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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report date:
1992

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Staatstoezicht op de Volksgezondheid, Central Veterinary, Public Health Inspectorate, The Netherlands
Type of assay:
micronucleus assay

Test material

Constituent 1
Reference substance name:
Vinasses, residue of fermentation
EC Number:
932-215-9
Molecular formula:
Not applicable (a generic molecular formula cannot be provided for this specific UVCB substance).
IUPAC Name:
Vinasses, residue of fermentation
Details on test material:
- Name of test material (as cited in study report): Aromex liquor
- Physical state / appearance: brown liquid
- Analytical purity: 100%
- Composition of test material, percentage of components: dry matter: 67.6%; NaCl 2.7%; Nitrogen 4.2%, Ash content 19.1%, K 63600 ppm, Na 21700 ppm; Ca 4010 ppm; Mg 225 ppm, Fe 175 ppm
- Lot/batch No.: PR 91-237
- Permissable storage time: 6 months
- Storage condition of test material: at 4°C in the dark

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
Swiss mice (Charles River CD-1 strain)
TEST ANIMALS
- Source: Charles River Wiga GmbH, Sulzfeld, F.R. Germany
- SPF conditions
- Age at study initiation: young adult
- Weight at study initiation: males: 25 to 35.7 g; females: 22.5 to 30.2 g
- Assigned to test groups randomly: yes (but just before dosing one female was replaced by a reserve mouse in order to equalize the mean initial body weights of the three groups
- Fasting period before study: 2 - 4 hours just prior to treatment
- Housing: in Makrolon, sterilized cages with a grid cover of stainless steel and with bedding of sterilized softwood chips. Males were housed individually and females up to five per cage.
- Diet (ad libitum): Institute's cereal based, open formula diet for rats and mice
- Water (ad libitum): tap water
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5 - 22.5 (experimental room); 20.5 - 21.5(quarantine room)
- Humidity (%): 50 - 63 (experimental room); 62 - 81 (quarantine room)
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: none
Duration of treatment / exposure:
one single application (at 24, 48 and 72 hours after treatment the animals were killed)
Frequency of treatment:
one single dose
Post exposure period:
24, 48 and 72 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
10 mL/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
15
Control animals:
yes, sham-exposed
Positive control(s):
Mitomycin C
- Justification for choice of positive control(s): known mutagen
- Route of administration: intraperitoneally
- Doses / concentrations: 1.5 mg in 20 mL saline/kg bw

Examinations

Tissues and cell types examined:
bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: previous test with Aromex liquor in mice of the same strain and age: 20 mL/kg bw resulted in the death of all animals, while 10 mL/kg bw was tolerated well and did not provoke signs of intoxications during the observation period (7 days).

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): 24, 48 and 72 hours, the negative control group was treated in an identical way with tap water, animals were weighed on day 0, just prior to gavage, they were inspected 1-4 hours after treatment for signs of ill health or reactions to treatment, and once every day thereafter till the end of the experimental period.

DETAILS OF SLIDE PREPARATION: The femurs were dissected, and bone marrow was flushed from the femurs with foetal calf serum using a syringe and needle. The collected cells were mixed gently with the serum and the cell suspension was centrifuged at c. 300 x g for five minutes. After removal of the excess serum the cell pellets were resuspended in the remaining fluid and processed into glass-drawn smears were prepared from each animal. The smears were air-dried, fixed in methanol, stained according to May-Grünwald Giemsa and mounted with a coverslipping mounting medium.

METHOD OF ANALYSIS: All slides were randomly coded by a person not involved in the study. The incidence of MPE (micronucleated polychromatic erythrocytes) and MNE (micronucleated normochromatic erythrocytes) and the total numbers of poly- and normochromatic erythrocytes were recorded in atotal of at least 2000 and maximally 3000 erythrocytes per animal in such a way that a minimum of 1000 PE was observed, if feasible. Slides (one slide per animal), were read by moving from the beginning of the smear (label end) to the leading edge in horizontal lines taking care that areas selected for evaluation were evenly distributed over the whole smear.

Statistics:
Statistical analysis was carried out in two stages. In the first step of the procedure overall significance tests were carried out as follows: the fraction of MPE, MNE and ME per counted number of PE, NE, and E, respectively, were analysed with a generalized linear model using a binominal error-distribution, and the numbers of PE per 1000 E were analized with linear regression techniques (e.g. Draper and Smith, 1981). Both methods assess the influence of sex, treatment, exposure time and various interactions on the total variation in the data. As a second stage, a posteriori comparisons of treatment groups with the negative control group were carried out with asymptomic t-tests if either the main effect of treatment or the treatment by sex interaction was significant.
For all calculations involved, the Genstat 5 statistical package was used (1987).

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
other: no vehicle used
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no induction

Any other information on results incl. tables

Table 1: Results of micronucleus test with Aromex liquor (group mean numbers)

Treatment

Dose/ kg bw

Males

Females

24 h

48 h

72 h

24 h

48 h

72 h

Mean number of MPE/1000 PE

Tap water

10 mL

1.7

1.0

1.0

1.0

1.0

1.3

Aromex liquor

10 mL

0.7

1.0

1.2

1.1

0.8

1.6

MC

1.5 mg

40.6***

37.3***

12.1***

42.5***

38.7***

13.0***

Mean number of MNE/1000 NE

Tap water

10 mL

1.4

0.8

1.1

0.2

1.2

0.9

Aromex liquor

10 mL

0.4

0.8

1.1

0.9

0.6

1.3

MC

1.5 mg

5.4***

12.1***

13.8***

3.9***

14.5***

11.4***

Mean number of ME/1000 E

Tap water

10 mL

1.5

0.9

1.0

0.7

1.1

1.1

Aromex liquor

10 mL

0.6*

0.9

1.2

1.0

0.8

1.3

MC

1.5 mg

24.8***

21.2***

13.0***

22.6***

22.0***

12.0***

Mean number of PE/1000 E

Tap water

10 mL

553

598

609

587

586

534

Aromex liquor

10 mL

548

584

587

536

585

567

MC

1.5 mg

556

364***

357***

492*

335***

365***

PE = polychromatic erythrocytes

NE= normochromatic erythrocytes

E = erythrocytes

MPE,,= micronucleated PE, NE, E, respectively

MC = mitomycin C

* p<0.05; *** p<0.001

Compared with concomitant controls the ME count per 1000 E was statistically significantly lower in the male test animals 24 hours after treatment. This finding was not considered to be of any biological significance as the value was within the range normally seen in control animals of the same strain and age.

The mean number of PE per 1000 E was fully comparable between control and the aromex liquor treated group, indicating that treatment with Aromex liquor did not result in cytotoxicity for the bone marrow cells.

The considerable increase in the incidence of micronucleated erythrocytes and the decrease in the PE counts per 1000 E in positive control animals, demonstrated the sensitivity of the test system.

The results did not provide any evidence of chromosomal damage and/or damage to the mitotic apparatus in bone marrow cells of mice treated orally with a high dose (10 mL/kg bw) of Aromex liquor.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative