Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD TG 474), but as read-across from supporting substance maximum reliability is 2 Read-across hypothesis: for details please see read-across report in IUCLID section 13

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
GLP compliance:
yes
Remarks:
: according to the GLP regulations of the German Chemicals Act and the OECD principles of GLP
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Butan-1-ol
EC Number:
200-751-6
EC Name:
Butan-1-ol
Cas Number:
71-36-3
Molecular formula:
C4H10O
IUPAC Name:
butan-1-ol
Details on test material:
- Name of test material (as cited in study report): n-Butanol
- Physical state: colorless liquid
- Storage condition of test material: room temperature (N2 conditions)
- Stability under test conditions: stability throughout the study period has been verified by reanalysis

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River GmbH, WIGA, Sulzfeld, Germany
- Age at study initiation: 5-8 weeks
- Weight at study initiation: 26.9 g (mean)
- Assigned to test groups randomly: yes
- Housing: during acclimation period: in groups of 5, separated by sex, Makrolon cages, type MIII; during test: individually, Makrolon cages, type MI
- Diet: standardized pelleted feed (Kliba Haltungsdiät, Klingentalmühle AG, Kaiseraugst, Switzerland); ad libitum
- Water: drinking water; ad libitum
- Acclimation period: about 1 week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12


Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: olive oil
- Justification for choice of solvent/vehicle: due to the limited solubility of the test item in water; olive oil had been demonstrated to be suitable in the MN-assay and historical control data are available
- Concentration of test material in vehicle: 5 g / 100 mL; 10 g / 100 mL; 20 g / 100 mL
- Amount of vehicle: 10 ml/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- solution of the test substance in the vehicle was prepared immediately before dosing
Duration of treatment / exposure:
- 24 h (all dose groups and control group)
- 48 h (highest dose group and control group)
Frequency of treatment:
- single application
Post exposure period:
- none
Doses / concentrations
Remarks:
Doses / Concentrations:
500, 1000, 2000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
- cyclophosphamide (CPP; 2 males, 3 females) and vincristine sulphate (VCR; 3 males, 2 females)
- Justification for choice of positive control(s): CPP causes clastogenicity; VCR is a known spindle poison
- Route of administration: CPP: orally; VCR: intraperitoneally
- Doses: 20 mg CPP/kg bw ; 0.15 mg VCR/kg bw (both dissolved in purified water; application volume: 10 ml/kg bw)
- Exposure duration: 24 h

Examinations

Tissues and cell types examined:
- polychromatic erythrocytes and normocytes isolated from the bone marrow of the femora
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
- testing up to the highest recommended dose in OECD TG 474


TREATMENT AND SAMPLING TIMES:
- sampling of bone marrow of the two femora of each animal 24 or 48 hours after dosing


DETAILS OF SLIDE PREPARATION:
- bone marrow smear was prepared on microscopic slides and dried in the air
- STAINING:
- 5 minutes modified May Grünwald solution (Wrights solution): eosin, methylene blue
- rinsed in purified water and finally stained for 15 minutes in Giemsa solution
- embedded in Corbit Balsam


METHOD OF ANALYSIS:
In general, 2,000 polychromatic erythrocytes (PCE) from each of the male and female animals of every test group are evaluated and investigated for micronuclei (MN). The normochromatic erythrocytes (NCE) which occur are also scored. The following parameters are recorded:
• Number of polychromatic erythrocytes.
• Number of polychromatic erythrocytes containing micronuclei.
The increase in the number of micronuclei in polychromatic erythrocytes of treated animals as compared with the solvent control group provides an index of a chromosome-breaking (clastogenic) effect or of a spindle activity of the substance tested.
• Number of normochromatic erythrocytes.
• Number of normochromatic erythrocytes containing micronuclei.
The number of micronuclei in normochromatic erythrocytes at the early sacrifice intervals shows the situation before test substance administration and may serve as a control value. A substance-induced increase in the number of micronuclei in normocytes may be found with an increase in the duration of the sacrifice intervals.
• Ratio of polychromatic to normochromatic erythrocytes.
An alteration of this ratio indicates that the test substance actually reached the target.
Individual animals with pathological bone marrow depression may be identified and excluded from the evaluation.
• Number of small micronuclei (d < D/4) and of large micronuclei (d >/= D/4) (d = diameter of micronucleus, D = cell diameter).
The size of micronuclei may give an indication an the possible mode of action of the test substance, i.e. a clastogenic or a spindle poison effect.
Slides were coded before microscopic analysis.

Acceptance criteria:
The mouse micronucleus test is to be considered valid if the following criteria are met:
The quality of the slides allowed the identification and evaluation of a sufficient number of analyzable cells, i.e. >/= 2,000 polychromatic erythrocytes per animal.
• The proportion of cells with micronuclei in negative control animals was within the normal range of the historical control data.
• The two positive control chemicals induced a significant increase in the number of cells containing small and large micronuclei.


Evaluation criteria:
The test chemical is to be considered positive in this assay if the following criteria are met:
A dose-related and significant increase in the number of micronucleated polychromatic erythrocytes at the 24 hour interval.
• The proportion of cells containing micronuclei exceeded both the values of the concurrent negative control range and the negative historical control range.
A test substance is generally considered negative in this test system if:
• There was no significant increase in the number of micronucleated polychromatic erythrocytes at any dose above concurrent control frequencies and at any time.
• The frequencies of cells containing micronuclei were within the historical control range.
Statistics:
The statistical evaluation of the data was carried out using the program system MUKERN (BASF AG).
The number of micronuclei in polychromatic erythrocytes was analyzed.
A comparison of the dose group with the vehicle control was carried out using the Wilcoxon test for the hypothesis of equal medians. Here, the relative frequencies of cells with micronuclei of each animal were used. If the results of this test were significant, labels (* for p

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
: in the mid and high dose group (1000 and 2000 mg/kg bw)
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- pretest for determination of acute oral toxicity
- Dose range: 2000 mg/kg bw
- Clinical signs of toxicity in test animals: abdominal position, irregular respiration, staggering, squatting posture and narcotic like state
- no mortality,
- Rationale for exposure: recommended highest dose by OECD TG 474


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: none
- Ratio of PCE/NCE (for Micronucleus assay): see below
- Statistical evaluation: no statistical significant increase in micronuclei was observed
- no clinical signs of toxicity in the animals of the vehicle control, 500 mg/kg bw and positive control group;
- 1000 mg/kg bw: piloerection after about 30 minutes until sacrifice of the animals;
- 2000 mg/kg bw: animals were in poor condition: abdominal position, irregular respiration, staggering, squatting posture, narcotic like state
-

Any other information on results incl. tables

Read-across justification:for details please see read-across report in IUCLID section 13

The single oral administration of olive oil in a volume of 10 mL/kg body weight led to 1.5% polychromatic erythrocytes containing micronuclei after the 24-hour sacrifice interval or to 0.8% after the 48-hour sacrifice interval.

After the single administration of the highest dose of 2,000 mg/kg body weight, 0.7% polychromatic erythrocytes containing micronuclei were found after 24 hours and 0.6% after 48 hours.

In the two lower dose groups, rates of micronuclei of about 0.4% (1,000 mg/kg group) and 0.6% (500 mg/kg group) were detected after a sacrifice interval of 24 hours in each case.

An 11.6% increase in polychromatic erythrocytes was observed in animals treated with 20 mg/kg cyclophosphamide, the positive control agent. This expected increase consisted mainly of small micronuclei indicative of clastogenicity.

Vincristine, a spindle poison agent, produed a 60.9%increase of micronuclei in polychromatic erythrocytes. A significant portion of this increase,

7.7% was attributable to large micronuclei.

The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the negative control or in the various dose groups at any of the sacrifice intervals.

Thus, the test substance, n-Butanol, did not lead to any increase in the rate of micronuclei. The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d < D/4) or large micronuclei (d >/= D/4) did not deviate from the vehicle control value at any of the sacrifice intervals.

No inhibition of erythropoiesis induced by the treatment of mice with n-Butanol was detected; the ratio of polychromatic to normochromatic erythrocytes was always in the same range as that of the control values in all dose groups.

Table 1:Polychromatic and normochromatic erythocytes (males and females)

 

Interval: 24 hours

Interval: 48 hours

 

Total No. of

MN (o/oo) in

Total No. of

MN (o/oo) in

 

PCEs

NCEs

PCEs

NCEs

PCEs

NCEs

PCEs

NCEs

Vehicle Olive Oil

20,000

7942

 1.5

0.3

20,000

7963

0.8

0.6

 

 

 

 

 

 

 

 

 

 500 mg/kg

20,000

6912

 0.6

0.0

 

 

 

 

1000 mg/kg

20,000

8570

 0.4

0.1

 

 

 

 

2000 mg/kg

20,000

7959

 0.7

0.4

20,000

8995

0.6

0.3

 

 

 

 

 

 

 

 

 

20 mg/kg CPP

10,000

4610

11.6**

0.4

 

 

 

 

0.15 mg/kg VCR

10,000

4721

60.9**

0.0

 

 

 

 

Wilcoxon test (one-sided): *: p </= 0.05, ** p </= 0.01

Pairwise comparison of each dose group with the vehicle control group.

Table 2:Polychromatic erythrocytes: differentiation between small and large micronuclei (males and females)

 

Interval: 24 hours

Interval: 48 hours

 

Total No. of PCEs

Cells (o/oo) with

Total No. of PCEs

Cells (o/oo) with

 

 

MN.d<D/4

MN.d=/>D/4

 

MN.d<D/4

MN.d=/>D/4

Vehicle Olive Oil

20,000

 1.5

0.0

20,000

0.8

0.0

 

 

 

 

 

 

 

 500 mg/kg

20,000

 0.6

0.0

 

 

 

1000 mg/kg

20,000

 0.3

0.1

 

 

 

2000 mg/kg

20,000

 0.7

0.0

20,000

0.5

0.1

 

 

 

 

 

 

 

20 mg/kg CPP

10,000

11.5**

0.1

 

 

 

0.15 mg/kg VCR

10,000

53.2**

7.7**

 

 

 

Wilcoxon test (one-sided): *: p </= 0.05, **: p </= 0.01

Pairwise comparison of each dose group with the vehicle control group.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Butan-1-ol did not induce micronuclei in erythrocytes of mice after single oral application up to the maximum tolerable dose of 2000 mg/kg bw. Under the experimental conditions of this assay butan-1-ol has no chromosome damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis.
Executive summary:

The substance n-Butanol was tested for clastogenicity and for the ability to have spindle poison effects in NMRI mice using the micronucleus test method. For this purpose, the test substance, dissolved in olive oil, was administered once orally to male and female animals at dose levels of 500 mg/kg, 1,000 mg/kg and 2,000 mg/kg body weight in a volume of 10 mL/kg body weight in each case.

As a negative control, male and female mice were administered merely the vehicle, olive oil, by the same route, which gave frequencies of micronucleated polychromatic erythrocytes within the historical control range.

Both of the positive control chemicals, i.e. cyclo­phosphamide for clastogenicity and vincristine for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei.

Animals which were administered the vehicle or the positive control substances cyclophosphamide or vincristine did not show any clinical signs of toxicity.

The administration of the test substance led to evident signs of toxicity in the highest dose groupof 2,000 mg/kg body weight.

The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the highest dose group of 2,000.mg/kg body weight and in the vehicle controls. In the test groups of 500 mg/kg and 1,000 mg/kg bodyweight and in the positive control groups, the 24-hour sacrifice interval was investigated only. After staining of the preparations, 2,000 poly­chromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2,000 polychromatic erythrocytes were also registered. In addition, the number of normochromatic erythrocytes with micronuclei and the ratio of polychromatic to normochromatic erythrocytes were determined.

According to the results of the present study, the single oral administration of n-Butanol did not lead to any increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was always in the same range as that of the negative control in all dose groups and at all sacrifice intervals (Engelhardt and Hoffmann, 1998).

No inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected.

Thus, under the experimental conditions chosen here,the test substance n-Butanol does not have any chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis.