groups of mice were given i.p. dose of test material at 1000 mg/kg. Animals were killed 24, 48 or 72 hours later, the bone marrow extracted and smear preparations made and stained.
Duration of treatment / exposure:
not applicable (following single i.p.; 24, 48 and 72 hours observation period before sacrifice).
Frequency of treatment:
one single i.p. dose
Post exposure period:
24, 48 or 72 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
1000 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: i.p.
- Doses / concentrations: 50 mg/kg

Examinations

Tissues and cell types examined:
Erythrocytes from the bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A range-finding study was performed to determine a suitable dose level and route of administration for the micronucleus study.


TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Three groups, each of ten mice (five males and five females) were dosed once only with test material at the maximum tolerated dose (1000 mg/kg). 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 dosed with the vehicle alone and the fourth group was dosed 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 dosing.


DETAILS OF 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.


METHOD OF ANALYSIS:
Stained bone marrow smears were coded and examined blind 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. 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 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 comnined.
Evaluation criteria:
A comparison was made between the number of micronucleated polychromatic erythrocytes occuring in each of the three test material groups and the number occuring 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 when compared to each of the three vehicle control groups.
If these criteria are not demonstrated, then the test material is considered to be non-genotoxic under the conditions of the test.
A positive response for bone marrow toxicity is demonstrated when the dose group mean normochromatic to polychromatic ratio is shown to be statistically significantly different from the concurrent vehicle control group.
Statistics:
The data were analysed using Student's t-test (two-tailed) and any significant results were confirmed using the one-way analysis of variation.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
At 1000 mg/kg, clinical signs were observed: hunched posture, lethargy, decreased respiratory rate, ptosis and ataxia. The same signs were observed at 2000 mg/kg + deaths (preliminary study).
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING TOXICITY STUDY
- Dose range: 3000 and 5000 mg/kg (oral); 500, 1000, 2000, 3000 and 5000 mg/kg (i.p.)
- Solubility: no data
- Clinical signs of toxicity in test animals: No clinical signs were observed in animals dosed orally with ITC 288 and there were no premature deaths. In animals dosed with ITC 288 via the intra-peritoneal route the following clinical signs were observed; hunched posture, lethargy, decreased respiratory rate, ptosis and ataxia, and premature deaths were seen at and above 2000 mg/kg. With no clinical signs observed at 500 mg/kg (i.p.), and clinical signs but no premature deaths with ITC 288 at 1000 mg/kg, the dose level selcted as the maximum tolerated dose for use in the micronucleus study was 1000 mg/kg via the intraperitoneal route.


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): There was no significant increase in the frequency of micronucleated PCE's in any of the test material dose groups when compared to their concurrent vehicle control groups. There was no significant increase in the frequency of micronucleated NCE's in any of the test material dose groups when compared to their concurrent vehicle control groups
- Ratio of PCE/NCE (for Micronucleus assay): There was no significant change in the NCE/PCE ratio in any of the test material dose groups when compared to their concurrent vehicle control group.

Any other information on results incl. tables

Table 7.6.2/3: Results of in vivo micronucleus test with ITC 288/S

 

Control

ITC 288/S (1000 mg/kg)

Control

ITC 288/S (1000 mg/kg)

Control

ITC 288/S (1000 mg/kg)

Number of cells evaluated

2000

2000

2000

2000

2000

2000

Sampling time (h)

 

24 h

 

48 h

72h

Number of erythro-cytes

normo­chromatic

1000

1000

1000

1000

1000

1000

poly­chromatic

1000

1000

 1000

1000

1000

1000

polychromatic with micronuclei

 0.9+/-1.

 0.6+/-0.8

 0.3 +/-0.7

0.4+/-0.5

0.2 +/-0.4

 0.1+/-0.3

Ratio of erythro­cytes

Normochromatic with micronuclei

0.3+/-0.6

0.4+/-0.8

 0+/-0

0.6+/-1.2

0.1+/-0.4

0.1+/-0.4

polychromatic erythrocytes/Normochromatic erythrocytes (PCE/NCE)

1.5 +/- 0.15

1.20+/-0.30

 1.3 +/-0.33

1.3 +/-0.33

1.5 +/-0.19

1.20+/-0.14

 

- Number of PCE with Micronuclei per 1000 PCE (positive control 24 hour sampling time): 30.6 +/- 11.4

- PCE/NCE Ratio (positive control 24 -hour sampling time): 0.9 +/- 0.34

- Number of NCE with Micronuclei per 1000 NCE (positive control 24 hour sampling time): 1.0 +/- 1.4

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of this test, ITC 288 was considered to be non-genotoxic.
Executive summary:

In a micronucleus test (Durward R, 1993), groups of ten mice (five males and five females) were given a single i.p. dose of ITC 288 at 1000 mg/kg. Animals were killed 24, 48, or 72 hours later, the bone marrow extracted and smear preparations made and stained. Polychromatic and normochromatic erythrocytes were scored for the presence of micronuclei. Further groups of mice were dosed with distilled water or cyclophosphamide, to serve as vehicle and positive control respectively. There was no evidence of an increase in the incidence of micronucleated polychromatic or normochromatic erythrocytes (NCE or PCE) in animals dosed with ITC 288 when compared to the concurrent vehicle control groups. No significant change in the NCE/PCE ratio was observed after dosing with ITC 288.The positive control material induced the appropriate responses. Under the conditions of this test, ITC 288 was considered to be non-genotoxic.

This study is classified as acceptable. This study satisfies the requirement for Guideline OECD 474 for in vivo Mammalian Erythrocyte Micronucleus Test.