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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro:

Ames test (OECD 471): negative (± S9 mix)

MNT test (OECD 487): negative (± S9 mix)

CA test in Human Lymphocytes (OECD 473): negative (± S9 mix)

HPRT test (OECD 476): negative (± S9 mix)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
from 2012-07-04 to 2013-02-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, GL study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
- Type and identity of media: DMEM:F12 (Dulbecco's modified eagle medium/Ham's F12 medium, mixture 1:1) supplemented with 200 mM GlutaMax
- Properly maintained: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Mammalian Microsomal Fraction S9 Mix
Test concentrations with justification for top dose:
pre-test on cytotoxicity: up to 2500.0 µg/mL (approx. 10 mM)

Further concentrations please see Table 1

The highest treatment concentration in this study, 2500.0 µg/mL (approx. 10 mM) was chosen with regard to the molecular weight and the purity of the test item and with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol; final concentration of ethanol in the culture medium was 0.5 % (v/v)
- Justification for choice of solvent: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
Without metabolic activation
Positive control substance:
mitomycin C
other: Demecolcin
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
With metabolic activation
Positive control substance:
cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48h
- Exposure duration: 4h with and without S9 mix (experiment 1) or 4h with S9 mix and 20h without S9 mix (experiment II) (see Table 1)
- Expression time (cells in growth medium): cells exposed for 4h have 16h recovery period before fixation, no recovery period for 20h exposure cells
- Fixation time (start of exposure up to fixation or harvest of cells): 20h with Cytochalasin B (4 µg/mL)
- Cells were prepared 40 hrs after start of the exposure

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B (4 µg/mL)
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED:
cytotoxic effect the CBPI: ca 500 cells per culture and cytotoxicity is expressed as % cytostasis
micronuclei effects: 1000 - 2000 binucleate cells per culture. The frequency of micronucleated cells was reported as % micronucleated cells

DETERMINATION OF CYTOTOXICITY
- percentages of reduction in the CBPI (cytokinesis-block proliferating index) in comparison with the controls (% cytostasis) by counting 500 cells per culture in duplicate
- Exposure time 4 hrs (with and without S9 mix), cells were prepared 40 hrs after start of the exposure


Evaluation criteria:
cytotoxic effect: percentages of reduction in the CBPI (cytokinesis-block proliferating index) in comparison with the controls (% cytostasis)

micronuclei effects: 1000 binucleate cells per culture. The frequency of micronucleated cells was reported as % micronucleated cells

criteria for the evaluation of micronuclei:
The micronuclei were counted in cells showing a clearly visible cytoplasm area. The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus. At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. The frequency of micronucleated cells was reported as % micronucleated cells. To describe a cytotoxic effect the CBPI was determined in approximately 500 cells per culture and cytotoxicity is expressed as % cytostasis. A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.


A test item can be classified as non-mutagenic if:
- the number of micronucleated cells in all evaluated dose groups is in the range of the laboratory historical control data and/or
- no statistically significant or concentration-related increase in the number of micronucleated cells is observed.

A test item can be classified as mutagenic if:
- the number of micronucleated cells is not in the range of the historical laboratory control data and
- either a concentration-related increase of micronucleated cells in three test groups or a statistically significant increase of the number of micronucleated cells is observed.
Statistics:
For statistical significance: Chi square test
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolarity: decreased in Experiment I
- Evaporation from medium: no
- Water solubility: Phase separation in Experiment I at 87.0 µg/mL and above without S9 mix and at 28.4 µg/mL and above with S9 mix; In Experiment II at 266.6 µg/mL without S9 mix and at 28.4 µg/mL and above with S9 mix
- Precipitation: no
- Other confounding effects: no

RANGE-FINDING/SCREENING STUDIES:

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: no

Table2: Summary of results of the in vitro micronucleus test in human lymphocytes with AMBERWOOD F

Exp.

Preparation

Test item

Proliferation

Cytostasis

Micronucleated

 

interval

concentration

index

in %*

cells

 

 

in µg/mL

CBPI

 

in %**

Exposure period 4 hrs without S9 mix

I

40 hrs

Solvent control1

2.07

 

0.40

 

 

Positive control2

1.87

18.7

 4.00S

 

 

49.7

1.90

16.1

0.45

 

 

266.6PS

1.86

19.5

0.25

 

 

816.3PS

1.56

47.9

0.50

 

 

1428.6PS

1.43

59.4

0.40

Exposure period 20 hrs without S9 mix

II

40 hrs

Solvent control1

1.76

 

0.60

 

 

Positive control3

1.43

43.8

 3.65S

 

 

16.2

1.68

10.7

0.95

 

 

28.4

1.58

23.5

0.55

 

 

49.7

1.17

77.9

0.60

*     For the positive control groups and the test item treatment groups the values are related to the solvent controls

**    The number of micronucleated cells was determined in a sample of 2000 binucleated cells

PS    Phase separation occurred at the end of treatment

S     The number of micronucleated cells is statistically significantly higher than corresponding control values

1     Ethanol            0.5 % (v/v)
2
           MMC                1.0 µg/mL
3
           Demecolcin  150.0 ng/mL

Table 2, cont.: Summary of results of the in vitro micronucleus test in human lymphocytes with AMBERWOOD F

Exp.

Preparation

Test item

Proliferation

Cytostasis

Micronucleated

 

interval

concentration

index

in %*

cells

 

 

in µg/mL

CBPI

 

in %**

Exposure period 4 hrs with S9 mix

I

40 hrs

Solvent control1

1.91

 

0.25

 

 

Positive control2

1.48

47.4

 6.55S

 

 

16.2

1.84

7.4

0.35

 

 

266.6PS

1.80

12.1

0.45

 

 

466.5PS

1.72

20.5

0.30

 

 

816.3PS

1.53

42.1

0.35

II

40 hrs

Solvent control1

1.65

 

0.55

 

 

Positive control3

1.20

68.5

 3.55S

 

 

16.2

1.68

n.c.

0.25

 

 

466.5PS

1.56

13.7

0.45

 

 

816.3PS

1.57

12.5

0.70

 

 

1428.6PS

1.30

54.5

0.70

*     For the positive control groups and the test item treatment groups the values are related to the solvent controls

**    The number of micronucleated cells was determined in a sample of 2000 binucleated cells

PS    Phase separation occurred at the end of treatment

S     The number of micronucleated cells is statistically significantly higher than corresponding control values

1     Ethanol   0.5 % (v/v)
2
           CPA      12.5 µg/mL
3
           CPA      20.0 µg/mL

Conclusions:
negative
Under the experimental conditions reported, the test item (ethoxymethoxy)cyclododecane did not induce micronuclei in human lymphocytes in vitro when tested up to cytotoxic or the highest evaluable concentrations
Executive summary:

The test item, dissolved in ethanol, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix.

Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure periods were 4 hours with S9 mix and 20 hours without S9 mix. The cells were prepared 40 hours after start of treatment with the test item.

In each experimental group two parallel cultures were analysed. 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. To determine a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is described as % cytostasis.

The highest treatment concentration in this study, 2500.0 µg/mL (approx. 10 mM) was chosen with regard to the molecular weight and the purity (97.7 %) of the test item and with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test.

No precipitation of the test item in the culture medium was observed. No relevant influence on pH value was observed. Osmolarity was decreased in Experiment I. Phase separation was observed in Experiment I at 87.0 µg/mL and above in the absence of S9 mix and at 28.4 µg/mL and above in the presence of S9 mix. In Experiment II phase separation was observed at 266.6 µg/mL in the absence of S9 mix and at 28.4 µg/mL and above in the presence of S9 mix.

In Experiment I and II in the absence of S9 mix and in Experiment II in the presence of S9 mix cytotoxicity was observed at the highest evaluated concentration (59.4, 77.9 and 54.5 % cytostasis, respectively). In Experiment I in the presence of S9 mix no clear cytotoxicity was observed up to the highest applied concentration.

In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying micronuclei was observed. The micronucleus rates of the cells after treatment with the test item (0.25 –0.95 % micronucleated cells) were close to the range of the solvent control values (0.25 –0.60 % micronucleated cells) and within the range of the laboratory historical control data.

In both experiments, either Demecolcin (150.0 ng/mL), MMC (1.0 µg/mL) or CPA (12.5 or 20.0 µg/mL) were used as positive controls and showed distinct increases in cells with micronuclei.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei in human lymphocytes in vitro when tested up to cytotoxic or the highest evaluable concentrations.

 

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2012-04-30 to 2012-08-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to OECD TG 471
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Experiment I (pre-experiment, plate incorporation test): 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Experiment II (pre-incubation test):
TA1535, TA98, WP2 uvra: 33, 100, 333, 1000, 2500 and 5000 µg/plate
TA1537, TA100: 1, 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.
Untreated negative controls:
yes
Remarks:
concurrent untreated
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 h

NUMBER OF CELLS EVALUATED: 10^8-10^9 cells/mL

DETERMINATION OF CYTOTOXICITY
- Method: a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
at 5000 µ/plate (-S-9mix) and 2500-5000 µg/plate (+S-9mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate Experiment II
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate Experiment I
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 2500 µg/plate Experiment I and 5000 µg/plate Experiments I and II
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
at 5000 µ/plate (-S-9mix) and 2500-5000 µg/plate (+S-9mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 333-5000 µg/plate Experiment I
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1000-5000 µg/plate Experiment I and 2500-5000 µg/plate Experiment II
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
at 5000 µg/plate (-S-9mix) and 2500-5000 µg/plate (+S-9mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Reduced background growth was observed in strain TA100 without S9 mix from 333 – 5000 µg/plate in experiment I. No reduction of the background growth was observed in the remaining strains and in experiment II.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups at the following concentrations (µg/plate):

Strain

Experiment I

Experiment II

 

without S9 mix

with S9 mix

without S9 mix

with S9 mix

TA1535

/

/

/

5000

TA1537

5000

2500, 5000

/

5000

TA98

/

/

/

/

TA100

333 – 5000

1000 – 5000

/

2500 – 5000

WP2 uvrA

/

/

/

/

/ no toxic effects observed

Conclusions:
negative
It can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.
Executive summary:

This study was performed to investigate the potential of the substance to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and

TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II,

TA 1535, TA 98, WP2 uvrA: 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II, TA 1537, TA 100: 1; 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Reduced background growth was observed in strain TA 100 without S9 mix from 333 - 5000 µg/plate in experiment I. No reduction of the background growth was observed in the remaining strains and in experiment II.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed in strains TA 1537 and TA 100 with and without S9 mix in experiment I. In experiment II, toxic effects were observed in strains TA 1535, TA 1537, and TA 100 with S9 mix.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.

Therefore, the substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2012-06-26 to 2013-04-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to guidelines
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: Minimal Essential Medium (MEM) containing Hank's salts supplemented with 10 % foetal bovine serum (except during 4 hour treatment), neomycin (5 µg/mL) and amphotericin B (1 %)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Experiment I:
4-hour exposure group (-S9 mix): 0.2, 0.3, 0.6, 1.3, 2.5
4-hour exposure group (+S9 mix): 10.0, 20.0, 80.0, 160.0, 320.0

Experiment II:
24-hour exposure group (-S9 mix): 1.3, 2.5, 5.0, 10.0, 20.0
4-hour exposure group (+S9 mix): 20.0, 40.0, 80.0, 160.0, 320.0
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties and its non-toxicity to the cells. The final concentration of ethanol in the culture medium was 0.5 % v/v.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with S9mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 h
- Exposure duration: 4 h (±S9 mix), 24 h (-S9 mix)
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): about 8 days
- Fixation time (start of exposure up to harvest of cells): 7 or 8 days

SELECTION AGENT (mutation assays): 6-Thioguanine
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): 10 % methylene blue in 0.01 % KOH solution

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 1.5x10^6

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points. A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concenentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory's historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 2.5 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above 20.0 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 20.0 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: no

RANGE-FINDING/SCREENING STUDIES: yes

COMPARISON WITH HISTORICAL CONTROL DATA: yes
Remarks on result:
other: 4-hour exposure (Experiment 1)
Conclusions:
negative
The test item was shown to be non-mutagenic to V79 cells at the HPRT locus under the conditions of the test.
Executive summary:

The study was performed to investigate the potential of the substance to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The test methods described are designed to be compatible with the OECD Guidelines for Testing of Chemicals No. 476 "In Vitro Mammalian Cell Gene Mutation Tests" and Method B17 of Commission Regulation (EC) No 440/2008 of 30 May 2008. The study is comprised of a pre-experiment and two independent main experiments. In the pre-experiment the cell cultures were treated with the test item for 4 hours with metabolic activation and for 4 and 24 hours without metabolic activation. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The maximum concentration of 2500 µg/mL used in the pre-experiment was equal to a molar concentration of about 10 mM. The concentration range of the main experiments was limited by cytotoxic effects and phase separation. Ethanol was used as solvent. The maximum concentration of 2500 µg/mL used in the pre-experiment was equal to a molar concentration of about 10 mM. The concentration range of the main experiments was limited by cytotoxic effects and phase separation. Ethanol was used as solvent. The dose range of the test item was selected based on the results of a preliminary cytotoxicity test and were as follows:

Exposure Group

Concentration of test item (µg/mL)

4-hour

(-S9 mix)

0.04

0.1

0.2

0.3

0.6

1.3

2.5

5.0

10.0

20.0

4-hour

(+S9 mix)

10.0

20.0

40.0

80.0

1600

320.0

 

 

 

 

24-hour

(-S9 mix)

1.3

2.5

5.0

10.0

20.0

30.0

 

 

 

 

4-hour

(+S9 mix)

10.0

20.0

40.0

80.0

160.0

320.0

 

 

 

 

The evaluated experimental points and the results are summarised in Table 1 (see Attached document).

No substantial and reproducible dose dependent increase of the mutation frequency was observed in either of the main experiments.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. It can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the substance is considered to be non-mutagenic in this HPRT assay.

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:
2014-07-23 to 2014-11-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
According to OECD guideline 473 Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
Qualifier:
according to
Guideline:
other: • Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
- Type and identity of media: Dulbeccos's modified Eagle's medium/Ham's F12 medium
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9
Test concentrations with justification for top dose:
With metabolic activation:
Experiment IA: 15.8, 27.6, 48.2, 84.4, 147.7, 258.6, 452.5, 791.8, 1385.7, 2425.0 µg/mL
Experiment IIA: 6.3, 12.5, 25.0, 50.0, 60.0, 70.0, 80.0, 90.0, 100.0, 200.0 µg/mL
Experiment IIB: 10.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0, 600.0, 1000.0, 2425.0 µg/mL

Without metabolic activation:
Experiment IA: 5.1, 9.0, 15.8, 27.6, 48.2, 84.4, 147.7, 258.6, 452.5, 791.8, 1385.7, 2425.0 µg/mL
Experiment IB: 10.0, 25.0, 50.0, 200.0, 400.0, 800.0, 1000.0, 1200.0, 1400.0, 1600.0, 2000.0, 2425.0 µg/mL
Experiment IIA: 5.1, 9.0, 15.8, 27.6, 48.2, 84.4, 147.7, 258.6, 452.5, 791.8, 1385.7, 2425.0 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: solubility and relatively low cytotoxicity in accordance to the OECD Guideline 473
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Details on test system and experimental conditions:
Four independent experiments were performed. In Experiment IA the exposure period was 4 hours with and without S9 mix. In Experiment IB the exposure period was 4 hours without S9 mix. In Experiment IIA the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. In Experiment IIB the exposure period was 4 hours with S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item. Evaluation of two cultures per dose group.
METHOD OF APPLICATION: in culture medium

DURATION
- Exposure duration: 4 hours (+/- S9 mix) and 22 hours (- S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: about 1.5

NUMBER OF CELLS EVALUATED: At least 100 per culture, except for the positive control in Experiment IIA, in the absence of S9 mix, where only 50 metaphases were evaluated due to strong clastogenic effects

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index


Evaluation criteria:
Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe 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. At least 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides, except for the positive control in Experiment IIA, in the absence of S9 mix, where only 50 metaphases were evaluated due to strong clastogenic effects.
Only metaphases with characteristic chromosome numbers of 46 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.
In addition, the number of polyploid cells in 500 metaphases per culture was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype). Additionally the number of endomitotic cells scored at the evaluation of polyploid cells was noticed and reported (% endomitotic metaphases).
Statistics:
Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The test item Amberwood F, dissolved in ethanol, was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix.
Four independent experiments were performed. In Experiment IA the exposure period was 4 hours with and without S9 mix. In Experiment IB the exposure period was 4 hours without S9 mix. In Experiment IIA the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. In Experiment IIB the exposure period was 4 hours with S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item.
In each experimental group two parallel cultures were analysed. At least 100 metaphases per culture were evaluated for structural chromosomal aberrations, except for the positive control in Experiment IIA, in the absence of S9 mix, where only 50 metaphases were evaluated due to strong clastogenic effects. 1000 cells were counted per culture for determination of the mitotic index.
The highest treatment concentration in this study, 2425.0 µg/mL (approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the OECD Guideline for in vitro mammalian cytogenetic tests.
No visible precipitation of the test item in the culture medium was observed.
Phase separation was observed in Experiment IA at 48.2 µg/mL and above in the absence and presence of S9 mix, in Experiment IB at 200.0 µg/mL and above in the absence of S9 mix, in Experiment IIA at 27.6 µg/mL and above in the absence of S9 mix and at 50.0 µg/mL and above in Experiment IIA and IIB in the presence of S9 mix.
No relevant influence on pH was observed. In Experiment IIA osmolarity was decreased in a concentration range of 147.7 to 2425.0 µg/mL.
In Experiment IA and IIA in the absence of S9 mix, cytotoxicity was observed at the highest evaluated concentration (48.0 and 50.2 % of control, respectively). In Experiment IA in the presence of S9 mix and in Experiment IB in the absence of S9 mix, concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage. In Experiment IIB in the presence of S9 mix no cytotoxicity was observed up to the highest applied concentration.
In Experiment IA in the absence of S9 mix, statistically significant increases in chromosomal aberrations were observed after treatment with 27.6 and 1385.7 µg/mL (3.0 and 5.5 % aberrant cells, excluding gaps) (Table 8). The second value exceeded the range of the laboratory historical solvent control data (0.0 – 3.0 % aberrant cells, excluding gaps), but no dose-dependency was observed. In Experiment IB this finding could not be confirmed.
In Experiment IIA in the absence of S9 mix and in Experiment IA and IIB in the presence of S9 mix no biologically relevant increases in the number of cells carrying structural chromosome aberrations were observed (see Table 9, Table 11, Table 13, and Table 15). In these experiments the aberration rates of the cells after treatment with the test item (0.0 – 3.0 % aberrant cells, excluding gaps) slightly exceeded the range of the solvent control values (1.5 – 2.0 % aberrant cells, excluding gaps), but were within the range of the laboratory historical solvent control data (see Appendix 2).
No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.
In both experiments, either EMS (550.0 or 770.0 µg/mL) or CPA (15.0 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Table2     Summary of results

Exp.

Preparationinterval

Test itemconcentration
in µg/mL

Mitotic indices
in %
of control

Aberrant cells
in %

 

 

 

incl. gaps*

excl. gaps*

carrying exchanges

 

Exposure period 4 hrs without S9 mix

IA

22 hrs

Solvent control1

100.0

0.5

0.5

0.0

 

 

Positive control2

65.6

11.0

10.5S

1.5

 

 

27.6

113.9

3.5

3.0S

0.0

 

 

452.5PS

106.6

2.5

2.0

0.0

 

 

791.8PS

79.9

1.0

1.0

0.0

 

 

1385.7PS#

48.0

5.5

5.5S

0.0

IB

22 hrs

Solvent control1

100.0

1.5

1.5

0.0

 

 

Positive control2

62.5

12.0

10.5S

2.0

 

 

50.0

85.6

0.0

0.0

0.0

 

 

1200.0PS

78.4

2.0

2.0

0.0

 

 

1400.0PS

62.9

3.0

3.0

0.0

 

 

1600.0PS

74.9

0.5

0.0

0.0

 

 

2000.0PS

67.7

1.5

1.5

0.0

Exposure period 22 hrs without S9 mix

IIA

22 hrs

Solvent control1

100.0

1.5

1.5

0.0

 

 

Positive control3##

34.6

39.0

39.0S

10.0

 

 

15.8

120.6

1.5

1.5

0.0

 

 

84.4PS

118.5

2.5

2.5

0.0

 

 

147.7PS

97.9

2.5

2.5

0.0

 

 

258.6PS

50.2

1.0

1.0

0.0

*   Including cells carrying exchanges

#    Evaluation of 200 metaphases per culture

##  Evaluation of 50 metaphases per culture

PS  Phase separation occurred at the end of treatment

S    Aberration frequency statistically significant higher than corresponding control values

1    Ethanol       0.5 % (v/v)

2     EMS       770.0 µg/mL

3     EMS       550.0 µg/mL


Table 2, cont.  Summary of results

Exp.

Preparationinterval

Test itemconcentration
in µg/mL

Mitotic indices
in %
of control

Aberrant cells
in %

 

 

 

incl. gaps*

excl. gaps*

carrying exchanges

 

Exposure period 4 hrs with S9 mix

IA

22 hrs

Solvent control1

100.0

2.5

2.0

0.0

 

 

Positive control2

22.7

19.0

15.5S

1.5

 

 

15.8

80.0

1.5

1.5

0.0

 

 

27.6

98.4

0.5

0.0

0.0

 

 

48.2PS

73.7

3.0

2.5

0.0

IIB

22 hrs

Solvent control1

100.0

1.5

1.5

0.0

 

 

Positive control2

32.4

18.5

17.5S

6.0

 

 

25.0

106.1

1.5

1.5

0.0

 

 

600.0PS

98.4

2.5

2.5

0.0

 

 

1000.0PS

85.1

1.5

1.5

0.0

 

 

2425.0PS

84.5

2.5

2.5

0.0

*   Including cells carrying exchanges

PS  Phase separation occurred at the end of treatment

S    Aberration frequency statistically significant higher than corresponding control values

1    Ethanol       0.5 % (v/v)

2    CPA          15.0 µg/mL

Conclusions:
In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro. Therefore, Amberwood F is considered to be non-clastogenic in this chromosome aberration test, when tested up to cytotoxic or the highest evaluable concentration.
Executive summary:

The test item Amberwood F, dissolved in ethanol, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytesin vitroin four independent experiments. The following study design was performed:

 

Without S9 mix

With S9 mix

 

Exp. IA & IB

Exp. IIA

Exp. IA, IIA & IIB

Exposure period

 4 hrs

22 hrs

 4 hrs

Recovery

18 hrs

18 hrs

Preparation interval

22 hrs

22 hrs

22 hrs

In each experimental group two parallel cultures were analysed. Per culture at least 100 metaphases were evaluated for structural chromosomal aberrations, except for the positive control in Experiment IIA, in the absence of S9 mix, where only 50 metaphases were evaluated.

The highest applied concentration in this study (2425.0 µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 473.

Dose selection of the cytogenetic experiment was performed considering the toxicity data in accordance with OECD Guideline 473. The rationale for the dose selection is reported in section3.5.1. The chosen treatment concentrations are reported inTable 1and the results are summarised inTable 2.

In Experiment IA and IIA in the absence of S9 mix, cytotoxicity was observed at the highest evaluated concentration. In Experiment IA in the presence of S9 mix and in Experiment IB in the absence of S9 mix, concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage. In Experiment IIB in the presence of S9 mix no cytotoxicity was observed up to the highest applied concentration.

In Experiment IA in the absence of S9 mix, statistically significant increases in chromosomal aberrations were observed after treatment with 27.6 and 1385.7 µg/mL (3.0 and 5.5 % aberrant cells, excluding gaps). The second value exceeded the range of the laboratory historical solvent control data (0.0 – 3.0 % aberrant cells, excluding gaps), but no dose-dependency was observed. In Experiment IB this finding could not be confirmed. In Experiment IIA in the absence of S9 mix and in Experiment IA and IIB in the presence of S9 mix no relevant increases in chromosomal aberrations were observed at the concentrations evaluated for cytogenetic damage.

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with structural chromosome aberrations.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Genetic toxicity

Four genetic toxicity in vitro assays were conducted with the substance covering different modes of action of genetic toxicity. The substance did not produce significant genetic toxicity in a reliable bacterial reverse mutation assay with S. typhimurium and E. coli (2012b), in an in vitro micronucleus assay with human lymphocytes (2013b), in an in vitro Gene Mutation Assay in Chinese Hamster V79 Cells (V79/HPRT, 2013c) and in an in vitro chromosome aberration assay in human lymphocytes (2014). It is therefore concluded that the substance is not genotoxic. Further testing on the genetic toxicity of the substance is not required.

Justification for classification or non-classification

The substance was not genotoxic in a battery of four reliable in vitro assays. The substance is considered to be not genotoxic. The substance does not need to be classified for this endpoint.