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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 - 31 Oct 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1990
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted in 1983
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted in 2016
Deviations:
yes
Remarks:
only 1 dose level + controls, vehicle produces signs of toxicity, only 1000 erythrocytes/animal were scored for micronuclei, intraperitoneal injection as route of administration
GLP compliance:
yes
Type of assay:
other: mammalian erythrocyte micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Batch No.: 280

Test animals

Species:
mouse
Strain:
other: Bor: NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: F. Winkelmann, Borchen, Germany
- Age at study initiation: approximately 8 - 12 weeks
- Weight at study initiation: 29 - 44 g (males and females)
- Assigned to test groups randomly: yes
- Housing: 3 - 5 animals of the same sex per cage in Makrolon type I and II cages on soft wood granules bedding, type S 8/15 (Fa. Ssniff, Spezialdiäten GmbH, Soest, Germany)
- Diet: Altromin 1324 Standard Diet (Altromin GmbH, Lage, Germany), ad libitum
- Water: tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.5 - 22.5
- Humidity (%): 30 - 50
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle/solvent used: polyethylene glycol (PEG 400)
- Concentration of test material in vehicle: 125 mg/kg bw
- Amount of vehicle (if gavage or dermal): 5 mL/kg bw
Duration of treatment / exposure:
not applicable
Frequency of treatment:
single treatment
Post exposure period:
24, 48 and 72 h
Doses / concentrations
Dose / conc.:
125 mg/kg bw/day
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: intraperitoneal
- Doses / concentrations: 20 mg/kg bw

Examinations

Tissues and cell types examined:
Tissue: bone marrow
Cell type: bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The applied dose was chosen based on a preliminary dose range finding test: Groups of 5 animals (males and females) were intraperitoneally administered 100, 175 and 250 mg/kg bw test substance. Within 5 days 3/5 animals died in the 175 and 250 mg/kg bw groups. The following clinical signs were noted, starting in the 100 mg/kg bw group and lasting for more than 72 h: apathy, roughened fur, staggering gait, prone position, spasm, twitching and difficulty in breathing.

TREATMENT AND SAMPLING TIMES: sampling was performed 24, 48 or 72 h after administration of the test substance; sampling was performed 24 h after administration of the vehicle and positive control

DETAILS OF SLIDE PREPARATION: Slides were dried overnight or with heat for a short period if fresh smears were needed. The smears were stained automatically and "destained" with methanol, rinsed with deionized water, and left to dry. Smears were then coated by immersion with xylene and covered with a covering agent. The slide was left to dry.

METHOD OF ANALYSIS: Evaluation of the slides was performed using light microscopes at a magnification of about 1000. Per animal 1000 polychromatic erythrocytes were analysed for micronuclei. To identify animals with pathological bone marrow depressions and to show that the test substance actually reached the target as evident by possible alterations the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 1000 polychromatic ones. Additionally, the number of normochromatic erythrocytes showing micronuclei was established.
Evaluation criteria:
A test was considered positive if, at any of the intervals, there was a relevant and significant increase in the number of polychromatic erythrocytes showing micronuclei in comparison to the negative control. A test was considered negative if there was no relevant or significant increase in the rate of micronucleated polychromatic erythrocytes at any time. A test was also considered negative if there was a significant increase in that rate which, according to the laboratory's experience was within the range of negative controls. In addition, a test was considered equivocal if there was an increase of micronucleated polychromatic erythrocytes above the range of attached historical negative controls, provided the increase was not significant. In this case, a second test had to be performed at the most sensitive interval.
Statistics:
Wilcoxon's non-parametric rank sum test (number of polychromatic erythrocytes having micronuclei and the number of normochromatic erythrocytes), one-sided chi² -test (rate of normochromatic erythrocytes containing micronuclei if the micronuclear rate for polychromatic erythrocytes was already relevantly increased)

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 100, 175 and 250 mg/kg bw
- Clinical signs of toxicity in test animals: 3/5 animals died within 5 days after administration of 175 and 250 mg/kg bw; apathy, roughened fur, staggering gait, prone position, spasm, twitching and difficulty in breathing was observed in all dose groups lasting for more than 72 h

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and gender after administration of the test item.
- Ratio of PCE/NCE: The mean number of polychromatic erythrocytes was not substantially decreased after treatment with the test item as compared to the mean value of polychromatic erythrocytes of the vehicle control.
- Appropriateness of dose levels and route: The PCE/NCE ratio of the 24 and 48 h interval were clearly higher than the ratio of the negative control. In addition, 2 animals in the 72 h group showed ratios of 1000/3852 and 1000/3421 which are not normally found in negative controls. These findings demonstrate an effect of the test substance on bone marrow prolifration. According to the symptoms observed in the treated groups the compound was resorbed and present in the blood-stream and thus, able to reach the bone marrow. Therefore, it can be assumed that the test substance had reached the target organ.

OTHER
- Mortalities: 4/40 animals died during the test period: 24 h group: 1/5 male found dead after 24 h; 48 h group: 1/5 female found dead after 48 h; 72 h group: 1/5 male found dead after 48 h; Replacement group: 1/5 male found dead after 72 h
- Clinical signs: apathy, roughened fur, staggering gait, prone position, spasm, twitching and diarrhoea for up to 72 h after treatment

Any other information on results incl. tables

Table 1: Results of the in vivo micronucleus test

Experimental group

Time of sacrifice
[hours after treatment]

Number of evaluated polychromatic erythrocytes per animal

No. of normo­chromatic erythro­cytes per 1000 poly­chromatic erythro­cytes
[mean ± SD]

Micronucleated cells per 1000

normochromatic erythrocytes
[mean ± SD]

polychromatic erythrocytes
[mean ± SD]

Negative controla

24

1000

1179 ± 495

1.3 ± 1.1

1.2 ± 0.9

Test substanceb

24

1000

1319 ± 439

0.7 ± 0.9

1.3 ± 1.4

Test substanceb

48

1000

1595 ± 633

1.2 ± 0.9

0.7 ± 0.8

Test substanceb

72

1000

1524 ± 1185

0.7 ± 0.7

1.1 ± 0.7

Positive controlc

24

1000

881 ± 160

1.3 ± 1.2

16.1* ± 6.4

aPolyethyleneglycol 400 (as intraperitoneal injection)

b125 mg/kg bw (as intraperitoneal injection)

c20 mg/kg bw cyclophosphamide (as intraperitoneal injection)

* p < 0.01 in non-parametric Wilcoxon ranking test

Applicant's summary and conclusion

Executive summary:

The potential of the test substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse was assessed in a study performed according to OECD Guideline 474 (1983) and in compliance with GLP. The test substance was suspended in polyethylene glycol 400, which was also used as vehicle control. Five male and five female Bor: NMRI mice per test group received a single intraperitoneal injection of 125 mg/kg bw. The mice were sacrificed at 24, 48 and 72 h after treatment and the bone marrow cells were collected for micronuclei analysis. Animals administered the positive control (20 mg/kg bw cyclophosphamide) or the vehicle were sacrificed at 24 h after treatment. Per animal 1000 polychromatic erythrocytes (PCEs) were scored for micronuclei. The ratio between PCEs and normochromatic erythrocytes (NCEs) was determined in the same sample and expressed in NCEs per 1000 PCEs. Animals treated with 125 mg/kg bw test substance showed symptoms of toxicity which comprise apathy, roughened fur, staggering gait, prone position, spasm, twitching and diarrhoea. The symptoms lasted until sacrifice. 4/40 mice treated with the test substance died during the study period. The ratio of PCEs to NCEs was not altered in the groups which received the test substance. In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any gender used. The positive control caused a clear clastogenic effect which is shown by the significant increase of PCEs with micronuclei. Thus, under the conditions of this study, the test substance is considered to be non-mutagenic.