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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:
experimental study
Adequacy of study:
weight of evidence
Study period:
28 April 1997 to 28 July 1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to GLP. Cytotoxicity was only evaluated during a range finding study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report date:
1997

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: (Kanpogyo Notification No. 39, Yakuhatsu Notification No. 229, jointly issued with 59 Kikyoku Notification No. 85
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
other: About Standard for Investigation by Chromosome Aberration Test using Cultured Mammalian Cells”(Kihatsu No. 143 issued by the director general of Ministry of Labor’s Labor Standards Bureau on March 18, 1987
Deviations:
not specified
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
(2R)-2-methyloctane-1,8-diamine; (2S)-2-methyloctane-1,8-diamine
EC Number:
700-111-0
Cas Number:
148528-05-6
Molecular formula:
C9H22N2
IUPAC Name:
(2R)-2-methyloctane-1,8-diamine; (2S)-2-methyloctane-1,8-diamine
Details on test material:
- Name of test material (as cited in study report): 2-methyl-1,8-octanediamine (MODA)
- Substance type: Technical material
- Physical state: Colourless fluid
- Analytical purity: 99.94%
- Impurities (identity and concentrations): Not reported
- Composition of test material, percentage of components: N/A
- Purity test date: Not reported
- Lot/batch No.: 093538
- Expiration date of the lot/batch: Not reported
- Stability under test conditions: Not reported
- Storage condition of test material: at 20±4°C
- Other:

Method

Species / strain
Species / strain / cell type:
mammalian cell line, other: The fibroblast cell line, CHL/IU, derived from the lungs of newborn female Chinese hamsters.
Details on mammalian cell type (if applicable):
- Type and identity of media:
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: Not reported
- Periodically checked for karyotype stability: Not reported
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
The presence or absence of structural and numerical aberrations was determined at 12.5, 25, 50, and 100 µg/ml in the absence of metabolic
activation with the treatment periods of 24 and 48 hrs, at 44.4, 66.7, 100, and 150 µg/ml in the presence of metabolic activation without S9 Mix, and
at 444, 667, 1,000, and 1,500 µg/ml in the presence of metabolic activation with S9 Mix.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: physiological saline and DMSO
- Justification for choice of vehicle: solubility
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Physiological saline
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Migrated to IUCLID6: MMC; Lot Number ESH5706; biochemical grade
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Migrated to IUCLID6: Lot No. ECF6942; purity, 98%; analytical grade
Details on test system and experimental conditions:
METHOD OF APPLICATION: agar plate (plating method)

DURATION
- Preincubation period: not applicable
- Exposure duration:
Dose-ranging: 24 and 48 hours (direct method), 24 hours (metabolic activation method)
Main study: 24 and 48 hours (direct method), 6 hours (metabolic activation method)

- Expression time (cells in growth medium): 72 hours
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells):

Dose-ranging: 24 and 48 hours (direct method), 24 hours (metabolic activation method)
Main study: 24 and 48 hours (direct method), 24 hours (metabolic activation method)


SELECTION AGENT (mutation assays): not used
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): 3.0% Giemsa stain


NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: 100 per replicate


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: growth inhibition

OTHER EXAMINATIONS:
- Determination of polyploidy: Structural aberrations were classified into chromatid and chromosome gaps (gaps), chromatid break (ctb),
chromatid exchange (cte), chromosome break (csb), chromosome exchange (cse), and others (o) according to the Chromosomal Aberration Atlas6).
- Determination of endoreplication:
- Other:


OTHER:
Statistics:
The incidence of cells with chromosomal aberrations at each dose was analyzed and compared between the negative control group and each test
substance group by the ¿2-test. If significant differences were found, the binominal test was conducted between the background data from the
negative control group and data from each test substance group. If significant differences were observed, Cochran-Armitage’s trend test was
conducted to determine the presence or absence of dose-dependency. The test result was judged to be positive if all these tests showed significant
differences. Otherwise, the test result was regarded as negative. The D20 value was calculated if the test result was positive. Data obtained from
the positive control group were subjected to the ¿2-test and binominal test, and the test result was regarded as positive if both tests showed
significant differences.

Results and discussion

Test resultsopen allclose all
Species / strain:
other: The fibroblast cell line, CHL/IU, derived from the lungs of newborn female Chinese hamsters.
Metabolic activation:
without
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
not determined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: The fibroblast cell line, CHL/IU, derived from the lungs of newborn female Chinese hamsters.
Metabolic activation:
with
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
not determined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not reported
- Effects of osmolality: Not reported
- Evaporation from medium: Not reported
- Water solubility:
- Precipitation:
- Other confounding effects:


RANGE-FINDING/SCREENING STUDIES:
A dose-ranging study was undertaken according to the testing guidelines in the presence of metabolic activation (using S9 Mix with a treatment
period of 6 hrs and a recovery period of 18 hrs and without using S9 Mix) and the absence of metabolic activation (with a treatment period of 24 or 48hrs) in order to select doses of the test substance for the main study by determining whether or not cell growth was inhibited2)3). The highest dose of the test substance was set at 5,000 µg/mL, followed by 1,500, 500, 150, 50, 15, and 5 µg/mL with a common ratio of about 3. The test
substance and negative control (physiological saline (JP) used as solvent) were used.

The results of the range finding study are tabulated in the "Any other information on results incl. tables" section below.


Any other information on results incl. tables

Dose-range finding test results

Direct method

24-hr

treatment

Dose (µg/mL)

0

5

15

50

150

500

1,500

5,000

Cell growth (%)*

100

102.5

84

40

12

0

0

0

48-hr treatment

Dose (µg/mL)

0

5

15

50

150

500

1,500

5,000

Cell growth (%)*

100

82

76

35

7

0

0

0

Metabolic activation method

Without S9 Mix

Dose (µg/mL)

0

5

15

50

150

500

1,500

5,000

Cell growth (%)*

100

118

112

74

21

0

0

0

With S9Mix

Dose (µg/mL)

0

5

15

50

150

500

1,500

5,000

Cell growth (%)*

100

99

96.5

94

91

64

17

0

* mean of two dishes Direct method (without metabolic activation): In the test conducted with a treatment period of 24 hrs, the incidence of cells with structural aberrations in the MODA groups was significantly and dose-dependently higher than in the negative control group: 20.0% at 50 µg/mL and 35.0% at 100 µg/mL compared to 2.0% in the negative control group. Significant, dose-dependent increases were also noted when the treatment period was extended to 48hrs: 7.5% at 50 µg/mL and 52.5% at 100 µg/mL compared to 1.5% in the negative control group. Chromatid breaks (ctb) and chromatid exchanges (cte) were the main structural aberrations regardless of the treatment period (24 or 48 hrs). The incidence of cells with numerical aberrations was similar in the test substance and negative control groups: 0.5-2.0% vs 0.0% for the treatment period of 24 hrs and 0.5-3.0% vs 0.5% for the treatment period of 48 hrs. In addition, the incidence of C-mitoses, which were not counted as aberrations but which are thought to contribute to the inhibition of the spindle formation, was 38.5% and 14.0%, respectively, when cells were treated for 24 and 48 hrs in the highest dose group (100 µg/mL). The D20 value for cells with structural aberrations was calculated to be 59.1 and 50.8 µg/mL, respectively, when cells were treated for 24 and 48 hrs. Cells with structural aberrations showed significant increases in the incidence in the positive control group treated with 0.05 µg/mL of MMC: 35.5% and 45.5%, respectively, when cells were treated for 24 and 48 hrs. The incidence of cells with numerical aberrations was 0.5% and 0.0%, respectively, when cells were treated for 24 and 48 hrs. Metabolic activation method: The incidence of cells with structural aberrations in the MODA groups increased significantly and dose-dependently compared to the negative control group (1.5%) when S9 Mix was not added with the incidence being 12.5% at 44.4 µg/mL, 23.5% at 66.7µg/mL, 28.5% at 100µg/mL and 44.5% at 150µg/mL. When S9 Mix was not added, however, the incidence of cells with structural aberrations was 2.0-3.5%, and no significant differences were noted compared to the negative control group (1.0%). No metaphase chromosomes were observed at the highest dose group (1,500 µg/mL) when S9 Mix was added. The incidence of cells with numerical aberrations increased significantly and dose-dependently at 44.4 µg/mL (4.5%), 66.7µg/mL (6.0%), and 100µg/mL (7.0%) when S9 Mix was not added, but it decreased to 2.5% at 150 µg/mL. The trend test was conducted up to 100 µg/mL because the cell growth rate was 21% at 150 µg/mL in the dose-ranging study, suggesting the presence of marked cytotoxicity at this and higher concentrations. The incidence of cells with numerical aberrations significantly increased to 5.5% at 1,000 µg/mL when S9 Mix was added. When S9 Mix was not added, the incidence of C-mitoses, which were not regarded as aberrant but were regarded as a factor that contributes to the inhibition of spindle formation, was 15.0% at 100 µg/mL and 31.5% at 150 µg/mL. The D20 value for cells with structural aberrations was 64.9 µg/mL when S9 Mix was not added. The incidence of cells with structural aberrations was 1.5% in the positive control group (B(a)P 20 µg/mL) when S9 Mix was not added, but it increased significantly to 36.0% when S9 Mix was added. The incidence of cells with numerical aberrations was 0.5% regardless of whether or nor S9 Mix was added.

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
positive with metabolic activation
positive without metabolic activation

It was concluded that MODA inhibited cell division regardless of the presence or absence of S9 Mix and induced structural chromosomal aberrations in the absence of S9 Mix under the conditions of the present study. The responses were generated at highly cytotoxic doses and should be interpreted with caution. Therefore a new in vitro micronucleus study (OECD 487, 2015) was conducted with the aim to evaluate the genotoxicity in an appropriate concentration range and with the determination of the cytotoxicity simultaneously during the main study.
Executive summary:
A study was conducted at Shin Nippon Biomedical Laboratories, Ltd, Japan, in order to evaluate whether or not the substance 2-methyl-1,8-octanediamine (MODA) induces chromosomal aberrations for the Chinese hamster's lung-derived fibroblast cell line CHL/IU. The study was conducted to GLP and according to the Japenese Guidelines 'Kanpogyo Notification No. 39, Yakuhatsu Notification No. 229, jointly issued with 59 Kikyoku Notification No. 85' and to 'Kihatsu No. 143'. The presence or absence of structural and numerical aberrations was determined at 12.5, 25, 50, and 100 µg/mL in the absence of metabolic activation with the treatment periods of 24 and 48 hrs, at 44.4, 66.7, 100, and 150 µg/mL in the presence of metabolic activation without S9 Mix, and at 444, 667, 1,000, and 1,500 µg/mL in the presence of metabolic activation with S9 Mix. The incidence of cells with structural aberrations significantly increased compared with the negative control in the absence of metabolic activation with the treatment periods of 24 and 48 hrs, as well as in the presence of metabolic activation without S9 Mix. The incidence of cells with numerical aberrations significantly increased compared to the negative control at 44.4-100 µg/mL in the presence of metabolic activation without S9 Mix and at 1,000 µg/mL in the presence of metabolic activation with S9 Mix. In addition, C-mitoses, which are not considered as aberrations but are thought to be due to the inhibition of spindle formation, were observed in high dose groups in the absence of metabolic activation and mid to high dose groups in the presence of metabolic activation without S9 Mix. The incidence of cells with structural and numerical aberrations in the negative and positive control groups was within the standard ranges of this laboratory. From these findings, it was concluded that MODA inhibited cell division regardless of the presence or absence of S9 Mix and induced structural chromosomal aberrations in the absence of S9 Mix under the conditions of the present study.

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