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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:
03 Feb - 07 Mar 2000
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline study. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (Dec 2012)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.
Justification for type of information:
refer to category justification provided in IUCLID section 13

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

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 (incl. certificate)
Remarks:
DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
mouse
Strain:
other: Crl:CD-1TM(ICR)BR
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Limited, Margate, UK
- Age at study initiation: approx. 5-8 weeks
- Weight at study initiation: 25-30 g
- Housing: in groups of up to seven in solid-floor polypropylene cages with woodflakes bedding
- Diet: Rat and Mouse Expanded Diet No. 1 (Special Diets Services Limited, Witham, UK), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hour): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle/solvent used: sterile, distilled water
- Amount of vehicle (gavage): 10 mL/kg
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: dosing solutions were prepared freshly by dissolving the test substance in sterile water yielding final concentrations of 25, 12.5 and 6.25 mg/mL (corresponding to 250, 125 and 6.25 mg/kg bw, respectively).

Duration of treatment / exposure:
not applicable
Frequency of treatment:
single treatment
Post exposure period:
24 and 48 h (vehicle and high dose group)
24 h (low, mid dose and positive control group)
Doses / concentrationsopen allclose all
Dose / conc.:
62.5 mg/kg bw/day (nominal)
Remarks:
low dose
Dose / conc.:
125 mg/kg bw/day (nominal)
Remarks:
mid dose
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
high dose
No. of animals per sex per dose:
7 (vehicle and dose groups), 5 (positive control)
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Route of administration: oral
- Doses / concentrations: 5 mg/mL in sterile water (corresponding to 50 mg/kg bw )

Examinations

Tissues and cell types examined:
Femur bone marrow smears
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A range finding study in 2 male and 2 female mice was performed to find the maximum tolerated dose level, a suitable route of administration and to assess gender-specific differences in toxicity.

TREATMENT AND SAMPLING TIMES:
24 h sampling time: 0, 62.5, 125 and 250 mg/kg bw (test substance); 50 mg/kg bw (positive control)
48 h sampling time: 0 and 250 mg/kg bw (test substance)
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.

DETAILS OF SLIDE PREPARATION:
Immediately following sacrifice (i.e. 24 or 48 h following dosing), both femurs were dissected from each animal, aspirated with fetal calf serum and bone marrow smears were prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol and stained with May-Grünwald/Giemsa, allowed to air-dry and coverslipped using mounting medium.

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 was 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 and standard deviations.
Evaluation criteria:
A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test material groups and the number occurring in the corresponding vehicle control group.
A positive mutagenic response was demonstrated when a biologically relevant statistically significant, dose-related increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24 or 48-hour time points, when compared to their corresponding control group.
If these criteria were not demonstrated, then the test material 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 were analysed following a transformation using Student's t-test (two tailed) and any significant results were confirmed using the one way analysis of variance. Statistical significance was indicated at *p < 0.05, **p < 0.01 and ***p < 0.001.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
≥500 mg/kg bw (range finding toxicity study): all animals dosed i.p. died within the study period of 48 h; 250 mg/kg bw (main study): 2/7 animals died in the 48-h harvest time group
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 250, 500, 1000, 2000 mg/kg bw (intraperitoneal) and 2000 mg/kg bw (oral)
- Clinical signs of toxicity in test animals: in animals dosed with the test material via the intraperitoneal route premature deaths occurred at ≥ 500 mg/kg bw. Clinical signs were observed at ≥ 250 mg/kg bw (and increased in severity with increasing doses) and included: hunched posture, lethargy, pilo-erection, decreased respiratory rate, ptosis, ataxia, splayed gait, prostration, laboured respiration and pallor of the extremities.
- Sacrifice time: 48 h
- Dose selection: based on the results, the maximum tolerated dose of the test material was 250 mg/kg bw and thus selected as high dose for the main study.
- Other: no marked differences in the toxicity of the test substance were noted between male and female mice. Therefore, it was considered acceptable to use males only for the main study.

RESULTS OF DEFINITIVE STUDY
- Clinical signs: there were two premature deaths seen in the 48-h 250 mg/kg bw test material dose group. Clinical signs were observed in animals dosed with the test material at 250 mg/kg bw in both the 24 and 48 h groups. These included hunched posture, lethargy, pilo-erection, decreased respiratory rate, ptosis and ataxia.
- Induction of micronuclei: there was a small, statistically significant increase in the frequency of micronucleated PCEs in the 24-h 125 mg/kg bw test material dose group when compared to the respective control group. However, the response was within the current historical range for vehicle controls, the 24-h vehicle control PCE+MN value was very low and no individual animal values for micronucleated PCEs were greater than it would be considered acceptable for vehicle control animals. Therefore, the response was considered to be of no biological relevance. The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes, hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.
- Ratio of PCE/NCE: there was a statistically significant decrease in the PCE/NCE ratio of the 24-h 250 mg/kg bw test material group when compared to the concurrent vehicle control group. The response was part of a dose-related reduction in PCE/NCE ratios and was taken to indicate that cytotoxicity and exposure of the bone marrow had been achieved.
- Appropriateness of dose levels and route: it was considered that the loss of animals due to premature death did not affect the integrity of the study, because at least five analysable animals were available in each group, as recommended in the OECD test guideline No. 474.

Any other information on results incl. tables

Table 1. Results of the in vivo micronucleus assay

Treatment group

Dose (Conc) [mg/kg]

Sampling time

[h]

Number of PCE with MN

[per 2000 PCEs]

PCE/NCE ratio

Mean value

SD

Mean value

SD

Vehicle control

0

48

1.0

1.3

1.87

0.29

 

0

24

0.7

0.8

1.72

0.65

Positive control

50

24

54.8***

15.9

1.62

0.41

Test substance

250 (a)

48

2.2

1.6

1.25

0.80

 

250

24

1.3

1.4

0.98*

0.28

 

125

24

2.1**

0.9

1.42

0.26

 

62.5

24

0.9

0.9

2.12

1.25

Positive control: cyclophosphamide

*p<0.05, **p<0.01, ***p < 0.001, Student's t-test (two tailed) and one way analysis of variance

(a): data from 5 animals

PE: polychromatic erythrocytes

NCE: normochromatic erythrocytes

SD: standard deviation

Applicant's summary and conclusion

Conclusions:
The test material has been tested according to OECD 474 and under GLP conditions. No significant increase in the incidence of micronucleated polychromatic erythrocytes was observed in the bone marrow of male mice 24 or 48 hours after a single oral dose of up to and including 250 mg/kg bw. A reduction in the PCE/total erythrocyte ratio was only observed in the 24 hour positive control group. It is concluded that the test substance is negative for the induction of micronuclei under the conditions of the study.