Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames test: non mutagenic (2 studies, OECD 471, K, rel. 1 & rel.2)
- hprt/CHO: non mutagenic (OECD 476, K, rel. 1)
- CA/HL test: non clastogenic (OECD 473, K, rel. 1)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2006-07-01 to 2006-09-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD test guideline No. 471 and in compliance with GLP
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Program (inspection date: 2005-08-30)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine gene for Salmonella and tryptophan gene for E.coli
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (S9 fraction from Sprague-Dawley rats pre-treated with phenobarbitone/B-naphtoflavone)
Test concentrations with justification for top dose:
Preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate.
Range-finding test: 50, 150, 500, 1500 and 5000 μg/plate.
Main test: 50, 150, 500, 1500 and 5000 μg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The test material was immiscible in sterile distilled water and dimethyl sulphoxide at 50 mg/mL but was fully miscible in acetone at the same concentration in solubility checks performed in-house.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Remarks:
See Table 7.6.1/1
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Remarks:
See Table 7.6.1/1
Positive control substance:
benzo(a)pyrene
other: 2-Aminoanthracene
Remarks:
With S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: ca. 48 hours at 37°C

NUMBER OF REPLICATIONS: triplicate plates per dose level

DETERMINATION OF CYTOTOXICITY
- Method: growth assessment of the bacterial background lawn


OTHER: ACCEPTANCE CRITERIA: The reverse mutation assay was considered valid if the following criteria were met:
1. All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (according to historical control 2004 & 2005).
2. The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
3. All tester strain cultures should be in the approximate range of 1 to 9.9 billion bacteria per mLl.
4. Each mean positive control value should be at least two times the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
5. There should be a minimum of four non-toxic test material dose levels.
6. There should be no evidence of excessive loss of plates due to contamination.
Evaluation criteria:
Dose-related increase in revertant frequency over the dose range tested and/or reproducible at one or more concentrations in at least one bacterial strain with or without metabolic activation.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Statistics:
Statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not applicable
- Effects of osmolality: not applicable
- Evaporation from medium: no data
- Water solubility: Test material was solubilised in acetone to improve solubility
- Precipitation: a precipitate (oily in appearance) was observed at 5000 µg/plate, this did not prevent the scoring of revertant colonies.
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: The test material was non-toxic to the bacterial background lawns of the strains of bacteria used (TA100 and WP2uvrA-). The test material formulation and S9-mix used in this experiment were both shown to be sterile.

COMPARISON WITH HISTORICAL CONTROL DATA: All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-tnix and the sensitivity of the bacterial strains.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level, although substantial decreases in revertant colony frequency were observed to all of the Salmonella strains dosed in the presence of S9 at 5000 µg/plate, The test material was, therefore, tested up to the maximum recommended dose level of 5000 ug/plate
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Preliminary test:

The numbers of revertant colonies for the toxicity assay were:

With or without metabolic activation

Strain

Dose (µg/plate)

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

-

TA100

103

91

108

110

93

99

85

88

70

87

98P

+

TA100

110

110

92

89

96

93

97

89

90

111

46P

-

WP2uvrA-

20

13

21

14

23

18

25

15

21

21

20P

+

WP2uvrA-

29

25

26

30

29

26

30

30

23

26

20P

Mutation test:

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation. See "Tables of results" in "Attached background material"

Conclusions:
The test material is not mutagenic with and without metabolic activation in S. thyphimurium strains TA1535, TA1537 TA98 & TA100, and E.coli WP2 uvrA-.
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and E.coli strain WP2 uvrA- were exposed the test material diluted in acetone both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors) using the plate incorporation method. The dose range for the range-finding test was determined in a preliminary toxicity assay and was 15 to 5000 µg/plate. The experiment was repeated on a separate day using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test material formulations.

The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn although substantial decreases in revertant colony frequency were observed to all of the Salmonella strains dosed in the presence of S9 at 5000 µg/plate. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A precipitate (oily in appearance) was observed at 5000 µg/plate, this did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation.

 

Under the test condition, the test material is not mutagenic with and without metabolic activation in S. thyphimurium strains TA1535, TA1537 TA98 & TA100, and E.coli WP2 uvrA-.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From 1994-04-20 to 1994-05-12
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to the OECD guideline No 471 (1983), therefore E. coli WP2 uvrA- or S. thyphimurium TA 102 were not tested as currently recommended.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
E. coli WP2 uvrA- or S. thyphimurium TA 102 were not tested
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Program (Inspection date: 1994-01-31)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine gene
Species / strain / cell type:
other: TA1535, TA100, TA1537, TA1538 and TA98
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction from Sprague-Dawley rat which had received a single i.p. injection of Aroclor 1254
Test concentrations with justification for top dose:
Preliminary toxicity study: 312.5, 625, 1250, 2500 and 5000 µg/plate
Mutation study - Experiment 1: 8, 40, 200, 1000 and 5000 µg/plate
Mutation study - Experiment 2: 312.5, 625, 1250, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: not soluble in water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Remarks:
See Table 7.6.1/1
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
other: 4-Nitro-o-phenylenediamine
Remarks:
Without S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Remarks:
See Table 7.6.1/1
Positive control substance:
benzo(a)pyrene
other: 2-aminoanthracene
Remarks:
With S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: approx. 48 hours at 37°C

NUMBER OF REPLICATIONS: triplicate plates per dose level, 2 experiments

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
For a substance to be considered positive in this test system, it should have induced a dose-related and statistically significant increase in mutation rate in one or more strains of bacteria in the presence and/or absence of the 59 microsomal enzymes in both experiments at sub-toxic dose levels. If the two experiments give conflicting results or equivocal results are obtained then a third experiment may be used to confirm the correct response.
Statistics:
All data are statistically analysed using the methods recommended by the UKEM5 (5) and normally Dunnett's method of linear regression is used to evaluate the result. To be Considered negative the number of induced revertants compared to spontaneous revertants should be less than two fold at each dose level employed, the intervals of which should be between 2 and 5 fold and extend to the limits imposed by toxicity, solubility or up to the maximum recommended dose of 5000 µg/plate. In this case the limiting factor was the maximum recommended dose.
Key result
Species / strain:
other: TA1535, TA1537, TA1538, TA98 and TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: A very small, statistically significant increase was observed at three of the dose levels plated out, the response was, however, weak and non-repeatable in either the first or second experiment and can be considered spurious. See Table 7.6.1/2.

COMPARISON WITH HISTORICAL CONTROL DATA: not done

ADDITIONAL INFORMATION ON CYTOTOXICITY: no toxicity was exhibited to any of the strains of Salmonella used up to 5000 µg/plate.

MUTATION STUDY: See "Table of Results" in "Attached background material".

Table 7.6.1/2: Mean number of revertant colonies for the toxicity assay

Strain

Dose (µg/plate)

0

312.5

625

1250

2500

5000

TA100

115.0

149.0

169.5

177.0

161.0

269.0

 

Conclusions:
The test material is not mutagenic with and without metabolic activation in S. thyphimurium strains TA1535, TA1537, TA1538, TA98 & TA100.
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 (1983) and in compliance with GLP, S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were exposed the test material diluted in acetone both in the presence and absence of metabolic activation system (S9 fraction from the livers of Aroclor 1254 treated rats) using the plate incorporation method. The dose range was determined in a preliminary toxicity assay and was 8 to 5000 µg/plate for the first experiment. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test material formulations. In this case the dose range was 312.5 to 5000 µg/plate.

The vehicle and positive controls induced the appropriate responses in the corresponding strains.

The test material caused no visible reduction in the growth of the bacterial lawn at any of the dose levels employed in any of the strains of Salmonella tested. It was, therefore, tested up to the maximum recommended dose of 5000 µg/plate.

No significant increase in the numbers of revertant colonies was recorded for any of the bacterial strains with any dose of the test material, either with or without metabolic activation. 

It is therefore concluded that, under the test conditions, the test material is not mutagenic with and without metabolic activation in S. thyphimurium strains TA1535, TA1537, TA1538, TA98 & TA100.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 1995-02-23 to 1995-07-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD test guideline No. 473 and in compliance with GLP
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
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Program (inspection date: 1994-01-31)
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
- Storage condition of test material: room temperature
Target gene:
not applicable
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
- Type and identity of media: Eagle's minimal essential medium, (supplemented with sodium bicarbonate, L-glutamine, penicillin streptomycin, amphotericin Band 15 % foetal calf serum) at 37 °C with 5 % CO2 in air. The lymphocytes in fresh -heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA) at 90 µg/mL final concentration.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data but not relevant
- Periodically checked for karyotype stability: no data but not relevant
- Periodically "cleansed" against high spontaneous background: not relevant but all donors used are validated for spontaneous aberration rate
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction from male Sprague-Dawley rats receiving intraperitoneally 500 mg/kg bw of Aroclor 1254
Test concentrations with justification for top dose:
- First experiment: 5, 10, 20, 40, 80 and 160 (+S9 only) µg/mL
- Second experiment: 10, 20, 40, 60, 80 µg/mL (-S9) / 20, 40, 80 , 160 and 320 µg/mL (+S9 )
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: not soluble in water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Remarks:
500 µg/mL in DMSO
Positive control substance:
ethylmethanesulphonate
Remarks:
Without S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Remarks:
25 µg/mL in medium without serum
Positive control substance:
cyclophosphamide
Remarks:
With S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
Cells cultured for 48 hours before exposure.
- Exposure duration: Experiment 1: 4h (with S9), 20h (without S9)
Experiment 2: 4h (with S9), 20h and 44h (without S9)
- Expression time (cells in growth medium): Experiment 1: 16h (after wash-off S9)
Experiment 2: 16h and 40h (after wash-off S9)
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours in Experiment 1, 20 and 44 hours in Experiment 2.

SPINDLE INHIBITOR (cytogenetic assays): Demecolcine (Colcemid) 0.1 µg/mL
STAIN (for cytogenetic assays): 5 % Gurrs Giemsa R66 for five minutes

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 per duplicate culture, 200 per dose level

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (2000 lymphocytes)

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes (included in polyploid cell frequency)
Evaluation criteria:
A positive response was recorded for a particular treatment if the % cells with aberrations markedly exceeded that seen in the concurrent control,
either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.
Statistics:
The frequency of cells with aberrations (both including and excluding gaps) and the frequency of polyploid cells was compared with the concurrent vehicle control value using Fisher's Exact test or Chi-squared test.
Key result
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: not measured
- Effects of osmolality: maximum dose lower than 10 mM, so no osmolarity effects
- Evaporation from medium: the test material is not volatile
- Water solubility: The test material was not sufficiently soluble in water for water to be used as the vehicle.
- Precipitation: none observed

RANGE-FINDING/SCREENING STUDIES: The molecular weight of the test material was calculated to be 284 and therefore the maximum dose level was originally set at 2840 µg/mL which was calculated to be equivalent to 10 mM. Following an initial experiment in which the test material proved to be toxic the first experiment was repeated with 160 µg/mL as the maximum dose level.

COMPARISON WITH HISTORICAL CONTROL DATA: Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures, these are currently in the range of 0 to 3.5% cells with structural aberrations including gaps, 0 to 2.5% cells excluding gaps and 0 to 1% polyploidy. In the Experiment 1, the frequencies of cells with chromosome aberrations in the presence of S9 were not within this expected range. This was thought by the author to be an artefactual effect induced by the S9.

ADDITIONAL INFORMATION ON CYTOTOXICITY: In Experiment 1, haemolysis was seen at 80 and 160 µg/mL, and the test material induced toxicity to the lymphocytes as indicated by the mitotic index values. In Experiment 2, haemolysis was seen with the test material at and above 80 µg/mL. See Table 7.6.1/1 to 7.6.1/6.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 7.6.1/1 Results of chromosome analysis (mean) – Experiment 1 without metabolic activation (20-hour harvest)

 

Control

10 µg/mL

20 µg/mL

40 µg/mL

EMS 500

Cytotoxicity

no

no

no

yes

yes

Number of cells scored

200

200

200

200

100

                                                   Aberrations per 100 cells

Gaps

2.0

5.5

2.5

5.0

40

Chromatid aberrations

breaks

0.5

1.5

0.0

2.5

73

exchanges

0.0

0.0

0.0

0.0

17

Chromosome aberrations

breaks

1.0

0.5

0.0

0.5

7.0

exchanges

0.0

0.0

0.0

0.0

1.0

Frequency of aberrant cell

+ Gaps

3

6.5

2.5

7.0

72

- Gaps

1.5

2.0

0.0

3.0***

58***

Mitotic index(% of control)

100

84

60

0

31

Mitotic index(mean)

3.95

3.33

2.38

-

1.23

Polyploidy(%)

0.5

0.0

0.0

0.0

0.0

*** = P < 0.001

 

Table 7.6.1/2 Results of chromosome analysis (mean) – Experiment 1 with metabolic activation (20-hour harvest)

 

Control

40 µg/mL

80 µg/mL

160 µg/mL

CP 25

Cytotoxicity

no

no

no

no

yes

Number of cells scored

200

200

200

200

200

                                                   Aberrations per 100 cells

Gaps

1.5

1.0

0.5

1.0

7.5

Chromatid aberrations

breaks

1.0

0.0

1.0

1.5

16

exchanges

0.0

0.0

0.0

0.0

3.5

Chromosome aberrations

breaks

2.5

0.5

0.0

2.5

2.5

exchanges

0.0

0.0

0.0

0.0

0.0

Frequency of aberrant cell

+ Gaps

4.5

1.5

1.5

3.5

21***

- Gaps

3.0

0.5

1.0

2.5

16.5***

Mitotic index(% of control)

100

99

70

56

41

Mitotic index (mean)

5.43

5.38

3.78

3.05

2.23

Polyploidy(%)

1.0

1.5

2.0

1.5

0.0

*** = P < 0.001

 

Table 7.6.1/3 Results of chromosome analysis (mean) – Experiment 2 without metabolic activation (20-hour harvest)

 

Control

20 µg/mL

40 µg/mL

60 µg/mL

EMS 500

Cytotoxicity

no

no

no

yes

yes

Number of cells scored

200

200

200

200

150

                                                   Aberrations per 100 cells

Gaps

2.0

2.0

2.5

2.0

30.7

Chromatid aberrations

breaks

0.5

0.5

1.0

2.0

27.3

exchanges

0.5

0.0

0.0

0.0

11.3

Chromosome aberrations

breaks

0.5

0.0

0.0

0.0

11.3

exchanges

0.0

0.0

0.0

0.0

0.0

Frequency of aberrant cell

+ Gaps

3.0

2.5

3.5

3.5

46***

- Gaps

1.0

1.0

2.0

1.5

32***

Mitotic index(% of control)

100

94

74

50

34

Mitotic index(mean)

3.23

3.03

2.40

1.60

1.10

Polyploidy(%)

0.0

0.5

0.5

0.5

0.0

*** = P < 0.001

 

Table 7.6.1/4 Results of chromosome analysis (mean) – Experiment 2 with metabolic activation (20-hour harvest)

 

Control

40 µg/mL

80 µg/mL

160 µg/mL

CP 25

Cytotoxicity

no

no

no

no

no

Number of cells scored

200

200

200

200

200

                                                   Aberrations per 100 cells

Gaps

3.0

0.0

3.5

3.0

8.0

Chromatid aberrations

breaks

1.0

0.0

0.5

0.5

3.5

exchanges

0.0

0.0

0.0

0.0

1.0

Chromosome aberrations

breaks

0.5

0.0

0.5

1.0

1.0

exchanges

0.0

0.0

0.0

0.0

0.5

Frequency of aberrant cell

+ Gaps

3.0

0.0

4.5

3.5

11**

- Gaps

1.0

0.0

1.0

1.0

5.0

Mitotic index(% of control)

100

135

170

86

58

Mitotic index (mean)

3.65

4.93

6.20

3.15

2.13

Polyploidy(%)

0.0

0.0

0.0

0.0

0.0

** = P < 0.01

 

Table 7.6.1/5 Results of chromosome analysis (mean) – Experiment 2 without metabolic activation (44-hour harvest)

 

Control

40 µg/mL

                                               Mean

Cytotoxicity

no

no

Number of cells scored

200

200

 

Aberrations per 100 cells

Gaps

1.0

1.5

Chromatid aberrations

breaks

0.0

2.0

interchanges

0.0

0.0

Chromosome aberrations

breaks

2.5

2.0

interchanges

0.0

0.0

Frequency of aberrant cell

+ Gaps

2.0

5.0

- Gaps

1.0

4.0

Mitotic index(% of control)

100

53

Mitotic index (mean)

2.80

1.48

Polyploidy(%)

0.0

1.0

 

Table 7.6.1/6 Results of chromosome analysis (mean) – Experiment 2 with metabolic activation (44-hour harvest)

 

 

Control

1600 µg/mL

                                               Mean

Cytotoxicity

no

no

Number of cells scored

200

200

 

Aberrations per 100 cells

Gaps

1.5

2.0

Chromatid aberrations

breaks

0.5

0.5

interchanges

0.0

0.0

Chromosome aberrations

breaks

1.5

2.0

interchanges

0.0

0.0

Frequency of aberrant cell

+ Gaps

3.5

3.0

- Gaps

2.0

1.5

Mitotic index(% of control)

100

126

Mitotic index (mean)

4.65

5.88

Polyploidy(%)

0.0

0.0

 

Conclusions:
Under the test conditions, the test material was considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

In an in vitro chromosome aberration test performed according to OECD guideline No 473 and in compliance with GLP, human primary lymphocyte cultures were exposed to the test material diluted in ethanol. Four treatment conditions were used, i.e. 4 hours exposure with the addition of an induced rat liver homogenate metabolising system at 10 % in standard co-factors with cell harvest after 16 and 40-hour expression periods and 20 and 44 hour continuous exposures in the absence of activation. In Experiment 1 the dose range for evaluation was selected from a series of 5 dose levels on the basis of toxicity.

In Experiment 1, both of the vehicle control cultures had frequencies of cells with chromosome aberrations that were considered to be acceptable although the values seen in the presence of S9 were not within the expected range. This was thought to be an artefactual effect induced by the S9. In Experiment 2, all vehicle controls gave frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control treatments gave significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material induced no statistically significant increases in the frequency of cells with aberrations excluding gaps or polyploidy cells.

Under the test conditions, the test material was considered to be non-clastogenic to human lymphocytes in vitro.

This study is considered as acceptable and satisfies the requirement for in vitro mammalian chromosome aberration assay.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2012-08-02 to 2012-10-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD test guideline No. 476 and in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
1998
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Monitoring Programme (inspected on 2011-08-11)
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
Storage condition of test material: Room temperature, dry area, unopened containers, optimum temperature 11-25°C
Target gene:
HPRT locus
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Cells: CHO-KI cells were originally obtained from BIBRA. The cells are stored at -196°C, in heat-inactivated foetal calf serum (HiFCS) containing 10% dimethyl sulphoxide (DMSO).
- Type and identity of media:
Ham’s Nutrient Mixture F12, supplemented with 2 mM L glutamine and 50 µg/mL gentamicin. The resulting medium is referred to as H0.
H0 medium supplemented with 10% HiFCS referred to as H10, is used for general cell culture, e.g. when growing cells up from frozen stocks.
The selective medium, in which only HPRT deficient cells will grow, consisted of H10 supplemented with 6-thioguanine (6-TG) at a final concentration of 10 µg/mL.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes, assumed to be stable
- Periodically "cleansed" against high spontaneous background: yes, 4 days prior to exposure, spontaneous mutants were eliminated from the stock cultures by incubating the cells in H10 containing 15 µg/mL hypoxanthine, 0.3 µg/mL amethopterin and 4 µg/mL thymidine for three days.
All cell cultures are maintained at 37°C in a atmosphere of 5% CO2 in air.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (see Table 7.6.1/1)
Test concentrations with justification for top dose:
Preliminary toxicity test: 22.19, 44.38, 88.75, 177.5, 355, 710, 1420, 2840 µg/mL (up to 10 mM).
Main test 1 (-S9 mix): 5, 10, 20, 40, 60, 80 and 100 µg/mL.
Main test 1 (+S9 mix): 10, 20, 80, 100, 150, 200, 250 and 300 µg/mL.
Main test 2 (-S9 mix): 10, 50, 70, 80, 90, 100 and 110 µg/mL.
Main test 2 (+S9 mix): 10, 70, 100, 150, 175, 200, 225 and 250 µg/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol (final concentration 1% v/v)
- Justification for choice of solvent/vehicle: Prior to commencing testing, the solubility of the test substance in a vehicle compatible with this test system was assessed. The test item was found to be soluble at 284 mg/mL in ethanol.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Remarks:
250 µg/mL in DMSO
Positive control substance:
ethylmethanesulphonate
Remarks:
in the absence of S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Remarks:
5 µg/mL in DMSO
Positive control substance:
3-methylcholanthrene
Remarks:
in the presence of S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
The cells were incubated for approximately 20 hours at 37°C, in an atmosphere of 5% CO2 in air, prior to exposure to the test substance on Day 1.
- Exposure duration: 3 hours.
- Expression time (cells in growth medium): 7 days, at 37°C, in a humidified atmosphere of 5% CO2 in air.

SELECTION AGENT (mutation assays): 6-thioguanine (6-TG)

NUMBER OF REPLICATIONS: duplicate cultures for each concentration of the test compound and positive controls, four individual cultures for solvent controls.

NUMBER OF CELLS EVALUATED: 10E6 cells from each culture were seeded to allow expression of the mutant phenotype.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency (200 cells/plate)
Evaluation criteria:
The demonstration of a statistically significant increase in mutant frequency following exposure to the test substance;
Evidence of a relationship, over at least two dose levels, in any increase in mutant frequency;
Demonstration of reproducibility in any increase in mutant frequency;
The mean mutant frequency should fall outside the upper limit of the historical solvent control of 20 mutants per 10E6 survivors with a corresponding survival rate of 20% or greater.
Statistics:
The statistical significance of the data was analysed by weighted analysis of variance, weighting assuming a Poisson distribution following the methods described by Arlett et al. (1989). Tests were conducted for a linear concentration-response relationship of the test substance, for non-linearity and for the comparison of positive control to solvent control.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
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 fluctuations in pH of the medium were observed at 2840 µg/mL of more than 1.0 unit compared with the vehicle control
- Effects of osmolality: The osmolality of the test substance in medium was tested at 2840 µg/mL; no fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed compared with the vehicle control.
- Evaporation from medium: not volatile
- Water solubility: not soluble in water
- Precipitation: In the preliminary toxicity test, precipitate was seen at 177.5 and 88.75 μg/mL and above post dosing in the absence and presence of S9 mix, respectively. Precipitate was seen at 88.75 μg/mL and above at the end of treatment in both the absence and presence of S9 mix. In the main mutation test 1 (+S9), precipitate was seen at 100 μg/mL and above post dosing and at 150 μg/mL and above at the end of treatment. In the main mutation test 2 (+S9), precipitate was seen at 100 μg/mL and above post dosing and at the end of treatment.
- Other confounding effects: none.

RANGE-FINDING/SCREENING STUDIES:
In the preliminary toxicity test, a four-hour exposure from 22.19 to 2840 μg/mL The test item, in the absence or the presence of S9 mix, resulted in Day 1 relative survivals of 85 to 15 % and 78 to 46 % respectively (Table 1). Precipitate was seen at 177.5 and 88.75 μg/mL and above post dosing in the absence and presence of S9 mix, respectively. Precipitate was seen at 88.75 μg/mL and above at the end of treatment in both the absence and presence of S9 mix. Concentrations for the main test were based upon these data.

COMPARISON WITH HISTORICAL CONTROL DATA:
- test 1 (-S9): Several of the test item test cultures also had mutation frequencies that exceeded the historical control maximum. However, there was no dose-response relationship or statistical significance so these data were considered to be acceptable and not to be indicative of a positive response.
- test 1 (+S9): Several of the test item test cultures also had mutation frequencies that exceeded the historical control maximum. However, there was no statistical significance so these data were considered to be acceptable and the apparent dose-related increases not to be indicative of a positive response.
- test 2 (-S9): Several of the test item test cultures also had mutation frequencies that exceeded the historical control maximum. Reproducibility in the mutation frequencies between replicate cultures at 70 and 80 μg/mL was noted to be inconsistent. However, there was no statistical significance so these data were considered to be acceptable and the apparent dose-related increases not to be indicative of a positive response.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- test 1 (-S9): Exposure to test item resulted in Day 1 relative survivals of between 116 and 24 %. The Day 8 cloning efficiencies were 101 to 95 % relative to the solvent controls.
- test 1 (+S9): Exposure to test item resulted in Day 1 relative survivals of between 106 and 23 % . The Day 8 cloning efficiencies
were 113 to 81 % relative to the solvent control.
- test 2 (-S9): Exposure to test item resulted in Day 1 relative survivals of between 104 and 17 %. The Day 8 cloning efficiencies were 113 to 91 % relative to the solvent control.
- test 2 (+S9): Exposure to test item resulted in Day 1 relative survivals of between 115 and 23 % . The Day 8 cloning efficiencies were 100 to 89 % relative to the solvent control.

Table 7.6.1/2: Summary table

Test Article

Dose Level

µg/mL

Test 1

Test 2

3 hr Treatment ‑S9‑mix

3 hr Treatment +S9‑mix

3 hr Treatment ‑S9‑mix

3 hr Treatment +S9‑mix

Mean Relative Survival (%)

Mean Mutant Frequency (x106)

Mean Relative Survival (%)

Mean Mutant Frequency (x106)

Mean Relative Survival (%)

Mean Mutant Frequency (x106)

Mean Relative Survival (%)

Mean Mutant Frequency (x106)

Ethanol

0

100

16.73

100

18.90

100

15.05

100

9.44

Test item

10

 

 

106

15.03

104

15.47

115

10.04

Test item

20

116

23.62

 

 

 

 

 

 

 Test item

50

 

 

 

 

52

20.55

 

 

Test item

60

108

17.27

 

 

 

 

 

 

Test item

70

 

 

 

 

31

28.49

113

19.15

Test item

80

64

33.38

67

19.45

17

36.50

 

 

Test item

100

24

5.50

 

 

 

 

801

16.77

Test item

150

 

 

611

20.43

 

 

511

13.77

Test item

175

 

 

 

 

 

 

231

13.70

Tes item

200

 

 

231

24.72

 

 

 

 

Ethyl methansulphonate

250

62

332.01**

 

 

84

347.95**

 

 

3-Methylcholanthrene

5

 

 

91

309.08**

 

 

88

419.36**

**        Statistically significant: p < 0.01

1         Precipitate observed by eye at the end of treatment

Conclusions:
The test item did not induce any toxicologically significant or dose-related increases in the mutant frequency at the HPRT locus in CHO cells at any dose level, either in the presence or absence of metabolic activation, in both experiments.
Executive summary:

In an in vitro mammalian cell mutation assay performed according to the OECD test guideline No. 476 and in compliance with GLP, Chinese hamster ovary cells (CHO-K1) were exposed to the test item diluted in Ethanol, in duplicate in the presence and absence of metabolic activation (S9 -mix). The entire test was repeated to confirm the result of the first test. Three-hour exposures were used both with and without activation (S9) in both tests. The dose range of the test item, plated out for expression of mutant colonies, was based on the results of a preliminary toxicity test, in which relative survival values were from 85 to 15%, and 78 to 46%, after exposure to concentrations from 22.19 to 2840 mg/mL in the absence and presence of S9 mix respectively (up to the maximum recommended level of 10 mM).

In the absence of S9 mix, in Test 1, cells were exposed to concentrations from 5 to 100 µg/mL. Day 1 survival values ranged from 116 to 24% relative to the solvent control. The test item did not statistically significantly increase the mutant frequency. 

In the presence of S9 mix, in Test 1, cells were exposed to concentrations from 10 to 300 µg/mL. Day 1 survival values ranged from 106 to 23% for relative to the solvent control. The test item did not statistically significantly increase the mutant frequency.  frequency.

In the absence of S9 mix, in Test 2, cells were exposed to concentrations from 10 to 110 µg/mL. Day 1 survival values ranged from 104 to 17% relative to the solvent control. The test item did not statistically significantly increase the mutant frequency. 

In the presence of S9 mix, in Test 2, cells were exposed to concentrations from 10 to 250 µg/mL. Day 1 survival values ranged from 115 to 23% relative to the solvent control. The test item did not statistically significantly increase the mutant frequency. 

 

The positive control treatments, both in the absence and presence of metabolic activation, induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test item did not induce any toxicologically significant or dose-related increases in the mutant frequency at the HPRT locus in CHO cells at any dose level, either in the presence or absence of metabolic activation, in both experiments.

 

This study is considered as acceptable and satisfies the requirement for the mammalian cell gene mutation endpoint.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Table 7.6/1: Summary of genotoxicity tests

Test n°

Test / Guideline

Reliability

Focus

Strains tested

Metabolic activation

Test concentration

Statement

1

 

Safepharm, 2006

Ames Test

(OECD 471)

K, rel. 1

Gene mutation

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

-S9

+S9

Up to 5000 µg/plate

-S9 : non mutagenic

+S9 : non mutagenic

2

 

Toxicol, 1994

Ames Test

(OECD 471)

S, rel. 2

Gene mutation

TA 1535

TA 1537

TA 1538

TA 98

TA 100

-S9

+S9

Up to 5000 µg/plate

-S9 : non mutagenic

+S9 : non mutagenic

3

 

HLS, 2013

CHO hprt test (OECD 476)

K, rel. 1

Gene mutation

Chines hamster ovary cells 

-S9

+S9 

Up to cytotoxicity 

-S9: not mutagenic

+S9: not mutagenic 

4

 

Safepharm, 1995

HL CAT

(OECD 473)

K, rel. 1

Chromosomal aberration

Human Lymphocytes

 -S9

+S9

Up to cytotoxicity

-S9 : non clastogenic

+S9 : non clastogenic

 

Gene mutation Assays (Tests n° 1-3):

Two Bacterial Reverse mutation Assays (Ames test) were performed according to OECD 471 test guidelines with the test material (See Table 7.6/1). Test n°1 was selected as the key study. Test 2 was used as supporting data because neither E. coli WP2 uvrA- nor S. Thyphimurium were used. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains in both tests, with any dose of the test material, either with or without metabolic activation. Both tests indicate that the test material does not induce gene mutations in bacteria whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies. The substance is therefore considered as non-mutagenic according to both Ames tests.

Inability to produce gene mutation was confirmed in mammals using an in vitro forward mutation assay in Chinese hamster ovary cells (CHO/hprt test)(Test n°3). None of the dose levels up to the cytotoxicity limit with the test material, either in the presence or absence of metabolic activation, induced significant mutant frequency increases in the initial or repeat tests. The test material does not induce forward mutations at the hprt locus in CHO cells under activation and non-activation conditions whereas both positive control chemicals (with and without metabolic activation) induced significant mutant frequency increases. The substance is therefore considered as negative for inducing forward mutations at the hprt locus in CHO cells under activation and non-activation

 

Chromosomal aberration (Test n°4)

The clastogenic potential of the test material was determined using an in vitro chromosome aberration test in human lymphocytes, which measures the potential of a substance to increase the incidence the of structural chromosome aberrations in cultured human lymphocytes.

None of the dose levels up to the cytotoxicity limit with the test material, either with or without metabolic activation, induced significant increases in the frequency of cells with aberrations in either of two experiments. The test material does not induce structural aberrations in the chromosomes of human lymphocytes under activation and non-activation conditions, whereas both positive control chemicals (with and without metabolic activation) induced significant increases in the frequency of aberrant cells. The substance is therefore considered as negative for inducing chromosomal mutations in human lymphocyte cells under activation and non-activation conditions used in this assay.

Justification for classification or non-classification

Harmonised classification:

The substance has no harmonized classification according to the Regulation (EC) No. 1272/2008 (CLP).

Self-classification:

Based on the available information, no self-classification is proposed according to the CLP or the GHS.