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Genetic toxicity in vitro

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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was condusted in compliance with the principles of GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
mutant histidine gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 mix was made from the livers of male Sprague Dawley rats, which received a single intraperitoneal injection of 500 mg/kg bw Aroclor 1254, dissolved in corn oil, 5 days prior to sacrifice. The S9 mix comprised 10% S9 fraction.
Test concentrations with justification for top dose:
plate incorporation assay:
0, 50, 160, 500, 1600, 5000 µg/plate with and without S9 mix
preincubation assay:
0, 50, 160, 500, 1600, 5000 µg/tube with and without S9 mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionized water (positiv control Mitomycin C); DMSO (test item and other positive controls)
- Justification for choice of solvent/vehicle: test item formed colorless clear solution
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
-S9: Na-azide (only TA 1535), nitrofurantoin (only TA 100), 4-nitro-1,2-phenylene diamine (TA 1537 and TA 98), mitomycin C (only TA 102 in plate incorporation assay), cumene hydroperoxide (only TA 102 in preincubation assay): + S9: 2-aminoanthracene
Details on test system and experimental conditions:
- Composition of test material, percentage of components: 100%
Evaluation criteria:
A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100 and TA 98 this increase should be about twice that of negative controls, whereas for TA 1537, at least a threefold increase should be reached. For TA 102 an increase of about 100 mutants should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgment.
In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.
Statistics:
no statistics perfomed; evaluation based on criteria mentioned above
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium 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
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Test item precipitation occurred at 5000 µg per plate/tube. No bacteriotoxic effects were produced up to 5000 µg/plate in the plate incorporation test. In the preincubation assay bacteriotoxic effects were observed at 5000 µg/tube only in TA 1537.
No indication of mutagenic effects could be found for the test item at doses of up to 5000 µg per plate in any of the Salmonella typhimurium strains used, without and with metabolic activation, under the experimental conditions applied.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Executive summary:

The test item was evaluated in an Ames Test on Salmonella typhimurium strains TA 1535, TA 100, TA, 1537, TA 98, and TA 102, performed according to OECD TG 471. Doses of up to and including 5000 µg per plate did not produce bacteriotoxic effects. The test material was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test. Positive controls increased the mutant counts to well over those of the negative controls, thus demonstrated the system´s sensitivity.

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was condusted in compliance with the principles of GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
(1997)
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT locus
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix from the liver of phenobarital/beta-naphthoflavone induced male Wistar rats.
Test concentrations with justification for top dose:
The highest concentration used in the pre-test (5.3 μL/mL) was chosen with regard to the current OECD guideline. Test item concentrations between 19.5 μg/mL and 5.3 μL/mL were used to evaluate toxicity in the presence and absence of metabolic activation (4 and 24 hours treatment). Relevant cytotoxic effects were observed at the highest concentration without metabolic activation following 4 hours treatment. No precipitation or phase separation occurred up to the maximum concentration with and without metabolic activation.In the pre-experiment the two highest concentrations were neutralised with 2 N sodium hydroxide. There was no relevant shift of osmolarity of the medium even at the maximum concentration of the test item. In the main experiments the pH was adjusted with 2 N sodium hydroxide at the two highest concentrations.

Based on the results of the pre-experiment, the maximum concentration of the main experiments was again, set to 5.3 μL/mL or 5.0 μL/mL considering the purity of the test item. The individual concentrations were generally spaced by a factor of 2. A slightly larger step was used between the two highest concentrations.

Doses applied in the gene mutation assay with:

exposure S9
period mix concentrations in µg/mL
Experiment I
4 hours - 156.3(x) 312.5 625.0 1250 2500 5.3 µL/mL
4 hours + 156.3(x) 312.5 625.0 1250 2500 5.3 µL/mL

Experiment II
24 hours - 156.3(x) 312.5 625.0 1250 2500 5.3 µL/mL
4 hours + 156.3(x) 312.5 625.0 1250 2500 5.3 µL/mL

(x) = concentration not chosen for mutation rate analysis
Vehicle / solvent:
Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ethylmethane sulphonate; 7,12-dimethylbenz(a)anthracene
Remarks:
ethylmethane sulfonate used as positive control in cultures without S9-mix (150 µg/mL); dimethylbenzanthracene used as positive control in cultures with S9-mix (1.1 µg/mL).
Details on test system and experimental conditions:
Influence of test item in a concentration of 5.3 µL/mL on pH and osmolality was assessed.
Three or four days after treatment 1.5x10 E6 cells per experimental point were subcultivated in 175 cm² flasks containing 30 mL medium. Fowowing the expression time of 7 days five 80 cm² cell culture flasks were seeded with about 3 - 5x10 E5 cells each in medium containing 6-Thioguanine (11 µg/ml). Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability. The cultures were incubated for about 8 days. The colonies were stained with 10 % methylene blue in 0.01 % KOH solution.
Stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.
Evaluation criteria:
A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points. A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as folIows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment. The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed. However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate in the range normally found (3.3 -33.2 mutants per 106 cells) a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
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:
No precipitation or phase separation occurred up to the maximum concentration with and without metabolic activation.
A relevant cytotoxic effect indicated by a relative cloning efficiency I and/or relative cell density below 50% in both parallel cultures solely occurred at the maximum concentration of 5.3 μL/mL following 4 hours treatment in the absence of metabolic activation.
No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiments up to the maximum concentration. A single, isolated increase of the induction factor, exceeding the threshold of 3 times the mutation frequency of the corresponding solvent control occurred at 625 μg/mL in the first culture of the second experiment without metabolic activation. This increase was judged as biological irrelevant fluctuation as it was neither reproduced in the parallel culture under identical experimental conditions nor at any other, even higher concentration of both parallel cultures.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequency. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in the first culture of the first experiment with metabolic activation and in the second culture of the second experiment with metabolic activation. However, both trends were judged as biologically irrelevant since they were not reproduced and the mutation frequency did not exceed the threshold described above.
In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 10.6 up to 33.1 mutants per 106 cells; the range of the groups treated with the test item was from 10.5 up to 59.7 mutants per 106 cells.
EMS (150 μg/mL) and DMBA (1.1 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolarity: no relevant shift even ath the maximum concentration of the test item. The pH was adjusted with 2 N sodium hydroxide at the two highest concentrations.
Remarks on result:
other: strain/cell type: Chinese hamster lung fibroblasts (V79)
Remarks:
Migrated from field 'Test system'.
Executive summary:

The test item was tested in an in vitro gene mutation assay in V79 cells (HPRT) according to OECD TG 476. The vehicle control and appropriate positive controls with known mutagens demonstrated the sensitivity of the test system and the activity of the metabolic activation system. No substantial and reproducible dose dependent increase of the mutation frequency was observed for the test item in the cultures with and without S9 mix when tested up to cytotoxic concentrations. Based on these results the test substance is considered to be non-mutagenic in this V79/HPRT test under the conditions tested.

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: other: chromosome breakage (structural chromosomal aberrations) and misdistribution of chromosomes
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was condusted in compliance with the principles of GLP
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test), 2010
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
not applicable
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
Liver homogenates were isolated in house from the livers of Aroclor 1254-induced male Sprague-Dawley rats. Before treatment S9 was thawed and mixed with co-factor solution to result in a final protein concentration of 2% S) in cultures.
Test concentrations with justification for top dose:
pulse treatment (4h): 250, 500, 1000, 2000, 3000, 4000 and 5000 µl/mL Without/with S9 mix
continuous treatment (24h): 100, 250, 500, 1000, 2000, 3000 and 4000 µl/mL without S9 mix
Without S9 mix relevant cytotoxic effects were observed at 1000 μg/mL and above after 4 hours treatment and at 500 μg/mL and above after 24 hours treatment. With S9 mix relevant cytotoxic effects were observed at 1000 μg/mL and above.
based on the results of the relative increase in cell count (RICC) or the proliferation index (PI) three concentrations of the test item were selected for the reading of slides: pulse treatment without/with S9 mix: 1000, 2000 and 5000 µl/mL; continuous treatment: 500, 1000 and 3000 µl/mL
Vehicle / solvent:
as vehicle for the test item DMSO was used
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
without metabolic activation: Mitomycin C (0.1 or 0.05 µg/ml) and vinblastine sulfate (0.2 and 0.25 µg/ml); with metabolic activation: cyclophosphamide (2 µg/ml)
Details on test system and experimental conditions:
The used V79 cell line has a stable karyotype with a modal chromosome number of 22. Cells were routinely checked for mycoplasma contamination and karyotype stability.
Evaluation criteria:
The evaluation of results was performed as follows:
- The test item is classified as mutagenic if one of the test substance concentrations induces a micronucleus frequency that is three times higher than the micronucleus frequency of the concurrent solvent control.
- The test item is classified as mutagenic if there is a reproducible concentration-related increase in the micronucleus frequency. Such an evaluation may be considered independently of the enhancement factor for induced micronucleus frequencies.
- In the evaluation of the test results historical control data obtained in the laboratory and scientific plausibility is taken into consideration.
- Any positive test result should be evaluated for its biological relevance.
Statistics:
So far no satisfactory mathematical methods are available for statistical analysis of mammalian cell mutagenicity experiments such as the in vitro micronucleus assay. Assessment was performed according to the evaluation criteria.
Species / strain:
Chinese hamster lung fibroblasts (V79)
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:
The results of the solvent control confirmed the spontaneous micronucleus frequency which is characteristic for V79 cells.
Appropriate positive control items (mitomycin C, vinblastine sulfate, cyclophosphamide) showed the expected increase in micronucleus frequencies which demonstrates the ability and the sensitivity of the test system to detect cytogenetic damage.
Without S9 mix relevant cytotoxic effects were observed at 1000 μg/mL and above after 4 hours treatment and at 500 μg/mL and above after 24 hours treatment. With S9 mix relevant cytotoxic effects were observed at 1000 μg/mL and above. Precipitation in the medium did not occur.
In addition, alteration in cell morphology was observed at 3000 μg/ml and above. No biologically relevant increase in the frequency of micronucleus containing V79 cells treated with Ester PSA+DEG in the absence (4 hours and 24 hours treatment) or in the presence of S9 mix (4 hours treatment).



Summary of the results 4 hours treatment without and with S9 mix

 

Test substance

Concentration

Cytotoxicity (RICC)
(mean, %)

Cytotoxicity (PI)
(mean, %)

MN (mean, %)

>= 6 MN
(mean, %)

(µg/mL)

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

DMSO (sc)

1 % (v/v)

0

0

0

0

0.8

0.6

0

0

test item

250

17.3

27.2

3.8

-4.3

---

---

---

---

500

26.6

13.9

10.4

-7.9

---

---

---

---

1000

10.1

42.4#

11.4

-10.8

1.00

1.0

0

0

2000

15.8

32.2#

19.4

2.0

0.8

0.9

0

0

3000

18.0

45.6

14.6

-0.3

---

---

---

---

4000

29.5

36.7

11.6

-1.4

---

---

---

---

5000

13.7

39.2#

13.3

1.3

0.5

0.3

0

0

Mitomycin C (pc)

0.1

43.9#

-

29.7#

-

12.8*

-

0

-

Vinblastine sulfate salt (pc)

0.08

71.9

-

18.7

-

---

-

---

-

0.10

60.4

-

24.3

-

---

-

---

-

0.12

63.3#

-

22.7

-

7.6*

-

0

-

0.14

64.7

-

23.0

-

---

-

---

-

Cyclophosphamide (pc)

2.0

-

69.6#

-

27.6

-

13.4*

-

0

Summary of the results 24 hours treatment without S9 mix

 

Test substance

Concentration
(µg/mL)

Cytotoxicity (RICC)
(mean, %)

Cytotoxicity(PI)
(mean, %)

MN
(mean, %)

>= 6 MN
(mean, %)

 

 

DMSO (sc)

1 % (v/v)

0

0

0.5

0

 

 

 

100

7.6

0.9

---

---

 

 

 

250

18.8

3.9

---

---

 

 

 

500

32.4#

14.0

0.4

0

 

 

 

test item

1000

45.9#

18.3

1.5

0

 

 

 

2000

35.3

21.8

---

0

 

 

 

3000

66.5#

18.8

0.4

---

 

 

 

4000

65.3

22.8

---

---

 

 

 

Mitomycin C (pc)

0.05

73.5#

30.4#

18.8*

0.6

 

 

 

Vinblastine sulfate salt

0.012

42.4#

-3.9

4.2*

0

 

 

 

MN        = micronucleus containing cells

>= 6 MN   = cells containing 6 or more micronuclei

pc          = positive control

sc          = solvent control

---           = not scored

-            = not tested

#            = relevant increase in cytotoxicity

*            = relevant increase in micronucleated cells

Executive summary:

The test item was examined for mutagenic activity (chromsome breakage and misdistribution of chromosomes) in the in vitro micronucleus test using Chinese Hamster V79 cells in accordance to OECD Guideline 487. The negative control and appropriate positive controls with known mutagens demonstrated the suitability and sensitivity of the test system. The test item showed no biologically relevant increase in the frequency of micronucleus containing V79 cells in the absence (both pulse and continuous treatment) or in the presence of S9 mix (pulse treatment) when tested up to cytotoxic concentrations.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In vitro gene mutation study in bacteria:

The test item was evaluated in an Ames Test on Salmonella typhimurium strains TA 1535, TA 100, TA, 1537, TA 98, and TA 102, performed according to OECD TG 471. Doses of up to and including 5000 µg per plate did not produce bacteriotoxic effects. The test material was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test. Positive controls increased the mutant counts to well over those of the negative controls, thus demonstrated the system´s sensitivity.

In vitro micronucleus study:

The test item was examined for mutagenic activity (chromsome breakage and misdistribution of chromosomes) in the in vitro micronucleus test using Chinese Hamster V79 cells in accordance to OECD Guideline 487. The negative control and appropriate positive controls with known mutagens demonstrated the suitability and sensitivity of the test system. The test item showed no biologically relevant increase in the frequency of micronucleus containing V79 cells in the absence (both pulse and continuous treatment) or in the presence of S9 mix (pulse treatment) when tested up to cytotoxic concentrations.

In vitro gene mutation study in mammalian cells

The test item was tested in an in vitro gene mutation assay in V79 cells (HPRT) according to OECD TG 476. The vehicle control and appropriate positive controls with known mutagens demonstrated the sensitivity of the test system and the activity of the metabolic activation system. No substantial and reproducible dose dependent increase of the mutation frequency was observed for the test item in the cultures with and without S9 mix when tested up to cytotoxic concentrations. Based on these results the test substance is considered to be non-mutagenic in this V79/HPRT test under the conditions tested.


Justification for selection of genetic toxicity endpoint
No study was selected since all three in vitro mutagenicity studies were negative.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available study results (negative in Ames test, HPRT test and micronucleus test, in vitro) a classification according to Regulation (EC) No 1272/2008, Annex I is not warranted.