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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
To address toxicological endpoints as part of the REACH registration of Cyclamen Aldehyde Extra (Target substance) It is proposed to read across to Florhydral (Source substance) The use of read-across works within the spirit of REACH and the stated aim of the legislation to reduce animal testing where possible. The Target Substance and Source Substance have been characterised in using the categories and databases present in the OECD QSAR toolbox . From the profiling in this table , it can be seen that the two substances share structural similarities and also ‘mechanistic action’ similarities which are both general and endpoint specific. Therefore read across is justified.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Florhydral
- IUPAC name: ß-methyl-3-(1-methylethyl)benzenepropanal
- CAS number: 125109-85-5
- EC number: 412-050-4
- Molecular formula (if other than submission substance): C13H18O
- Molecular weight (if other than submission substance): 190
- Smiles notation (if other than submission substance): CC(C)C1=CC(=CC=C1)C(C)CC=O
- InChl (if other than submission substance):
InChI: InChI=1S/C13H18O/c1-10(2)12-5-4-6-13(9-12)11(3)7-8-14/h4-6,8-11H,7H2,1-3H3
InChI key: InChIKey=OHRBQTOZYGEWCJ-UHFFFAOYSA-N
- Structure: image attached below
Batch No.: 110001
Aggregate State
at room temperature: Liquid
Colour: Colourless
Purity: 98.8%
Analytical Method: GC
Storage: In the refrigerator at 2°C to 8°C, light protected
Expiration Date: January 26, 2005


Method

Target gene:
Chinese hamster lung fibroblasts (V79)
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Cell Cultures
Large stocks of the V79 cell line (supplied by Laboratory for Mutagenicty Testing LMP Technical University Damstadt, D-64287 Damstadt) were stored in liquid nitrogen in the cell bank of RCC Cryotest Cell Research GmbH allowing the repeated use of the same cell culture batch in experiments. Before freezing each batch was screened for cytoplasm contamination and checked for karyotype stability. Consequently, the parameters of the experiments remain similar because of standardized characteristics of the cells.
Thawed stock cultures were propagated at 37°C in 80 cm2 plastic flasks (GREINER D-72632 Frickenhausen). About 5x105 cells per flask were seeded into 15mL of MEM (Minimal Essential Medium; DEROMED; D-12247 Berlin) supplemented with 10% fetal calf serum (FCS; PAA Laboratories GmbH, D-35091 Cölbe). The cells were subculture d twice weekly. The cell cultures were incubated at 37°C in a humidified atmosphere with 1.5carbon dioxide (98.5% air).
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
see test concentrations table in the "any other information on methods including tables" section below
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: On the day of the experiment (immediately before treatment), the test item was dissolved in ethanol (E. Merck, D-64293 Damstadt; purity 99.8%). The final concentration of ethanol in the cultue medium was 0.5% (v/v). The solvent was chosen to its solubility properties and its relative nontoxicity to the cell cultures.
Controls
Untreated negative controls:
yes
Remarks:
culture medium
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Remarks:
ethylmethane sulfonate -S9 and cyclophosphamide +S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 4, 18 and 28 hours

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 per replicate (200 total)

DETERMINATION OF CYTOTOXICITY
- Method:
Evaluation of Cell Numbers
For evaluation of cytotoxicity indicated by reduced cell numbers, two additional cultures per test item and solvent control group, not treated with colcemid, were set up in parallel. These cultures were stained after 18 hours and 28 hours, respectively, in order to determine microscopically the cell number within 10 defined fields per coded slide. The cell number of the treatment groups is given in percentage compared t the respective solvent control.
Analysis of Metaphase Cells
Evaluation of the cultures was performed (according to standard protocol of the “Arbbeits gruppe der Industrie, Cytogenetik” using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. 100 well spread metaphase plates per culture were scored for cytogenic damage on coded slides, except for the positive control in experiment II, in the absence of S9 mix, at preparation interval 28hrs, where only 50 metaphase plates were scored, Only metaphases with characteristic chromosome numbers of 22±1 were included in the analysis. To describe the effect the mitotic index (% cells in mitosis) was determined (% poluploid metaphases; in case of this aneuploid cell line polyploidy means a near tetraploid kayotype). Additionally the number of endomitotic cells scored at the evaluation of polyploidy cells was noticed and reported (% endomitotic metaphases).
Evaluation criteria:
The test item would be classified as non-clastogenic if:
-the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of oir historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps).
and/or
- no significant increase of the number of structural chromosome aberrations is observed.

The test item would be classified as clastogenic if:
- the number of induced structural chromosome aberrations is not in the range of historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps).
and
-either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

Although the inclusion of the structural chromosome aberrations is the purpose of th study, it was important also to include the polyploids and endoreduplications. the following criteria therefore was also considered:
The test item could be classified as aneugenic if:
-the number of induced numerical aberations is not in the range of historical control data (0.0 - 8.5 % polyploid cells)
Statistics:
Statistical significance was confirmed by means of the Fisher's exact test (9) (p<0.05). However, both biological and statistical significance should be considered together. If the criteria mentioned above for the test item were not clearly met, the classificiation with regard to the historical data and the biological relevance would be discussed and/or a confirmatory experiment would be performed.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test item Florhydral, dissolved in ethanol was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in the absence amd the presence of metabolic activation by S9 mix.
Two independent experiments were performed. In experiment I, the exposure period was 4 hours with and without metabolic activation. In experiment II the exposure period was 4 hours with S9 mix and 18 hours without S9 mix. The chromosomes were prepared 18 hours (exp. I and II) and 28 hours (Exp. II) after the start of treatment with the test item.
In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations, except for the positive control in Experiment OO in the absence of S9 mix, at preparation interval 28 hours, where only 50 metaphase plates were scored.
In a range finding pre-test on toxicity cell numbers 24 hrs after start of treatment were scored as an indicator for cytotoxicity. Concentrations between 15.2 1950 µg/ml were applied. Clear toxic effects were observed after 4 hours treatment with 60.9 µg/ml and above in the absence of S9 mix and with 243.8 µg/ml and above in the presence of S9 mix. In addition, 24 hours continuous treatment with 30.5 µg/ml and above in the absence of S9 mix induced strong toxic effects (see Table 4 below).
In the pre-experiment, precipitation of the test item in culture medium was observed after treatment with 121.9 µg/m: and above in the absence of S9 mix. It was difficult to determine the oily test item drops on the surface of the culture medium. No relevant influence of the test item on the pH value or osmolarity was observed (solvent control 418mOsm, pH 7.3 versus 377mOsm and pH 7.2 at 1950 µg/ml).

Toxic effects indicated by reduced cell numbers (see Table 5 below) of below 50% of control were observed in Experiment I, in the presence of S9 mix, after 4 hours treatment with 150 µg/ml (16% of control). However, in all other experimental parts in the absence and the presence of S9 mix, concentrations showing clear cytotoxicity were not scorable for chromosomal damage. (see tables 5,6 and 10 below)
In Both experiments, in the absence and the presence of S9 mix, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed (see tables 8, 9, 11 and 12 below). The aberration rates of the cells after treatment with the test item (0.-4.0% aberrant cells, exclusive gaps) were close to the range of the solvent control values (0.5-3.5% aberrant cells, exclusive gaps) and within the range of historical control data: 0.0 – 4% aberrant cells, exclusive gaps. In experiment I in the presence of S9 mix a slight increase in the number of cells carrying exchanges (2.5%) was observed after 4 hours treatment with 150 µg/ml. However, the single value slightly exceeding the testing laboratory’s historical control data range (0.0 – 2.0 % aberrant cells with exchanges) has to be regarded as biologically irrelevant.
Endomitotic metaphases were observed in Experiment I after 4 hours treatment in the absence and the presence of S9 mix (see table 7). Generally, no cells with endo-reduplicated chromosomes were observed in the control groups. But Experiment I in the Absence of S9 mix, a relevant increase of endomitoitic cells indicating that the test item influence the cell cycle progression if the cells was observed after treatment with 37.5 and 50 µg/ml (3.1% and 1.4%, respectively).
Tables 6 and 10 show the occurrence of polyploidy metaphases. In Experiment I in the absence and the presence of S9 mix, in Experiment II after 18 hours treatment in the absence of S9mix, and in Experiment II in the presence of S9 mix, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (1.4-5.6%) as compared it the rates of the solvent controls (1.5-3.6%). In Experiment II, in the absence of S9 mix, at the 28 hours preparation interval, after treatment with 3.75µg/ml and increase in the frequencies of polyploidy metaphases (7%) close to the borderline of the testing laboratory’s historical control data range. A relevant increase (19.8% polyploidy cells) clearly exceeding the testing laboratory’s historical control data range (0.0-8.5% polyploidy cells were observed (no quantified). Therefore, the next higher concentration (7.5µg/ml) was scored to corroborate the observation at 3.75µg/ml. A relevant increase (19.8% polyploidy cells) was observed. This finding may indicate that the test item has the potential to inhibit mitotic processes and to induce numerical chromosome aberrations.
In both experiments, EMS (400 and 300 µg/ml, respectively) and CPA (0.7 and 1.0µg/ml respectively) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.
In conclusion, it can be stated that under the experimental conditions reported, the test item Florhydral did no induce structural chromosomes aberrations in V19 cells (Chinese hamster cell line) when tested up to cytotoxic concentrations.
However, Florhydral is considered to inhibit mitotic processes and to induce numerical chromosome aberrations (increased rate of polyploidy cells), and to inhibit cell cycle progression (increased rate of cells with endoreduplicated chromosomes) in this chromosome aberration test the absence of metabolic activation.

Any other information on results incl. tables

Table 4: Cytotoxicity of Florhydral to cultures of Chinese hamster cell line V79

without S9 mix, 4 hours exposure

with S9 mix, 4 hrs exposure

without S9 mix, 24 hours exposure

Concentration (µg/ml)

Number of cells

% of solvent control

Concentration (µg/ml)

Number of cells

% of solvent control

Concentration (µg/ml)

Number of cells

% of solvent control

Solvent control

672

100

Solvent control

1067

100

Solvent control

762

100

15.2

781

116

15.2

895

84

15.2

397

52

30.5

653

97

30.5

890

83

30.5

371

49

60.9

160

24

60.9

814

76

60.9

72

9

121.9P

38

6

121.9P

772

72

121.9P

142

19

243.8P

154

23

243.8P

12

1

243.8P

154

20

487.5P

230

34

487.5P

494

46

487.5P

274

36

975P

193

29

975P

184

17

975P

203

27

1950P

168

25

1950P

588

53

1950P

209

27

Table 5: Number of cells in % of solvent control

without metabolic activation (S9 mix)
Experiment I: 4 hours exposure Experiment II: continuous exposure
Preparation interval Concentration (µg/ml) Cells in % of solvent control Preparation interval Concentration (µg/ml) Cells in % of solvent control
18 hours 6.3 81 18 hours 0.47 98
12.5 83 0.94 83
25.0 76 1.88 78
37.5 79 3.75 72
50.0 62 7.5 47
75.0 38 15 41
28 hours 23 98
0.47 99
0.97 80
1.88 82
3.75 62
7.5 60
with metabolic activation (S9 mix)
Experiment I: 4 hours exposure Experiment II: 4 hours exposure
Preparation interval Concentration (µg/ml) Cells in % of solvent control Preparation interval Concentration (µg/ml) Cells in % of solvent control
18 hours 37.5 113 28 hours 37.5 69
75.0 102 75.0 95
100.0 100 100.0 60
150.0 16 150.0 16
200.0 2 200.0 0
250.0 13 250.0 0

Table 6: Experiment I - Number of polyploid cells and mitotic index; preparation interval 18hrs with and without S9 mix

Treatment group Concentration per mL S( mix Exposure period/Recovery Polyploid cells * mitotic indices **
culture total % absolute mean % ***
1 2 1 2
Neg. Control - 4 / 14 hrs 10 14 24 2.4 13.0 13.5 13.3 100.0
Solv. Control 0.50% - 4 / 14 hrs 23 13 36 3.6 14.9 16.2 15.6 100.0
Pos. Control 400.0 µg - 4 / 14 hrs 7 9 16 1.6 13.6 16.5 15.1 113.6
Test item 6.3 µg - 4 / 14 hrs n.d. n.d. - - 16.2 16.4 16.3 104.8
" 12.5 µg - 4 / 14 hrs n.d. n.d. - - 15.5 15.2 15.4 98.7
" 25.0 µg - 4 / 14 hrs 21 16 37 3.7 14.8 16.0 15.4 99.0
" 37.5 µg - 4 / 14 hrs 32 24 56 5.6 13.7 13.9 13.8 88.7
" 50.0 µg - 4 / 14 hrs 19 16 35 3.5 15.7 17.5 16.7 107.1
" 75.0 µg - 4 / 14 hrs n.d. n.d. - - 0.0 0.0 0.0 0.0
Neg. Control + 4 / 14 hrs 12 8 20 2 17.6 18.6 18.1 100.0
Solv. Control 0.50% + 4 / 14 hrs 10 9 19 1.9 17.8 18.5 18.2 100.0
Pos. Control 0.7 µg + 4 / 14 hrs 9 8 17 1.7 13.1 11.2 12.2 67.1
Test item 37.5 µg + 4 / 14 hrs n.d. n.d. - - 20.9 21.9 21.4 117.9
" 75.0 µg + 4 / 14 hrs 16 17 33 3.3 14.4 16.7 15.6 85.7
" 100.0 µg + 4 / 14 hrs 10 11 21 2.1 16.7 15.6 16.2 89.0
" 150.0 µg + 4 / 14 hrs 9 21 30 3 16.8 17.1 17.0 93.4
" 200.0 µg + 4 / 14 hrs n.d. n.d. - - n.e. n.e. n.e. n.e.
" 250.0 µg + 4 / 14 hrs n.d. n.d. - - 15.0 19.8 17.4 95.9

Table 7: Experiment I -Number of endomitotic cells; preparation interval 18 hrs with and without S9 mix

Treatment group Concentration per mL S( mix Exposure period/Recovery Endomitotic cells*
culture total %
1 2
Neg. Control - 4 / 14 hrs 0 0 0 0.0
Solv. Control 0.50% - 4 / 14 hrs 1 0 1 0.1
Pos. Control 400.0 µg - 4 / 14 hrs 0 0 0 0.0
Test item 6.3 µg - 4 / 14 hrs 4 2 6 0.6
" 12.5 µg - 4 / 14 hrs 21 10 31 3.1
" 25.0 µg - 4 / 14 hrs 9 5 14 1.4
Neg. Control + 4 / 14 hrs 0 1 1 0.1
Solv. Control 0.50% + 4 / 14 hrs 2 0 2 0.2
Pos. Control 0.7 µg + 4 / 14 hrs 0 0 0 0.0
Test item 37.5 µg + 4 / 14 hrs 1 1 2 0.2
" 75.0 µg + 4 / 14 hrs 0 2 2 0.2
" 100.0 µg + 4 / 14 hrs 0 4 4 0.4

Table 8:Structural chromosome aberrations Experiment I- preparation interval 18 hrs without S9 mix: exposure period 4 hrs

Slide no. Cells scored % aberrant cells Aberrations
incl. Gaps * excl. Gaps* with exchanges gaps chromatid type chromosome typw other
gaps ig b f d ex ib if id cx ma cd
without S9 mix
Negative control
1 100 0 0 0 0 0 0 0 0 0 0 0 0
2 100 1 0 2 0 0 0 0 1 0 0 0 0
1+2 200 2.0 1.5 0.0 1 0 2 0 0 0 0 1 0 0 0 0
Solvent control : ethanol 0.5%
1 100 1 0 1 0 0 0 0 0 0 0 0 0
2 100 0 0 0 0 0 0 0 0 0 0 0 0
1+2 200 1.0 0.5 0.0 1 0 1 0 0 0 0 0 0 0 0 0
Positive control: EMS 400.0 µg/ml
1 100 3 0 4 0 0 6 1 0 0 0 1 0
2 100 2 0 1 1 0 5 1 2 0 0 0 0
1+2 200 11.5 9.5 5.0 5 0 5 1 0 11 2 2 0 0 1 0
Test item: 25.0 µg/ml
1 100 3 0 0 0 0 1 0 0 0 0 0 0
2 100 1 0 1 0 0 1 0 0 0 0 0 0
1+2 200 2.5 1.0 1.0 4 0 1 0 0 2 0 0 0 0 0 0
Test item: 37.5 µg/ml
1 100 0 0 1 1 0 0 0 0 0 0 0 0
2 100 2 0 1 0 0 2 0 0 0 0 0 0
1+2 200 3.0 2.0 0.5 2 0 2 1 0 2 0 0 0 0 0 0
Test item: 50.0 µg/ml
1 100 0 0 0 0 0 1 0 0 0 0 0 0
2 100 0 0 0 0 0 0 0 0 0 0 0 0
1+2 200 0.5 0.5 0.5 0 0 0 0 0 1 0 0 0 0 0 0

Table 9: Structural chromosome aberrations Experiment I- preparation interval 18 hrs with S9 mix: exposure period 4 hrs

Slide no. Cells scored % aberrant cells Aberrations
incl. Gaps * excl. Gaps* with exchanges gaps chromatid type chromosome typw other
gaps ig b f d ex ib if id cx ma cd
withS9 mix
Negative control
1 100 0 0 2 0 0 1 0 0 0 0 0 0
2 100 0 0 1 0 0 1 0 0 0 0 0 0
1+2 200 2.0 2.0 1.0 0 0 3 0 0 2 0 0 0 0 0 0
Solvent control : ethanol 0.5%
1 100 0 0 0 0 0 0 1 0 0 0 0 0
2 100 0 0 0 1 0 2 0 0 0 0 0 0
1+2 200 2.0 2.0 1.0 0 0 0 1 0 2 1 0 0 0 0 0
Positive control: CPA 0.7 µg/ml
1 100 1 0 2 2 0 6 1 0 0 0 0 0
2 100 0 1 5 2 0 4 8 5 0 0 0 0
1+2 200 13.5 13.0 5.0 1 1 7 4 0 10 9 5 0 0 0 0
Test item: 75.0 µg/ml
1 100 0 0 1 0 0 2 0 0 0 0 0 0
2 100 0 0 0 0 0 1 0 0 0 0 0 0
1+2 200 2.0 2.0 1.5 0 0 1 0 0 3 0 0 0 0 0 0
Test item: 100.0 µg/ml
1 100 0 0 0 0 0 0 0 0 0 0 0 0
2 100 0 0 1 0 0 1 0 0 0 0 0 0
1+2 200 1.0 1.0 0.5 0 0 1 0 0 1 0 0 0 0 0 0
Test item: 150.0 µg/ml
1 100 0 0 1 0 0 4 0 0 0 0 0 0
2 100 0 0 2 0 0 2 0 0 0 0 0 0
1+2 200 4.0 4.0 2.5 0 0 3 0 0 6 0 0 0 0 0 0

Table 10: Experiment II- Number of polyploid cells and mitotic index; preparation interval 18hrs without S9 mix; preparation interval 28hrs with S9 mix

Treatment group Concentration per mL S( mix Exposure period/Recovery Polyploid cells * mitotic indices **
culture total % absolute mean % ***
1 2 1 2
Neg. Control - 18/- hrs 8 8 16 1.6 11.5 11.5 11.9 100.0
Solv. Control 0.5% - 18/- hrs 7 8 15 1.5 11.9 11.9 12.9 100.0
Pos. Control 300.0 µg - 18/- hrs 5 10 15 1.5 4.4 4.4 5.1 42.9
Test item 0.47 µg - 18/- hrs 7 11 18 1.8 13.1 13.1 11.5 89.1
" 0.94 µg - 18/- hrs 7 8 15 1.5 9.7 9.7 11.0 84.9
" 1.88 µg - 18/- hrs 8 6 14 1.4 9.5 9.5 10.2 79.1
" 3.75 µg - 18/- hrs n.d. n.d. - - 2.1 2.1 1.8 13.6
" 7.5 µg - 18/- hrs n.d. n.d. - - 2.0 2.0 2.1 15.9
" 15.0 µg - 18/- hrs n.d. n.d. - - 35.8 35.8 34.5 267.1
Neg. Control - 28/- hrs 11 7 18 1.8 13.3 13.3 17.2 100.0
Solv. Control 0.5% - 28/- hrs 5 9 14 1.4 15.0 15.0 15.2 100.0
Pos. Control 300.0 µg - 28/- hrs 5 3 8 0.8 13.8 13.8 13.7 79.4
Test item 0.23 µg - 28/- hrs n.d. n.d. - - 13.5 13.5 16.4 107.9
" 0.47 µg - 28/- hrs n.d. n.d. - - 11.5 11.5 11.4 74.9
" 0.94 µg - 28/- hrs n.d. n.d. - - 13.2 13.2 13.2 87.1
" 1.88 µg - 28/- hrs n.d. n.d. - - 21.4 21.4 20.0 131.7
" 3.75 µg - 28/- hrs 37 33 70 7 20.1 20.1 18.0 118.8
" 7.5 µg - 28/- hrs 49 149 196 19.8 11.4 11.4 9.9 65.0
Neg. Control + 4/24 hrs 9 12 21 2.1 16.8 16.8 15.8 100.0
Solv. Control 0.5% + 4/24 hrs 11 15 26 2.6 17.9 17.9 18.1 100.0
Pos. Control 1.4 µg + 4/24 hrs 7 16 23 2.3 16.9 16.9 19.2 121.9
Test item 37.5 µg + 4/24 hrs 8 8 16 1.6 18.3 18.3 18.0 99.7
" 75.0 µg + 4/24 hrs 11 10 21 2.1 20.6 20.6 20.1 111.1
" 100.0 µg + 4/24 hrs 10 15 25 2.5 17.3 17.3 16.2 89.5
" 150.0 µg + 4/24 hrs n.d. n.d. - - 3.0 3.0 2.5 1.4
" 200.0 µg + 4/24 hrs n.d. n.d. - - 0.0 0.0 0.0 0.0
" 250.0 µg + 4/24 hrs n.d. n.d. - - 0.0 0.0 0.0 0.0

Table 11: Structural chromosome aberrations Experiemnt II- preparation interval 18 hrs without S9 mix; exposure period 18 hrs

Slide no. Cells scored % aberrant cells Aberrations
incl. Gaps * excl. Gaps* with exchanges gaps chromatid type chromosome typw other
gaps ig b f d ex ib if id cx ma cd
without S9 mix
Negative control
1 100 1 0 1 0 0 0 0 0 0 0 0 0
2 100 0 0 2 0 0 0 1 0 0 0 0 0
1+2 200 2.5 2.0 0.0 1 0 3 0 0 0 1 0 0 0 0 0
Solvent control : ethanol 0.5%
1 100 0 0 0 1 0 0 0 0 0 0 0 0
2 100 1 0 3 0 0 0 1 0 0 0 0 0
1+2 200 3.0 2.5 0.0 1 0 3 1 0 0 1 0 0 0 0 0
Positive control: EMS 300.0 µg/ml
1 100 1 0 16 0 0 6 6 1 0 0 6 0
2 100 0 0 13 3 0 15 0 2 0 0 1 0
1+2 200 22.5 22.5 7.5 1 0 29 3 0 21 6 3 0 0 7 0
Test item: 0.47 µg/ml
1 100 1 0 1 1 0 0 0 0 0 0 0 0
2 100 1 0 1 0 0 1 2 0 0 0 0 0
1+2 200 3.5 3.0 0.5 2 0 2 1 0 1 2 0 0 0 0 0
Test item: 0.94 µg/ml
1 100 0 0 1 0 0 0 0 0 0 0 0 0
2 100 1 0 0 0 0 0 0 0 0 0 0 0
1+2 200 1.0 0.5 0.0 1 0 1 0 0 0 0 0 0 0 0 0
Test item: 1.88 µg/ml
1 100 0 0 2 0 0 1 0 0 0 0 0 0
2 100 0 0 0 0 0 0 0 0 0 0 0 0
1+2 200 1.5 1.5 0.5 0 0 2 0 0 1 0 0 0 0 0 0

Table 12: Structural chromosome aberrations Experiment II; preparation interval 28 hrs without S9 mix; exposure period 28 hrs; preparation interval 28 hrs with S9 mix; exposure period 4 hrs

Slide no. Cells scored % aberrant cells Aberrations
incl. Gaps * excl. Gaps* with exchanges gaps chromatid type chromosome typw other
gaps ig b f d ex ib if id cx ma cd
without S9 mix
Negative control
1 100 0 0 0 0 0 0 1 0 0 0 0 0
2 100 0 0 2 0 0 0 0 0 0 0 0 0
1+2 200 1.5 1.5 0.0 0 0 2 0 0 0 1 0 0 0 0 0
Solvent control : ethanol 0.5%
1 100 0 0 3 0 0 0 0 0 0 0 0 0
2 100 0 0 0 0 0 0 0 0 0 0 0 0
1+2 200 1.5 1.5 0.0 0 0 3 0 0 0 0 0 0 0 0 0
Positive control: EMS 300.0 µg/ml
1 100 3 0 7 1 0 7 8 1 0 0 1 0
2 100 0 0 8 5 0 6 7 3 0 0 0 0
1+2 200 39.0 36.0 12.0 3 0 15 6 0 13 15 4 0 0 1 0
Test item: 3.75 µg/ml
1 100 0 0 0 0 0 0 0 1 0 0 0 0
2 100 0 0 2 0 0 3 0 0 0 0 0 0
1+2 200 1.5 1.5 1.0 0 0 2 0 0 3 0 1 0 0 0 0
with S9 mix
Negative control
1 100 0 0 0 1 0 1 0 0 0 0 1 0
2 100 0 0 0 0 0 0 0 0 0 0 0 0
1+2 200 2.0 1.5 0.5 0 0 0 1 0 1 0 0 0 0 1 0
Solvent control : ethanol 0.5%
1 100 0 0 3 0 0 0 0 0 0 0 0 0
2 100 0 0 2 0 0 0 0 2 0 0 0 0
1+2 200 5.0 3.5 0.0 0 0 5 0 0 0 0 2 0 0 0 0
Positive control: CPA 1.4 µg/ml
1 100 0 0 3 2 0 3 2 1 0 0 0 0
2 100 0 0 0 1 0 1 2 3 0 2 2 0
1+2 200 10.0 9.0 1.5 0 0 3 3 0 4 4 4 0 2 2 0
Test item: 37.5 µg/ml
1 100 0 0 1 1 0 0 0 0 0 0 0 0
2 100 0 0 1 1 0 0 0 1 0 0 1 0
1+2 200 4.0 3.0 0.0 0 0 2 2 0 0 0 1 0 0 1 0
Test item: 75.0 µg/ml
1 100 0 0 1 0 0 1 1 0 0 0 0 0
2 100 0 0 0 1 0 0 0 0 0 0 0 0
1+2 200 2.5 2.0 0.5 0 0 1 1 0 1 1 0 0 0 0 0
Test item: 100.0 µg/ml
1 100 0 0 0 0 0 0 0 0 0 0 0 0
2 100 0 0 3 0 0 0 0 0 0 0 0 0
1+2 200 1.5 1.5 0.0 0 0 3 0 0 0 0 0 0 0 0 0

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, it can be stated that under the experimental conditions reported, the test item Florhydral did not induce structural chromosomes aberrations in V19 cells (Chinese hamster cell line) when tested up to cytotoxic concentrations.
However, Florhydral is considered to inhibit mitotic processes and to induce numerical chromosome aberrations (increased rate of polyploidy cells), and to inhibit cell cycle progression (increased rate of cells with endoreduplicated chromosomes) in this chromosome aberration test the absence of metabolic activation.