Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial Reverse Mutation: Negative (non-mutagenic); OECD 471; Bowles (2011)

In Vitro Mammalian Chromosome Aberration Test: Negative (non-clastogenic); OECD 473; Bohnenberger (2012)

Mammalian Cell Gene Mutation Assay: Negative (non-mutagenic); OECD 476; Morris (2012)

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:
The experimental phase of this study was performed between 09 June 2011 and 15 August 2011.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
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 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
Meets the requirements of the Japanese Regulatory Authorities including METI, MHLW and MAFF, OECD Guidelines for Testing of Chemicals No. 471 "and the USA, EPA (TSCA) OPPTS harmonised guidelines.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine for Salmonella.
Tryptophan for E.coli
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable.
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/beta­naphthoflavone induced rat liver, S9
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
main test:
Experiment one: 50, 150, 500, 1500 and 5000 µg/plate
Experiment two: 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Sterile distilled water.
- Justification for choice of solvent/vehicle: The test material was fully soluble in sterile distilled water at 50 mg/ml in solubility checks performed in-house
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA100
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 1 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1535
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 2 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1537
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 2 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of WP2uvrA
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 10 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA98
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With S9 mix Migrated to IUCLID6: Benzo(a)pyrene: 5 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA98
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9 mix Migrated to IUCLID6: 4-Nitroquinoline-1-oxide: 0.2 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1537
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water.
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix Migrated to IUCLID6: 9-Aminoacridine: 80 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA100
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water.
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 3 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1535
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 5 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of WP2uvrA
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 2 µg/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) for Experiment 1 and 20 minutes pre-incubation at 37 deg C in Experiment 2.

DURATION
- Preincubation period for bacterial strains: 10h
- Exposure duration: Approximately 42 h
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.

Evaluation criteria:
Acceptance Criteria:

The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
All tester strain cultures should be in the approximate range of 1 to 9.9 x 109 bacteria per ml.
Each mean positive control value should be at least twice 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.
There should be a minimum of four non-toxic test material dose levels.
There should not be an excessive loss of plates due to contamination.

Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, 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.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.
Statistics:
Standard deviation
Statistical analysis of data as determined by UKEMS Dunnett's t test
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
Tested up to maximum recommended dose of 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
Tested up to maximum recommended dose of 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test item was fully soluble in sterile distilled water at 50 mg/ml in solubility checks performed in-house.
- Precipitation: No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

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

COMPARISON WITH HISTORICAL CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.

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-mix and the sensitivity of the bacterial strains.

ADDITIONAL INFORMATION ON CYTOTOXICITY: None
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Preliminary ToxicityTest

The test item was non-toxic to the strains of bacteria used (TA100 and WP2uvrA). The test item formulation and S9-mix used in this experiment were both shown to be sterile.

The numbers of revertant colonies for the toxicity assay were:

With (+) or without (-) S9-mix

Strain

Dose (µg/plate)

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

-

TA100

113

95

130

119

107

103

108

104

105

111

87

+

TA100

74

82

82

77

78

77

67

71

72

74

77

-

WP2uvrA

42

36

43

46

43

43

40

43

40

45

42

+

WP2uvrA

53

41

40

50

38

37

53

48

48

42

39

MutationTest

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile.

Results for the negative controls (spontaneous mutation rates) are presented in Table1(below) and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations for the test item, vehicle and positive controls both with and without metabolic activation, are presented in Attached Background Material.

Graphical data is also presented in Attached Background Material.

A history profile of vehicle and positive control values is presented in Attached Background Material.

The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

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 or exposure method.

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-mix and the sensitivity of the bacterial strains.


Table1               Spontaneous Mutation Rates (Concurrent Negative Controls

Range-finding Test

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

107

 

30

 

34

 

11

 

12

 

130

(110)

21

(22)

35

(30)

16

(15)

5

(8)

94

 

14

 

21

 

17

 

8

 

Main Test

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

104

 

18

 

23

 

44

 

8

 

95

(99)

25

(21)

20

(21)

19

(26)

8

(7)

97

 

20

 

20

 

15

 

5

 

Conclusions:
The test item was considered to be non-mutagenic under the conditions of this test.
Executive summary:

The method was designed to conform to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the, EPA (TSCA) OPPTS harmonised guidelines.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item, using both the Ames plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate (active ingredient). The experiment was repeated on a separate day (pre-incubation method) using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test item formulations.

The vehicle (sterile distilled water) 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 item caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate (active ingredient). No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

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

The test item was considered to be non-mutagenic under the conditions of this test.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Starting Date: June 20, 2011. Experimental Completion Date: August 19, 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
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
Target gene:
Not applicable.
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; mixture 1:1
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/beta­naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
With metabolic activation:
Experiment I: 33.9, 59.4, 103.9, 181.9, 318.3, 557.1, 974.9, 1706.1, 2985.7, 5225.0 µg/mL
Experiment IIA: 557.1, 974.9, 1706.1, 2985.7, 5225.0 µg/mL

Without metabolic activation:
Experiment I: 33.9, 59.4, 103.9, 181.9, 318.3, 557.1, 974.9, 1706.1, 2985.7, 5225.0 µg/mL
Experiment IIA: 103.9, 181.9, 318.3, 557.1, 974.9, 1706.1, 2985.7, 5225.0 µg/mL
Experiment IIB: 1000.0, 2000.0, 3000.0, 4000.0, 4500.0, 5225.0 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Deionised water
- Justification for choice of solvent/vehicle: The solvvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Used without metabolic activation Migrated to IUCLID6: (EMS)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Used with metabolic activation Migrated to IUCLID6: (CPA)
Details on test system and experimental conditions:
Three independent experiments were performed. In Experiment I the exposure period was 4 hours with and 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 22 hours without S9 mix. The chromosomes were prepared 22 hours (Exp. I, IIA & IIB) after the 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 In each experimental concentration two parallel cultures were analysed.


NUMBER OF CELLS EVALUATED: At least 100 metaphases per culture were scored for structural chromosomal aberrations, except for the positive control in Experiment IIA, in the absence of S9 mix, where only 50 metaphases were scored


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

The slides were evaluated (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100 x oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates . Only metaphases with 46 ± 1 centromer regions were included in the analysis. To describe a cytotoxic effect, the mitotic index (% cells in mitosis) was determined.


Evaluation criteria:
Evaluation of Results:
A test item is classified as non-mutagenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of the historical control data.
- no significant increase of the number of structural chromosome aberrations is observed.

A test item is classified as mutagenic if:
- the number of induced structural chromosome aberrations is not in the range of the historical control data and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criterion is valid:
The assay can indicate an aneugenic potential of the test item if:
- the number of induced numerical aberrations is not in the range of the historical control data.
Statistics:
Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05). Both biological and statistical significance should be considered together. If the above mentioned criteria for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
Species / strain:
lymphocytes: : (human)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The test item , dissolved in deionised water, 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.

Three independent experiments were performed. In Experiment I the exposure period was 4 hours with and 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 22 hours without S9 mix. The chromosomes were prepared 22 hours (Exp. I, IIA & IIB) after the start of treatment with the test item.

In each experimental group two parallel cultures were analysed. At least 100 metaphases per culture were scored for structural chromosomal aberrations, except for the positive control in Experiment IIA, in the absence of S9 mix, where only 50 metaphases were scored. 1000 cells were counted per culture for determination of the mitotic index.

The highest treatment concentration in this study, 5225.0 µg/mL was chosen with regard to the water content (4.3 %) of the test item and with respect to the OECD Guideline for in vitro mammalian cytogenetic tests.

No precipitation of the test item in the culture medium was observed. No relevant influence on osmolarity was observed, except for Experiment IIA, where osmolarity was slightly increased at the highest applied concentration. In Experiment I and IIA in the absence and presence of S9 mix, in a range of 974.9 and 5225.0 µg/mL and in Experiment IIB in the absence of S9 mix, in a range of 1000.0 and 5225.0 µg/mL the pH was adjusted to physiological values using small amounts of 2 N NaOH (Exp. I: solvent control: 305 mOsm, pH 7.2 versus 328 mOsm and pH 7.2 at 5225.0 µg/mL; Exp. IIA: solvent control: 260 mOsm, pH 7.2 versus 316 mOsm and pH 7.1 at 5225.0 µg/mL; Exp. IIB: solvent control: 275 mOsm, pH 7.3 versus 310 mOsm and pH 7.1 at 5225.0 µg/mL).

No relevant cytotoxicity indicated by reduced mitotic indices could be observed up to the highest applied concentration.

Neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. The aberration rates of the cells after treatment with the test item (0.5 - 3.8 % aberrant cells, excluding gaps) slightly exceeded the range of the solvent controls (0.5 - 1.5 % aberrant cells, excluding gaps) and the range of the laboratory historical solvent control data (0.0 - 3.0 % aberrant cells, excluding gaps). In Experiment IIA in the absence of S9 mix one single value (3.8 % aberrant cells, excluding gaps) slightly exceeded the range of the laboratory historical solvent control data (0.0 - 3.0 % aberrant cells, excluding gaps) after treatment with 5225.0 µg/mL. The values for Experiment IIA show a dose-dependent increase in chromosomal aberrations, but no statistical significance was observed. In the confirmatory Experiment IIB no increase in chromosomal aberration could be observed.

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 (660, 770 or 825 µg/mL) or CPA (7.5 or 15.0 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

In conclusion, it can be stated that under the experimental conditions reported, the test item Poly[oxy(methyl-1,2-ethanediyl)], alpha-[2-[bis(phosphonomethyl)amino]methylethyl]-omega-[2-[bis(phosphonomethyl)amino]methylethoxy]-sodium salt did not induce structural chromosomal aberrations in human lymphocytes in vitro, when tested up to the highest required concentration.

Summary of results of the chromosomal aberration study with Poly[oxy(methyl-1,2-ethanediyl)], alpha-[2-[bis(phosphonomethyl)amino]methylethyl]-omega-[2-[bis(phosphonomethyl)amino]methylethoxy]-sodium salt

Exp.

Preparation

Test item

Mitotic indices

Aberrant cells

 

 

interval

concentration

in %

in %

 

 

 

in µg/mL

of control

incl. gaps*

excl. gaps*

carrying exchanges

 

 

Exposure period 4 hrs without S9 mix

 

I

22 hrs

Solvent control1

100.0

0.5

0.5

0.0

 

 

 

Positive control2

74.7

12.5

12.5S

1.5

 

 

 

1706.1

90.1

1.0

1.0

0.0

 

 

 

2985.7

102.7

0.5

0.5

0.0

 

 

 

5225.0

104.0

0.5

0.5

0.0

 

 

Exposure period 22 hrs without S9 mix

IIA

22 hrs

Solvent control1

100.0

1.5

1.5

0.0

 

 

 

Positive control3#

45.7

36.0

36.0S

6.0

 

 

 

1706.1

72.0

0.5

0.5

0.0

 

 

 

2985.7

88.2

2.5

2.0

0.5

 

 

 

5225.0##

86.4

4.0

3.8

0.3

 

IIB

22 hrs

Solvent control1

100.0

1.0

1.0

0.0

 

 

 

Positive control4

42.0

11.5

11.5S

1.0

 

 

 

4000.0

94.0

2.0

1.5

0.0

 

 

 

4500.0

85.3

3.0

3.0

0.0

 

 

 

5225.0

86.0

2.5

2.0

0.0

 

*  Including cells carrying exchanges

#   Evaluation of 50 metaphases per culture

## Evaluation of 200 metaphases per culture

S  Aberration frequency statistically significant higher than corresponding control values

1   Deionised water 10.0 % (v/v)

2     EMS     825.0 µg/mL

3     EMS     770.0 µg/mL

4     EMS     660.0 µg/mL

 


Summary of results of the chromosomal aberration study with Poly[oxy(methyl-1,2-ethanediyl)], alpha-[2-[bis(phosphonomethyl)amino]methylethyl]-omega-[2-[bis(phosphonomethyl)amino]methylethoxy]-sodium salt

Exp.

Preparation

Test item

Mitotic indices

Aberrant cells

 

 

interval

concentration

in %

in %

 

 

 

in µg/mL

of control

incl. gaps*

excl. gaps*

carrying exchanges

 

 

Exposure period 4 hrs with S9 mix

I

22 hrs

Solvent control1

100.0

0.5

0.5

0.0

 

 

 

Positive control2

73.1

10.5

10.0S

1.5

 

 

 

1706.1

90.2

1.5

1.5

0.0

 

 

 

2985.7

97.9

0.5

0.5

0.0

 

 

 

5225.0

102.1

1.5

1.5

0.0

 

IIA

22 hrs

Solvent control1

100.0

1.5

1.0

0.0

 

 

 

Positive control3

59.3

23.5

23.0S

4.5

 

 

 

1706.1

99.0

2.0

1.5

0.0

 

 

 

2985.7

85.0

3.0

3.0

0.5

 

 

 

5225.0

77.3

1.0

1.0

0.0

 

*  Including cells carrying exchanges

S  Aberration frequency statistically significant higher than corresponding control values

1   Deionised water 10.0 % (v/v)

2   CPA        7.5 µg/mL

3   CPA      15.0 µg/mL

Conclusions:
It was concluded that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro when tested up to the highest required concentration. Therefore, the test item is considered to be non-clastogenic.
Executive summary:

In order to assess the potential clastogenicity of the test item, a study was conducted in accordance with OECD 473. The test item dissolved in deionised water, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in three independent experiments. The following study design was performed:

 

Without S9 mix

With S9 mix

 

Exp. I

Exp. IIA & IIB

Exp. I & IIA

Exposure period

 4 hrs

22 hrs

 4 hrs

Recovery

18 hrs

-

18 hrs

Preparation interval

22 hrs

22 hrs

22 hrs

In each experimental concentration 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 scored.

The highest applied concentration in this study (5225.0 µg/mL of the test item) was chosen with regard to the water content (4.3 %) 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.

In the absence and presence of S9 mix no cytotoxicity was observed up to the highest applied concentration.

Neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. However, in Experiment IIA in the absence of S9 mix one single value (3.8 % aberrant cells, excluding gaps) slightly exceeded the range of the laboratory historical solvent control data (0.0 - 3.0 % aberrant cells, excluding gaps) after treatment with 5225.0 µg/mL. The values of this experimental part show a dose-dependent increase in chromosomal aberrations, but no statistical significance was observed. In the confirmatory Experiment IIB no relevant increase in chromosomal aberration could be observed.

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 (p < 0.05) in cells with structural chromosome aberrations.

It was concluded that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro when tested up to the highest required concentration.  Therefore, the test item is considered to be non-clastogenic.

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:
The study was performed between 24 June 2011 and 05 December 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.
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)
Version / remarks:
Commission Regulation (EC) No. 440/2008 and the United Kingdom Environmental Mutagen Society (Cole et al, 1990).
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
.
Type of assay:
mammalian cell gene mutation assay
Target gene:
This study was designed to assess the potential mutagenicity of the test item on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary (CHO) cells.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Properly maintained: yes

- Periodically checked for Mycoplasma contamination: yes

- Periodically checked for karyotype stability: no

- Periodically "cleansed" against high spontaneous background: yes

Cell Line:
The Chinese hamster ovary (CHO-K1) cell line was obtained from ECACC, Salisbury, Wiltshire.

Cell Culture:
The stocks of cells were stored in liquid nitrogen at approximately -196°C. Cells were routinely cultured in Ham's F12 medium, supplemented with 5% foetal calf serum and antibiotics (Penicillin/Streptomycin at 100 units/100 µg per ml) at 37°C with 5% CO2 in air.

Cell Cleansing:
Cell stocks spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen down they were cleansed of HPRT- mutants by culturing in HAT medium for 4 days. This is Ham's F12 growth medium supplemented with Hypoxanthine (13.6 µg/ml, 100 µM), Aminopterin (0.0178 µg/ml, 0.4 µM) and Thymidine (3.85 µg/ml, 16 µM). After 4 days in medium containing HAT, the cells were passaged into HAT-free medium and grown for 4 to 7 days. Bulk frozen stocks of HAT cleansed cells were frozen down, with fresh cultures being recovered from frozen before each experiment.



Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbitone/beta-naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
The test item was considered to be a mixture with a molecular weight of greater than 500 and therefore the maximum recommended dose was 5000µg/ml. The test item had a water content of 4.3% and an allowance for this was made in the formulations (all concentrations of the test item stated throughout the report are of the active ingredient). The test item was dissolved in Hams F12 and appropriate dilutions made.

A dose range of 19.53 to 5000 µg/ml was used in the preliminary cytotoxicity test.

The dose ranges selected for Experiment 1 and Experiment 2 were based on the results of the preliminary cytotoxicity test and were as follows:-

Exposure Group Final concentration of test item (µg/ml)
4-hour without S9 0, 156.25, 312.5, 625, 1250, 2500, 5000
4-hour with S9 (2%) 0, 156.25, 312.5, 625, 1250, 2500, 5000
24-hour without S9 0, 156.25, 312.5, 625, 1250, 2500, 5000
4-hour with S9 (1%) 0, 156.25, 312.5, 625, 1250, 2500, 5000


Vehicle / solvent:
Vehicle(s)/solvent(s) used: Hams F12 cell culture media
Justification for choice of solvent/vehicle: The test material formed a solution at the required concentrations with the solvent suitable for dosing.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Hams F12
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Dimethyl benzanthracene (DMBA)
Remarks:
With metabolic activation (dose levels 0.5 and 1 µg/ml in Experiment 1 and 2)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Hams F12
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation (dose levels 500 and 705 µg/ml in Experiment 1, 200 and 300 µg/ml in Experiment 2). Migrated to IUCLID6: (EMS)
Details on test system and experimental conditions:
Preliminary Cytotoxicity Test:
A preliminary cytotoxicity test was performed on cell cultures plated approximately 24 hours before dosing at 3 x 10E6 cells/75 cm2 flask for the 4 hour exposure groups and at 1.5 x 10E6 cells/75 cm2 flask for the 24 hour exposure group. On dosing, the growth media was removed and replaced with serum free media (Ham's F12) for the 4 hour exposure groups and with Hams F12 with 1% serum for the 24 hour exposure group. One flask per dose level was treated for 4 hours with and without S9 metabolic activation and for 24-hours without metabolic activation, 9 dose levels using halving dilutions and vehicle controls were dosed. The dose levels of test item used were 19.53 to 5000 µg/ml and exposure was for 4 hours or 24 hours at 37°C. At the end of the exposure period the cultures were washed twice with phosphate buffered saline (PBS) before being trypsinised. Cells from each flask were suspended in Hams F12 with 5% FBS, a sample was removed from each dose group and counted using a Coulter counter. For each culture, 200 cells were plated out into three 25 cm2 flasks with 5 ml of Hams F12 with 5% FBS and incubated for 7 days at 37°C in an incubator with a humidified atmosphere of 5% CO2 in air. The cells were then fixed and stained and total numbers of colonies in each flask counted to give cloning efficiencies.
Results from the preliminary cytotoxicity test were used to select the test item dose levels for the mutagenicity test.

Mutagenicity Test:
Several days before starting each experiment, a fresh stock of cells was removed from the liquid nitrogen freezer and grown up to provide sufficient cells for use in the test. Cells were seeded at 3 x 10E6/75 cm2 flask for the 4 hour exposure groups of Experiment 1 and allowed to attached overnight before being exposed to the test or control items. In Experiment 2 the cells were seeded at 1.5 x 10E6 cells/75 cm2 flask for the 4-hour exposure and at 1.0 x 10E6 cells/75 cm2 flask for the 24 hour exposure group and allowed to attach for approximately 48 hours before being exposed to the test or control items. Duplicate cultures were set up, both in the presence and absence of metabolic activation, with six dose levels of test item, and vehicle and positive controls. Treatment was for 4 hours in serum free media (Ham's F12) at 37°C or for 24 hours in Hams F12 with 1% serum in an incubator with a humidified atmosphere of 5% CO2 in air. The dose range of test item was 156.25 to 5000 µg/ml in both the 4 hour exposure groups of Experiment 1 and the 4 hour with S9 and the 24 hour without S9 exposure groups of Experiment 2. At the end of the treatment period the flasks were washed twice with PBS, trypsinised and the cells suspended in Hams F12 with 5% serum. A sample of each dose group cell suspension was counted using a Coulter counter. Cultures were plated out at 2 x 10E6 cells/flask in a 225 cm2 flask to allow growth and expression of induced mutants, and in triplicate in 25 cm2 flasks at 200 cells/flask for an estimate of cytotoxicity. Cells were grown in Hams F12 with 5% serum and incubated at 37°C in an incubator with a humidified atmosphere of 5% CO2 in air.

Cytotoxicity flasks were incubated for 7 days then fixed with methanol and stained with Giemsa. Colonies were manually counted and recorded to estimate cytotoxicity.

During the 7 Day expression period the cultures were subcultured and maintained at 2 x 10E6 cells/225 cm2 flask on days 3 to 4 to maintain logarithmic growth. At the end of the expression period the cell monolayers were trypsinised, cell suspensions counted using a Coulter counter and plated out as follows:

i) In triplicate at 200 cells/25 cm2 flask in 5 ml of Hams F12 with 5% serum to determine cloning efficiency. Flasks were incubated for 7 days, fixed with methanol and stained with Giemsa. Colonies were manually counted, counts were recorded for each culture and the percentage cloning efficiency for each dose group calculated.
ii) At 2 x 10E5 cells/75 cm2 flask (5 replicates per group) in Ham's F12 growth media (5% serum), supplemented with 10 µg/ml 6-Thioguanine (6-TG), to determine mutant frequency. The flasks were incubated for 14 days at 37°C in an incubator with humidified atmosphere of 5% CO2 in air, then fixed with methanol and stained with Giemsa. Mutant colonies were manually counted and recorded for each flask.

The percentage of viability and mutation frequency per survivor were calculated for each dose group.

Fixation and staining of all flasks was achieved by aspirating off the media, washing with phosphate buffered saline, fixing for 5 minutes with methanol and finally staining with a 10% Giemsa solution for 5 minutes.

ASSAY ACCEPTANCE CRITERIA
An assay will normally be considered acceptable for the evaluation of the test results only if all the following criteria are satisfied. The with and without metabolic activation portions of mutation assays are usually performed concurrently, but each portion is, in fact, an independent assay with its own positive and negative controls. Activation or non-activation assays will be repeated independently, as needed, to satisfy the acceptance criteria.

i) The average absolute cloning efficiency of negative controls should be between 70 and 115% with allowances being made for errors in cell counts and dilutions during cloning and assay variables. Assays in the 50 to 70% range may be accepted but this will be dependent on the scientific judgement of the Study Director. All assays below 50% cloning efficiency will be unacceptable.
ii) The background (spontaneous) mutant frequency of the vehicle controls are generally in the range of 0 to 25 x 10-6. The background values for the with and without-activation segments of a test may vary even though the same stock populations of cells may be used for concurrent assays. Assays with backgrounds greater than 35 x 10-6 will not be used for the evaluation of a test item.
iii) Assays will only be acceptable without positive control data (loss due to contamination or technical error) if the test item clearly shows mutagenic activity. Negative or equivocal mutagenic responses by the test item must have a positive control mutant frequency that is markedly elevated over the concurrent negative control.
iv) Test items with little or no mutagenic activity, should include an acceptable assay where concentrations of the test item have reduced the clonal survival to approximately 10 to 15% of the average of the negative controls, reached the maximum recommended dose (10 mM or 5 mg/ml) or twice the solubility limit of the test article in culture medium. Where a test item is excessively toxic, with a steep response curve, a concentration that is at least 75% of the toxic dose level should be used. There is no maximum toxicity requirement for test items that are clearly mutagenic.
v) Mutant frequencies are normally derived from sets of five dishes for mutant colony count and three dishes for viable colony counts. To allow for contamination losses it is acceptable to score a minimum of four mutant selection dishes and two viability dishes.
vi) Five dose levels of test item, in duplicate, in each assay will normally be assessed for mutant frequency. A minimum of four analysed duplicate dose levels is considered necessary in order to accept a single assay for evaluation of the test item.
Evaluation criteria:
Please see assay acceptance criteria (details on test system and conditions section).
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
(non-mutagenic)
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Refer to the results section.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Preliminary Cytotoxicity Test:
A dose range of 19.53 to 5000 µg/ml was used in the preliminary cytotoxicity test. The results of the individual flask counts and their analysis are presented in the attached Table 1. It can be seen that there was a reduction in the cloning efficiencies (CE) in all three exposure groups. The toxicity was greatest in the 24-hour exposure group where there was a reduction in cloning efficiency of 80% at the maximum dose and a plateau of toxicity from 19.53 µg/ml, however the cloning efficiencies in this group were generally low at all dose levels of the test item. In both 4-hour exposure groups there was a dose related increase in toxicity with a 46% and a 36% decrease in cloning efficiency in the without S9 and with S9 exposure groups respectively at 5000 µg/ml. Precipitate was noted at the end of the exposure period in the 4-hour exposure group in the presence of S9 at and above 625 µg/ml and cloudy precipitate was noted at and above 78.13 µg/ml which reduced with increasing dose. Therefore, with toxicity being observed at dose levels beyond the onset of precipitation the maximum dose level selected for both Experiment 1 and 2 was the maximum recommended dose of 5000 µg/ml.

Mutagenicity Test - Experiment 1:
The dose levels of the controls and the test item are given in the table below:
Group Final concentration of test item (µg/ml)
4-hour without S9 0, 156.25, 312.5, 625, 1250, 2500, 5000, EMS 500 and 750
4-hour with S9 (2%) 0, 156.25, 312.5, 625, 1250, 2500, 5000, DMBA 0.5 and 1

No precipitate of the test item was seen at the end of exposure in either exposure group, however there was a reduction in the cloudiness of the 4-hour exposure group in the presence of S9 at and above 312.5 µg/ml, possibly due to the reduced pH causing a reaction with the calcium carbonate in the S9.

The Day 0 and Day 7 cloning efficiencies are presented in the attached Table 2 and Table 3. There was no reduction in the Day 0 and Day 7 cloning efficiency in the absence or presence of S9 and it can be seen that there was no marked toxicity with the test item when compared to the vehicle controls. The Day 0 and the Day 7 vehicle cloning efficiencies of the 4-hour exposure group in the absence of S9 and the ‘B’ replicates of the 4-hour exposure group in the presence of S9 did not achieve 70% but were considered to be acceptable as they did achieve at least 50% and there was no evidence of a positive response.

The mutation frequency counts and mean mutation frequency per survivor values are presented in the attached Table 2 and Table 3. There were no increases in mutation frequency per survivor which exceeded the vehicle control value by 20 x 10-6 with or without the presence of S9.

Mutagenicity Test - Experiment 2:

The dose levels of the controls and the test item are given in the table below:
Group Final concentration of test item (µg/ml)
24-hour without S9 0, 156.25, 312.5, 625, 1250, 2500, 5000, EMS 200 and 300
4-hour with S9 (1%) 0, 156.25, 312.5, 625, 1250, 2500, 5000, DMBA 0.5 and 1

No precipitate of the test item was seen at the end of exposure in either exposure group.

The Day 0 and Day 7 cloning efficiencies are presented in the attached Tables 4 and 5. It can be seen that, as in Experiment 1, there was no reduction in the Day 0 and Day 7 cloning efficiency in the presence of S9 and no toxicity of the test item was observed when compared to the vehicle controls. The 24-hour exposure group demonstrated a dose related increase in toxicity with a reduction in the Day 0 cloning efficiency of 58% at 5000 µg/ml when compared to the vehicle control. The Day 0 counts for the 24-hour exposure group did not achieve 70% but were considered to be acceptable as they did achieve the 50% minimum required and there was no evidence of a positive response.

The mutation frequency counts and mean mutation frequency per survivor values are presented in the attached Tables 4 and 5. There were no increases in mutation frequency per survivor which exceeded the vehicle control value by 20 x 10-6 with or without the presence of S9.

It can be seen that the vehicle control values were all within the maximum upper limit of 25 x 10-6 mutants per viable cell in both Experiment 1 and Experiment 2, and that the positive controls all gave marked increases in mutant frequency, indicating the test and the metabolic activation system were operating as expected.
Remarks on result:
other: strain/cell type: CHO
Remarks:
Migrated from field 'Test system'.

Please see attached background material for result tables:

Table 1: Preliminary Cytotoxicity Results

Table 2: Experiment 1 - 4 hour exposure without metabolic activation (S9)

Table 3: Experiment 1 - 4 hour exposure with metabolic activation (S9)

Table 4: Experiment 2 - 24 hour exposure without metabolic activation (s9)

Table 5: Experiment 2 - 4 hour exposure with metabolicactivation (S9)

Conclusions:
The test item did not induce any significant or dose-related increases in mutant frequency per survivor in either the presence or absence of metabolic activation in either of the two experiments. The test item was therefore considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of this test.
Executive summary:

The study was conducted to assess the potential mutagenicity of the test item on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary (CHO) cells. The test method used was designed to be compatible with the OECD Guidelines for Testing of Chemicals No. 476 In Vitro Mammalian Cell Gene Mutation Tests', Method B17 of Commission Regulation (EC) No 440/2008, the United Kingdom Environmental Mutagen Society (Cole et al, 1990) and the US EPA OPPTS 870.5300 Guideline. The technique used is a plate assay using tissue culture flasks and 6-thioguanine (6­TG) as the selective agent.

Chinese hamster ovary (CHO) cells were treated with the test item at six dose levels, in duplicate, together with vehicle (solvent) and positive controls. Four treatment conditions were used for the test, i.e. In Experiment 1, a 4 -hour exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration and a 4-hour exposure in the absence of metabolic activation (S9). In Experiment 2, the 4-hour exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

The dose ranges selected for Experiment 1 and Experiment 2 were based on the results of the preliminary cytotoxicity test and were as follows:-

 

Exposure Group

Final concentration of test item (µg active ingredient/ml)

4-hour without S9

0, 156.25, 312.5, 625, 1250, 2500, 5000

4-hour with S9 (2%)

0, 156.25, 312.5, 625, 1250, 2500, 5000

24-hour without S9

0, 156.25, 312.5, 625, 1250, 2500, 5000

4-hour with S9 (1%)

0, 156.25, 312.5, 625, 1250, 2500, 5000

 

The vehicle (solvent) controls gave mutant frequencies within the range expected of CHO cells at the HPRT locus.

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

The test item demonstrated no significant increases in mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment.

The test item was considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of the test.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

Not applicable, the substance is not considered mutagenic.

Additional information

OECD 471 - AMES Test

The potential mutagenicity of the substance was assessed in accordance with OECD 471 (AMES test).  Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item, using both the Ames plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate (active ingredient). The experiment was repeated on a separate day (pre-incubation method) using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test item formulations.

The vehicle (sterile distilled water) 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 item caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate (active ingredient). No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

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

The test item was considered to be non-mutagenic under the conditions of this test.

OECD 473 - CAT

In order to assess the potential clastogenicity of the test item, a study was conducted in accordance with OECD 473. The test item dissolved in deionised water, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in three independent experiments. The following study design was performed:

Without S9 mix

With S9 mix

 

Exp. I

Exp. IIA & IIB

Exp. I & IIA

Exposure period

 4 hrs

22 hrs

 4 hrs

Recovery

18 hrs

-

18 hrs

Preparation interval

22 hrs

22 hrs

22 hrs

In each experimental concentration 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 scored. The highest applied concentration in this study (5225.0 µg/mL of the test item) was chosen with regard to the water content (4.3 %) 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.

In the absence and presence of S9 mix no cytotoxicity was observed up to the highest applied concentration.  Neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. However, in Experiment IIA in the absence of S9 mix one single value (3.8 % aberrant cells, excluding gaps) slightly exceeded the range of the laboratory historical solvent control data (0.0 - 3.0 % aberrant cells, excluding gaps) after treatment with 5225.0 µg/mL. The values of this experimental part show a dose-dependent increase in chromosomal aberrations, but no statistical significance was observed. In the confirmatory Experiment IIB no relevant increase in chromosomal aberration could be observed.

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 (p < 0.05) in cells with structural chromosome aberrations.

It was concluded that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro when tested up to the highest required concentration.  Therefore, the test item is considered to be non-clastogenic.

OECD 476 - CHO HPRT

The study was conducted to assess the potential mutagenicity of the test item on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary (CHO) cells. The test method used was designed to be compatible with the OECD Guidelines for Testing of Chemicals No. 476 In Vitro Mammalian Cell Gene Mutation Tests', Method B17 of Commission Regulation (EC) No 440/2008, the United Kingdom Environmental Mutagen Society (Cole et al, 1990) and the US EPA OPPTS 870.5300 Guideline. The technique used is a plate assay using tissue culture flasks and 6-thioguanine (6­TG) as the selective agent.

Chinese hamster ovary (CHO) cells were treated with the test item at six dose levels, in duplicate, together with vehicle (solvent) and positive controls. Four treatment conditions were used for the test, i.e. In Experiment 1, a 4 -hour exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration and a 4-hour exposure in the absence of metabolic activation (S9). In Experiment 2, the 4-hour exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

The dose ranges selected for Experiment 1 and Experiment 2 were based on the results of the preliminary cytotoxicity test and were as follows:-

Exposure Group              Final concentration of test item (µg active ingredient/ml)

4-hour without S9              0, 156.25, 312.5, 625, 1250, 2500, 5000

4-hour with S9 (2%)              0, 156.25, 312.5, 625, 1250, 2500, 5000

24-hour without S9              0, 156.25, 312.5, 625, 1250, 2500, 5000

4-hour with S9 (1%)              0, 156.25, 312.5, 625, 1250, 2500, 5000

 

The vehicle (solvent) controls gave mutant frequencies within the range expected of CHO cells at the HPRT locus.

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

The test item demonstrated no significant increases in mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment.

The test item was considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of the test.

Justification for classification or non-classification

The substance did not meet the classification criteria in accordance with Regulation (EC) 1272/2008.