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Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay (Ames test; OECD 471): Negative, with/without metabolic activation

In vitro mammalian cell chromosome aberration test (OECD 473): Negative, with/without metabolic activation

In vitro mammalian cell gene mutation assay (OECD 476): Negative, with/without metabolic activation

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 July 2012-13 December 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine and Tryptophan
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S-9 rat liver induced with phenobarbital/β-naphthoflavone
Test concentrations with justification for top dose:
Range-finding test (TA 98, TA100): 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate
Main tests: 5000; 1581; 500; 158.1; 50; 15.81, 5 and 1.581 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: Substance is soluble in water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 4-nitro-1,2-phenylenediamine; 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) Experiment 1; preincubation Experiment 2

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: Triplicate plates per duplicate experiment

DETERMINATION OF CYTOTOXICITY
- Method: evaluation of background lawn of bacteria
Evaluation criteria:
Criteria for a Positive Response:
A test item is considered mutagenic if:
- a dose–related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- the number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions is more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.

According to the OECD guidelines, statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.

Criteria for a Negative Response:
A test article is considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.
Statistics:
Not applicable.
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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: None
- Effects of osmolality: None
- Evaporation from medium: None
- Water solubility: Soluble
- Precipitation: None
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES: Range-finder experiment performed to set the dose levels for the two main experiments.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes, all control data within the historical ranges.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Cytotoxicity varied slightly between bacterial strains; more toxic to strains TA1535 and TA1537.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Summary Table of the Initial Mutation Test

 

Concentrations
(µg/plate)

Mean
values of revertants / Mutation factor (MF)

Salmonella typhimuriumtester strains

Escherichia coli

TA98

TA100

TA1535

TA1537

WP2uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Untreated control

Mean

24.7

24.0

92.7

111.7

8.0

10.0

7.7

6.3

25.7

35.0

Distilled water control (50µL)

Mean

-

-

82.0

-

4.7

-

-

-

29.3

-

DMSO
control

Mean

23.3

24.3

-

118.7

-

7.3

5.3

6.0

-

27.0

Distilled water control (200µL)

Mean

25.3

28.7

82.0

102.3

8.7

8.3

9.0

8.3

30.0

32.7

5000

Mean

0.0

0.0

0.0

61.0

0.0

0.0

3.3

5.0

21.7

26.0

1581

Mean

0.0

3.0

12.7

60.7

0.0

4.0

2.0

1.0

27.7

31.3

500

Mean

8.0

18.7

38.0

115.3

2.0

8.0

4.0

11.3

30.3

41.7

158.1

Mean

13.7

17.0

73.3

123.3

8.7

7.3

9.0

10.7

27.3

36.7

50

Mean

20.0

17.7

112.0

125.7

6.7

9.0

8.0

8.3

25.7

43.7

15.81

Mean

17.7

20.7

111.0

120.3

8.0

10.0

6.7

5.7

38.7

38.7

5

Mean

17.0

24.3

107.7

113.7

10.3

16.7

10.0

11.0

31.3

36.0

1.581

Mean

20.3

23.3

118.0

122.7

7.7

10.3

7.3

6.7

30.0

38.3

NPD (4µg)

Mean

298.3

-

-

-

-

-

-

-

-

-

2AA (2µg)

Mean

-

2370.7

-

2386.7

-

198.0

-

203.3

-

-

2AA (50µg)

Mean

-

-

-

-

-

-

-

-

-

215.3

SAZ (2µg)

Mean

-

-

1292.0

-

1189.3

-

-

-

-

-

9AA (50µg)

Mean

-

-

-

-

-

-

394.7

-

-

-

MMS (2µL)

Mean

-

-

-

-

-

-

-

-

1048.0

-

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, the test item Lathanol LAL coarse had no mutagenic activity in the applied bacterium tester strains under the test conditions used in this study.
Executive summary:

The test item Lathanol LAL coarse was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/b-naphthoflavone-induced rats. The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method) and a Confirmatory Mutation Test (Pre-Incubation Method). Based on the results of the Solubility Test, the test item was formulated in Distilled water. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10µg/plate were examined in the Range Finding Test. Based on the results of the Range Finding Test, the test item concentrations in theInitial Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg test item/plate. Examined concentrations in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81, 5, 1.581 and 0.5μg test item/plate. In the Initial Mutation Test and Confirmatory Mutation Test, none of the observed revertant colony numbers were above the respective biological threshold value. There were no consistent dose-related trends and no indication of any treatment effect. In all test item treated groups, the numbers of revertant colonies were below the threshold of biological relevance when compared with the solvent controls, were within the historical control range and were within the range of normal biological variability of the test system.Inhibitory, cytotoxic effect of the test item was observed in the Initial Mutation Test in all examined bacterial strains without metabolic activation, and in all Salmonella typhimurium strains with metabolic activation. Similar inhibitory, cytotoxic effect of the test item was observed in the Confirmatory Mutation Test in all Salmonella typhimurium strains with and without metabolic activation. The mean values of revertant colonies of the solvent control plates were within the historical control range, the reference mutagens induced the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests.The tests were considered to be valid.

 

The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshift mutations in the genome of the strains used. In conclusion, the test item Lathanol LAL coarse had no mutagenic activity in the bacterium tester strains under the test conditions used in this study.

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:
2003-04-02 to 2003-06-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
cultured peripheral human lymphocytes
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
Derived from healthy adult male volunteers: blood samples taken by venapuncture using Venoject multiple blood collecting system with a suitable size sterile vessel containing sodium heparin. Lymphocyte culture started within 4 h of blood collection.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
Range finder: 100, 333, 1000, 3330 and 5000 µg/mL (+/- S9)
1st test: 100, 200, 300, 400, 500 and 750 µg/mL (+/- S9)
2nd test:
- S9: 100, 125, 150, 175, 200, 250 and 300 µg/mL (24 h exp, 24 h fixation)
- S9: 50, 75, 100, 125 150 and 175 µg/mL (48 h exp, 48 h fixation)
+ S9: 200, 300, 400, 500 and 600 µg/mL (3 h exp, 48 h fixation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl sulfoxide
- Justification for choice of solvent/vehicle: Not specified
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Vehicle only
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9: Mitomycin C; +S9: Cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 h
- Exposure duration: 3 h, 24h or 48 h
- Expression time (cells in growth medium): 20 - 22h (24 h fixation) or 44 - 46 h (48 h fixation)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h or 48 h

SPINDLE INHIBITOR (cytogenetic assays): colchicine (0.5 µg/mL medium)
STAIN (for cytogenetic assays): 5% (v/v) Giemsa solution in tap water

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 1000

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
Evaluation criteria:
Positive:
It induced a dose-related statistically significant (Chi-square test) increase in the number of cells with chromosome aberrrations
a statistically significant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.
Negative:
if none of the tested concentrations induced a statistically significant (Chi-square test) increase in the number of cells with chromosome aberrations.
Statistics:
The incidence of aberrant cells for each exposure group was compared to that of the solvent control using Chi-square statistics.
Species / strain:
lymphocytes:
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
- Other confounding effects: the test material is a surfactant and may have an aspecific, indirect cell damaging action which can result in chromosome aberrations

RANGE-FINDING/SCREENING STUDIES: No statistically significant or biologically relevant increase in chromosome aberrations with and without S9.
Cytotoxic concentration: 600 µg/mL
Only at the cytotoxic dose level of 600 µg/mL with S9 was a statistically significant increase in the number of aberrations observed. However, biological relevance of the observed increase may be doubted. The increase was not dose-related and observed at only one strongly cyctotoxic concentration ( mitotic index 36%). Furthermore the number of cells with aberrations is on the borderline of the historical control data range.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes, number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range.

ADDITIONAL INFORMATION ON CYTOTOXICITY: 600 µg.mL (+/- S9)
Remarks on result:
other: strain/cell type: human peripheral lymphocytes
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

The test material did not induce statistically significant or biologically relevant increases in the number of cells with chromosome aberrations with or without S9 below the limit of cyctoxicity
Executive summary:

In an OECD 473, EU Method B10 in vitro mammalian cell chromosome aberration test, cultured peripheral human lymphocytes in the absence and presence of metabolic activation (Arochlor induced rat liver S9) were exposed to test concentrations of up to 600 µg/mL (level of cyctoxicity) of the test material in dimethyl sulfoxide in two independant assays performed in duplicate. The vehicle alone served as the negative control and two positive controls (+ or -S9) tested the validity of the assay.

The validity criteria of the assay were fulfilled.

Only at the cytotoxic dose level of 600 µg/mL with S9 was a statistically significant increase in the number of aberrations observed. However, biological relevance of the observed increase may be doubted. The increase was not dose-related and observed at only one strongly cyctotoxic concentration (mitotic index 36%). Furthermore, the number of cells with aberrations is on the borderline of the historical control data range. In addition, the test material is a surfactant and may have an aspecific, indirect cell damaging action which can result in chromosome aberrations

The test material did not induce statistically significant or biologically relevant increases in the number of cells with chromosome aberrations with or without S9 below the limit of cyctoxicity.

The test material is not clastogenic with or without metabolic activation.

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:
10 September 2012 to 07 December 2012
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
PBNF rat liver S9
Test concentrations with justification for top dose:
2.5, 5, 10, 12.5, 20, 25, 40, 50, 60, 70, 80, 90, 100, 110, 120, 140
Vehicle / solvent:
RPMI 1640 culture medium
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: Not applicable
- Exposure duration: 3 and 24 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 2 weeks
- Fixation time (start of exposure up to fixation or harvest of cells): Not applicable

SELECTION AGENT (mutation assays): Trifluorothymidine
SPINDLE INHIBITOR (cytogenetic assays): Not applicable
STAIN (for cytogenetic assays): Not applicable

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: 2000 cels per well

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth
Evaluation criteria:
The test item was considered to be mutagenic in this assay if all the following criteria were met (based on M. Moore et al, 2006 [6]):
1. The assay is valid.
2. Statistically significant (p < 0.05) and biologically relevant increases in mutation frequency are observed in treated cultures compared to the corresponding negative (vehicle) control values at one or more concentrations.
3. The increases in mutation frequency are reproducible between replicate cultures and/or between tests (under the same treatment conditions).
4. There is a significant concentration-relationship as indicated by the linear trend analysis (p < 0.05).
5. The mutation frequency at the test concentration showing the largest increase is at least 126 mutants per 106 viable cells (GEF = the Global Evaluation Factor) higher than the corresponding negative (vehicle) control value.

Results, which only partially satisfied the acceptance and evaluation criteria, were evaluated on a case-by-case basis.
Statistics:
Statistical significance of mutant frequencies (total wells with clones) was performed using Microsoft Excel software. The control log mutant frequency (LMF) was compared to the LMF from each treatment dose, based on Dunnett's test for multiple comparisons and the data checked for a linear trend in mutant frequency with treatment dose using weighted regression. The test for linear trend was one-tailed, therefore negative trend was not considered significant. These tests required the calculation of the heterogeneity factor to obtain a modified estimate of variance.
Species / strain:
human lymphoblastoid cells (TK6)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: None
- Effects of osmolality: None
- Evaporation from medium: Not applicable
- Water solubility: Soluble
- Precipitation: None
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES: A range finding test was performed.

COMPARISON WITH HISTORICAL CONTROL DATA: Controls values within historical ranges

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Results tables attahched as pdf files.

Conclusions:
Interpretation of results (migrated information):
negative

No mutagenic effect of Lathanol LAL coarse was observed either in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.
Executive summary:

An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/-3.7.2 C cells at the tk locus to test the potential of Lathanol LAL coarse to cause gene mutation and/or chromosome damage. Treatment was performed for 3 hours with and without metabolic activation (±S9 mix) and for 24 hours without metabolic activation (-S9 mix).

RPMI-1640 medium was used as the vehicle of the test item in this study. The test item was examined at up to 5000 µg/mL in the Preliminary Toxicity Test. Based on the results of the preliminary experiment, the following test item concentrations were examined in the mutation assays:

Assay 1, 3-hour treatment with metabolic activation:140; 120; 110; 100; 90; 80; 70; 60; 50; 25 and 12.5 µg/mL

Assay 1, 3-hour treatment without metabolic activation:120; 110; 100; 90; 80; 70; 60; 50; 40; 20; 10; 5 and 2.5 µg/mL

Assay 2, 3-hour treatment with metabolic activation:140; 120; 110; 100; 90; 80; 70; 60; 50; 25 and 12.5 µg/mL

Assay 2, 24-hour treatment without metabolic activation:90; 80; 70; 65; 60; 55; 50; 40; 30; 20; 10; 5 and 2.5 µg/mL

Assay 3, 3-hour treatment with metabolic activation:140; 120; 110; 100; 90; 80; 70; 60; 50; 25 and 12.5 µg/mL

Assay 3, 24-hour treatment without metabolic activation:90; 80; 70; 65; 60; 55; 50; 40; 30; 20; 10; 5 and 2.5 µg/mL

In Assays 1-3, no insolubility was detected in the final treatment medium at the beginning and end of the treatment in any of the experiments. There were no large changes in pH or osmolality after treatment. In Assay 1, following a 3-hour treatment with metabolic activation, excessive cytotoxicity of the test item was observed at 140 µg/mL, such that all cells in that sample died during the treatment. An evaluation was made using data of the first surviving concentration of 120 µg/mL (relative survival value of 11 %) and the next seven lower concentrations (a total of eight samples). Nosignificant increase in the mutation frequency was observed at the evaluated concentrations. Nodose response to the treatment was indicated by the linear trend analysis. In Assay 1, following a 3-hour treatment without metabolic activation, excessive cytotoxicity of the test item was observed at concentrations of 120, 110 and 100 µg/mL, such that all cells in these samples died during the treatment. Therefore, an evaluation was made using data of the first surviving concentrations of 90 and 80 µg/mL (relative survival values of 9 and 39%, respectively) and the next six concentrations (a total of eight samples). No statistically significant increase in the mutation frequency was observed at the evaluated concentrations.Furthermore, no significant dose response to the treatment was indicated by the linear trend analysis. The result of this experiment was considered to be negative.

In Assay 2, no cytotoxicity was observed at the highest examined concentration after treatment. Therefore, the selected concentration range did not meet the requirements of the relevant OECD guideline. Thus the assay was considered invalid, and the experiments were terminated. Results of the invalid experiments are not reported, but all the data will be kept and archived in the raw data binder. An additional experiment (Assay 3) was started using the same experimental conditions.

In Assay 3, following a 3-hour treatment with metabolic activation, excessive cytotoxicity of the test item was observed at 140 µg/mL, such that all cells in that sample died during the treatment. Marked cytotoxicity of the test item was detected at 120 µg/mL (relative survival value of 1 %), therefore this concentration was excluded from the evaluation. An evaluation was made using data of the next concentrations of 110 and 100 µg/mL (relative survival values of 9 and 22 %, respectively) and six lower concentrations (a total of eight samples). No statistically significantincrease in the mutation frequency was observed at any of the evaluated concentrations. No dose response to the treatment was indicated by the linear trend analysis.

In Assay 3, following a 24-hour treatment without metabolic activation, excessive cytotoxicity of the test item was observed at 90 and 80 µg/mL, such that all cells in these samples died during the treatment. Marked cytotoxicity of the test item was detected at 70 µg/mL (relative survival value of 5 %), therefore this concentration was excluded from the evaluation. An evaluation was made using data of the next concentration of 65 µg/mL (relative survival value of 15 %) and seven lower concentrations (a total of eight samples). No statistically significant increases in the mutation frequency were observed at the evaluated concentrations. No significant dose response to the treatment was indicated by the linear trend analysis. The result of this experiment was considered to be negative and thus confirmed the results of the first main test.

The experiments were performed using appropriate untreated, negative (vehicle) and positive control samples in all cases. The spontaneous mutation frequency of the negative (vehicle) controls was in the recommended range in each test. The positive controls gave the anticipated increases in mutation frequency over the controls. The plating efficiencies for the solvent controls at the end of the expression period were within the acceptable range in all assays. The evaluated concentration ranges were considered to be adequate, as they covered the range from 80 to 90% cytotoxicity to no or little cytotoxicity. The number of test concentrations met the acceptance criteria. Therefore, the overall study was considered to be valid.

 

In conclusion, no mutagenic effect of Lathanol LAL coarse was observed either in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Bacterial Mutation Assay (Ames test)

The substance was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay [CiToxLAB Hungary Ltd. (2013b)].

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (WP2uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/b-naphthoflavone-induced rats. The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method) and a Confirmatory Mutation Test (Pre-Incubation Method).

Based on the results of the Solubility Test, the test item was formulated in Distilled water. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10µg/plate were examined in the Range Finding Test. Based on the results of the Range Finding Test, the test item concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg test item/plate. Examined concentrations in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81, 5, 1.581 and 0.5μg test item/plate.

In the Initial Mutation Test and Confirmatory Mutation Test, none of the observed revertant colony numbers were above the respective biological threshold value. There were no consistent dose-related trends and no indication of any treatment effect. In all test item treated groups, the numbers of revertant colonies were below the threshold of biological relevance when compared with the solvent controls, were within the historical control range and were within the range of normal biological variability of the test system. Inhibitory, cytotoxic effect of the test item was observed in the Initial Mutation Test in all examined bacterial strains without metabolic activation, and in all Salmonella typhimurium strains with metabolic activation. Similar inhibitory, cytotoxic effect of the test item was observed in the Confirmatory Mutation Test in all Salmonella typhimurium strains with and without metabolic activation. The mean values of revertant colonies of the solvent control plates were within the historical control range, the reference mutagens induced the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests. The tests were considered to be valid.

The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshift mutations in the genome of the strains used.

In conclusion, the substance had no mutagenic activity in the bacterium tester strains under the test conditions used in this study.

In vitro Chromosome Aberration Test

In an OECD 473, EU Method B10 in vitro mammalian cell chromosome aberration test, cultured peripheral human lymphocytes in the absence and presence of metabolic activation (Arochlor induced rat liver S9) were exposed to test concentrations of up to 600 µg/mL (level of cytotoxicity) of the test material in dimethyl sulfoxide in two independent assays performed in duplicate [Buskens CAF (2003)]. The vehicle alone served as the negative control and two positive controls (+ or -S9) tested the validity of the assay. The validity criteria of the assay were fulfilled. Only at the cytotoxic dose level of 600 µg/mL with S9 was a statistically significant increase in the number of aberrations observed. However, biological relevance of the observed increase may be doubted. The increase was not dose-related and observed at only one strongly cytototoxic concentration (mitotic index 36%). Furthermore, the number of cells with aberrations is on the borderline of the historical control data range. In addition, the test material is a surfactant and may have an aspecific, indirect cell damaging action which can result in chromosome aberrations. The test material did not induce statistically significant or biologically relevant increases in the number of cells with chromosome aberrations with or without S9 below the limit of cytotoxicity.

The test material is not clastogenic with or without metabolic activation.

In vitro Mammalian Cell Mutation Assay

An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/-3.7.2 C cells at the tk locus to test the potential of the substance to cause gene mutation and/or chromosome damage [CiToxLAB Hungary Ltd. (2013c)]. Treatment was performed for 3 hours with and without metabolic activation (±S9 mix) and for 24 hours without metabolic activation (-S9 mix).

RPMI-1640 medium was used as the vehicle of the test item in this study. The test item was examined at up to 5000 µg/mL in the Preliminary Toxicity Test. Based on the results of the preliminary experiment, the following test item concentrations were examined in the mutation assays:

Assay 1, 3-hour treatment with metabolic activation:140; 120; 110; 100; 90; 80; 70; 60; 50; 25 and 12.5 µg/mL

Assay 1, 3-hour treatment without metabolic activation:120; 110; 100; 90; 80; 70; 60; 50; 40; 20; 10; 5 and 2.5 µg/mL

Assay 2, 3-hour treatment with metabolic activation:140; 120; 110; 100; 90; 80; 70; 60; 50; 25 and 12.5 µg/mL

Assay 2, 24-hour treatment without metabolic activation:90; 80; 70; 65; 60; 55; 50; 40; 30; 20; 10; 5 and 2.5 µg/mL

Assay 3, 3-hour treatment with metabolic activation:140; 120; 110; 100; 90; 80; 70; 60; 50; 25 and 12.5 µg/mL

Assay 3, 24-hour treatment without metabolic activation:90; 80; 70; 65; 60; 55; 50; 40; 30; 20; 10; 5 and 2.5 µg/mL

In Assays 1-3, no insolubility was detected in the final treatment medium at the beginning and end of the treatment in any of the experiments. There were no large changes in pH or osmolality after treatment. In Assay 1, following a 3-hour treatment with metabolic activation, excessive cytotoxicity of the test item was observed at 140 µg/mL, such that all cells in that sample died during the treatment. An evaluation was made using data of the first surviving concentration of 120 µg/mL (relative survival value of 11 %) and the next seven lower concentrations (a total of eight samples). No significant increase in the mutation frequency was observed at the evaluated concentrations. No dose response to the treatment was indicated by the linear trend analysis. In Assay 1, following a 3-hour treatment without metabolic activation, excessive cytotoxicity of the test item was observed at concentrations of 120, 110 and 100 µg/mL, such that all cells in these samples died during the treatment. Therefore, an evaluation was made using data of the first surviving concentrations of 90 and 80 µg/mL (relative survival values of 9 and 39%, respectively) and the next six concentrations (a total of eight samples). No statistically significant increase in the mutation frequency was observed at the evaluated concentrations. Furthermore, no significant dose response to the treatment was indicated by the linear trend analysis. The result of this experiment was considered to be negative.

In Assay 2, no cytotoxicity was observed at the highest examined concentration after treatment. Therefore, the selected concentration range did not meet the requirements of the relevant OECD guideline. Thus the assay was considered invalid, and the experiments were terminated. Results of the invalid experiments are not reported, but all the data will be kept and archived in the raw data binder. An additional experiment (Assay 3) was started using the same experimental conditions.

In Assay 3, following a 3-hour treatment with metabolic activation, excessive cytotoxicity of the test item was observed at 140 µg/mL, such that all cells in that sample died during the treatment. Marked cytotoxicity of the test item was detected at 120 µg/mL (relative survival value of 1 %), therefore this concentration was excluded from the evaluation. An evaluation was made using data of the next concentrations of 110 and 100 µg/mL (relative survival values of 9 and 22 %, respectively) and six lower concentrations (a total of eight samples). No statistically significant increase in the mutation frequency was observed at any of the evaluated concentrations. No dose response to the treatment was indicated by the linear trend analysis.

In Assay 3, following a 24-hour treatment without metabolic activation, excessive cytotoxicity of the test item was observed at 90 and 80 µg/mL, such that all cells in these samples died during the treatment. Marked cytotoxicity of the test item was detected at 70 µg/mL (relative survival value of 5 %), therefore this concentration was excluded from the evaluation. An evaluation was made using data of the next concentration of 65 µg/mL (relative survival value of 15 %) and seven lower concentrations (a total of eight samples). No statistically significant increases in the mutation frequency were observed at the evaluated concentrations. No significant dose response to the treatment was indicated by the linear trend analysis. The result of this experiment was considered to be negative and thus confirmed the results of the first main test.

The experiments were performed using appropriate untreated, negative (vehicle) and positive control samples in all cases. The spontaneous mutation frequency of the negative (vehicle) controls was in the recommended range in each test. The positive controls gave the anticipated increases in mutation frequency over the controls. The plating efficiencies for the solvent controls at the end of the expression period were within the acceptable range in all assays. The evaluated concentration ranges were considered to be adequate, as they covered the range from 80 to 90% cytotoxicity to no or little cytotoxicity. The number of test concentrations met the acceptance criteria. Therefore, the overall study was considered to be valid.

In conclusion, no mutagenic effect of the substance was observed either in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No. 1272/2008 including ATP3.

Self-classification:

Based on the available data, no additional classification is proposed. All three in vitro genotoxicity assays were negative.