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Genetic toxicity in vitro

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Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: genome mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-06-01 to 2012-12-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study, detailed documentation
Qualifier:
according to guideline
Guideline:
other: OECD 487
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
Formation of micronuclei in human peripheral blood lymphocytes
Species / strain / cell type:
lymphocytes:
Metabolic activation:
with and without
Metabolic activation system:
Mammalian liver post-mitochondrial fraction (S9), prepared from Sprague Dawley male rats
Test concentrations with justification for top dose:
Range-finding: 0, 9.375, 18.75, 37.5, 75, 150, 300 µg/mL (final concentrations)
Definitive test. experiment 1, -S9: 75, 150, 300 µg/mL + vehicle (1% DMSO v/v ) + Cytosine arabinoside (pos. control)
Definitive test. experiment 1, +S9: 75, 150, 300 µg/mL + vehicle (1% DMSO v/v ) + Cytosine arabinoside (pos. control)
Definitive test. experiment 2, -S9: 7.5, 15, 30, 60, 120, 240, 480 µg/mL + vehicle (1% DMSO v/v ) + DMBA (pos. control)
Definitive test. experiment 2, +S9: 7.5, 15, 30, 60, 120, 240, 480 µg/mL + vehicle (1% DMSO v/v ) + DMBA (pos. control)
Appropriate amount of test item was weighted and diluted in DMSO not earlier than 30 min prior to test start. For dilution of final doses, sterile DMSO was used.
Vehicle / solvent:
- Vehicle/solvent used: DMSO+ culture medium
Untreated negative controls:
yes
Remarks:
culture medium
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Cytosine arabinoside was used as positive control without S9mix, Cyclophosphamide monohydrate was used as positive control with S9mix.
Details on test system and experimental conditions:
METHOD OF APPLICATION:
10exp5 cells in 75 µl

DURATION
- Exposure duration: 20 hr

METHOD OF APPLICATION: in medium;

DURATION
- Preincubation period: 24 hours
- Exposure duration: 20 hours
- Time with Cytochalasin B: 28 hours:
- Fixation time (start of exposure up to fixation or harvest of cells): 72 hours

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B
STAIN (for cytogenetic assays): 2% Giemsa-Romanowski for 10 minutes

NUMBER OF CELLS EVALUATED: 10exp5 cells

DETERMINATION OF CYTOTOXICITY
- Method: other: replication index (Number of binucleated cells, mulinucleated cells and toatal number)
Evaluation criteria:
For the untreated controls and solvent controls, the measured average number of micronuclei is related to the spontanous occurence of micronuclei. A two tailed Student's t-test revealed if the test substance can be considered as positive, negative or equivocal.
Statistics:
Student's t-test
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:
valid
Positive controls validity:
valid

Experiment 1: range-finding test without metabolic activation

Experiment 2:definitive test with and without metabolic activation

Conclusions:
Interpretation of results (migrated information):
negative

TC, dried did not induce micronuclei neither with nor without metabolic activation by S-9 mix in human lymphocytes. TC, dried was considered to be non-mutagenic.
Executive summary:

TC, dried was dissolved in DMSO in concentrations of 9.375 to 300 µg/ml in an in vitro micronucleus test with human lymphocytes according to OECD 487.

Up to and including the highest possible concentrations TC, dried, did not induce micronuclei neither with nor without metabolic activation by S-9 mix.

Therefore TC, dried was considered to be non-mutagenic.

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:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study, detailed documentation
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
TA100: his G46
TA98: his D3052
TA97: his D6610
TA102: his G428 (pAQ1)
TA1535: his G46
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
other: Strain is histidine-dependent
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
other: Strain is histidine-dependent
Species / strain / cell type:
S. typhimurium TA 97
Additional strain / cell type characteristics:
other: Strain is histidine-dependent
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
other: Strain is histidine-dependent
Species / strain / cell type:
S. typhimurium TA 1535
Additional strain / cell type characteristics:
other: Strain is histidine-dependent
Metabolic activation:
with and without
Metabolic activation system:
Mammalian liver post-mitochondrial fraction (S9), prepared from Sprague Dawley male rats.
Test concentrations with justification for top dose:
Range-Finding: 7 concentrations in the range of 0.0001-5.0 mg/plate
Confirmatory assay with preincubation: 0.00001-0.5 mg/plate
Appropriate amount of test item was weighted and diluted in DMSO not earlier than 30 min prior to test start. For dilution of final doses, sterile DMSO was used.
Vehicle / solvent:
- Vehicle/solvent used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48-72 hr

NUMBER OF REPLICATIONS: 3
Evaluation criteria:
- concentration-related increase over the tested range and reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation
- mutation factor >2
Statistics:
Student's t-test was used
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Untreated negative 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:
not specified
Untreated negative 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:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
valid

The results indicate that TC, dried neither produce statistically significant dose-related increase in the number of revertants nor a statistically significant and reproducible positive response at any of the test points.

Conclusions:
Interpretation of results (migrated information):
negative

TC, dried is considered to be non-mutagenic in bacterial gene mutation test system.
Executive summary:

TC, dried was dissolved in DMSO and tested in the in vitro gene mutation test according to OECD 471 (Ames-Test).

5 strains of Salmonella typhimurium were used: TA100, TA98, TA97, TA102, and TA1535.

The test was performed with and without metabolic activation with S-9 mix.

The test article did not induce mutagenicity up to and including concentrations of 5 mg/plate.

Therefore TC, dried is considered to be non-mutagenic.

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:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study, detailed documentation
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:
hprt locus of V79 Chinese Hamster lung cells
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
Mammalian liver post-mitochondrial fraction (S9), prepared from Sprague Dawley male rats
Test concentrations with justification for top dose:
Range-finding: 0, 0.75, 7.5, 15, 30, 60, 120, 240, 480 µg/mL (final concentrations)
Definitive test. experiment 1, -S9: 3.75, 7.5, 15, 30, 60, 120 µg/mL + vehicle (1% DMSO v/v ) + EMS (pos. control)
Definitive test. experiment 1, +S9: 7.5, 15, 30, 60, 120, 240, 480 µg/mL + vehicle (1% DMSO v/v ) + EMS (pos. control)
Definitive test. experiment 2, -S9: 7.5, 15, 30, 60, 120, 240, 480 µg/mL + vehicle (1% DMSO v/v ) + DMBA (pos. control)
Definitive test. experiment 2, +S9: 7.5, 15, 30, 60, 120, 240, 480 µg/mL + vehicle (1% DMSO v/v ) + DMBA (pos. control)
Appropriate amount of test item was weighted and diluted in DMSO not earlier than 30 min prior to test start. For dilution of final doses, sterile DMSO was used.
Vehicle / solvent:
- Vehicle/solvent used: DMSO
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
other: Ethyl methanesulphonate 600 µg/mL, 7,12-dimethyl-benz[a]anthracene 3 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION:
10exp6 cells in 5 mL Dulbecco's Modified Eagle's medium, with 4.5 g/L glucose supplemented with L-glutamine seeded in Petri dishes

DURATION
- Exposure duration: 3 hr

NUMBER OF REPLICATIONS: 2
Evaluation criteria:
- mutant frequency at one or more concentrations at least 3-fold greater than that of negative control
- concentration-related increase in mutant frequency
- effects described above were reproducible
Statistics:
Kruskat-Wallis test, p<0.01
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Experiment 1:

RPE was ca. 115.2% in absence of S-9 at 120 µg/mL and 72.6% in presence of S-9 (480 µg/mL.

Experiment 2:

at 480 µg/mL RPE was ca. 102.0% in absence of S-9 and ca. 82.6% in presence of S-9.

Conclusions:
Interpretation of results (migrated information):
negative

TC, dried did not induce mutation at the hprt locus of V79 Chinese Hamster lung cells in concentrations up to and including 480 µg/mL (limit of solubility).
Executive summary:

TC, dried was dissolved in DMSO in concentrations up to the limit of solubility at 480 µg/ml and tested in an in vitro mammalian cell gene mutation test according to OECD 476. The ability to induce mutagenicity was tested in this cytogenetic test at the hprt locus.

Up to and including the highest possible concentrations TC, dried did not induce mutagenic activity neither with nor without metabolic activation by S-9 mix. The maximum tested concentration was limited by solubility.

Therefore TC, dried was considered to be non-mutagenic.

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

Genetic toxicity in vivo

Description of key information

6,6',6''-(1,3,5-Triazine-2,4,6-triyltriimino)trihexanoic acid has no potential to cause chromosomal damage in vivo.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
(adopted 1997)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
other: Hsd:ICR (CD-1)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Italy S.r.l., San Pietro al Natisone (UD)
- Age at study initiation: 5-6 weeks
- Weight at arrival: 22-30 g
- Housing: 5 animals per cage in clear polycarbonate cages
- Diet: commercially available laboratory rodent diet (Altromin MT, Altromin, D-32770 Lage, Postfach 1120, Germany), ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 10
- Photoperiod: 12 hrs dark / 12 hrs light
Route of administration:
oral: gavage
Vehicle:
- Vehicle used: CMC (carboxymethyl cellulose, 0.5%)
- Concentration of test material in vehicle: 200 mg/mL; 100 mg/mL; 50 mg/mL
- Amount of vehicle: 10 mL/kg
- Batch No. (from BDH): 1072067
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Solutions of the test item, as received, were prepared immediately before use in carboxymethylcellulose 0.5%. Solutions were prepared on a weight/volume basis without connection for the displacement due to the volume of the test item.
Duration of treatment / exposure:
Not applicable.
Frequency of treatment:
Single treatment.
Post exposure period:
Control group: 24 and 48 h
500 mg/kg bw: 24 h
1000 mg/kg bw: 24 h
2000 mg/kg bw: 24 and 48 h
Positive control: 24 h
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Control group: 10 per sex
500 mg/kg bw: 5 per sex
1000 mg/kg bw: 5 per sex
2000 mg/kg bw: 10 per sex
Positive control: 5 per sex
Control animals:
yes, concurrent vehicle
Positive control(s):
Mitomycin-C (batch no. 31K2504; SIGMA)
- Route of administration: oral, gavage
- Doses / concentrations: 3 mg/kg bw
Tissues and cell types examined:
Erythrocytes of femur bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A toxicity test was performed to aid in the selection of dose-levels. Groups of two male and two female mice were dosed by oral gavage with the test item at 2000, 1000 and 500 mg/kg bodyweight. The animals were observed for 48 hours and sacrificed. Scoring was performed on slides prepared from the femurs of animals.

DETAILS OF SLIDE PREPARATION:
Animals were sacrificed at appropriate sampling times and the femurs of animals were removed and bone marrow cells obtained by flushing with fetal calf serum. The cells were centrifuged and a concentrated suspension was prepared to make smears on slides. These slides were air-dried and then stained with May-Gruenwald and Giemsa, and mounted with Eukitt. Three slides were made from each animal.

METHOD OF ANALYSIS:
The slides were randomly coded by a person not involved in the subsequent microscope scoring. The slides were examined under low power (x 16 objective) and one slide from each animal was selected according to staining and quality of smears. Where no depression of polychromatic erythrocytes was observed at least 2000 polychromatic cells per animal were examined for the presence of micronuclei at high power (x 100 objective, oil immersion). At the same time the numbers of normal and micronucleated normochromatic erythrocytes were also recorded.
Evaluation criteria:
The test item is considered to induce micronuclei if a statistically significant increase in the micronucleus incidence in polychromatic erythrocytes (at P<0.05) is observed in any treatment group, in the pooled data for both sexes, or for either sex considered separately. Where increases in the incidence of micronucleated PCE's are observed which are statistically significant, but fall within the range of negative control values within this laboratory, then concurrent and historical control data are used to demonstrate that these increases do not have biological significance.
Statistics:
Only counts obtained from polychromatic cells were subjected to statistical analysis. Using the original observations (and not the micronucleus frequencies per 1000 cells), a modified Chi-squared calculation was employed to compare treated and control groups.
The degree of heterogeneity within each group was first calculated and where this was significant it was taken into account in the comparison between groups. Variance ratios or Chi-squared values are taken to show the significance of any difference between each treated group and the controls.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
Reduced activity was observed in female animals on the day of the treatment and the day after. No other adverse reactions to treatment were noted. Treatment with the test item had no adverse effect on the proliferation of bone-marrow erythropoietic cells. Based on the above results, dose-levels of 500, 1000 and 2000 mg/kg bodyweight were selected for the Main assay.

RESULTS OF DEFINITIVE STUDY
No increases in the numbers of micronucleated PCE's were observed in any treatment group at any sampling time. Pronounced increases in the frequency of micronucleated PCE's were observed in the positive control group, indicating the correct functioning of the test system.
The ratio of mature to immature erythrocytes and the proportion of immature erythrocytes among total erythrocytes were analyzed to evaluate the bone marrow cell toxicity. Based on these results, no inhibitory effect on erythropoietic cell division was observed in male or female animals at any sampling time.

Table 1: Incidence of micronucleated PCEs and ratio of PCE/NCE of the in-vivo bone marrow micronucleus study.

Treatment

Dose level (mg/kg bw)

Incidence of micronucleated PCEs

PCE/(PCE+NCE)

% over the mean negative control value

 

male/female

Mean

SE

range

 

24 hr sampling time

Vehicle

10 mL/kg

0.6

0.2

0

1.5

100

Test item

500

0.9

0.3

0

3.0

99

Test item

1000

0.5

0.2

0

1.5

101

Test item

2000

0.8

0.3

0

2.5

98

Mitomycin C

3

10.6***

1.4

6.0

17.0

102

 48 hr sampling time

Vehicle

10 mL/kg

0.6

0.2

0

1.5

100

Test item

2000

0.8

0.3

0

3.0

98

***: Incidence significantly greater than control value at p < 0.001

Conclusions:
On the basis of the results obtained, it is concluded that the test article administered by oral gavage, does not induce micronuclei in the polychromatic erythrocytes of treated mice, under the reported experimental conditions.
Executive summary:

The test article's potential to cause chromosomal damage in vivo was investigated in a micronucleus test. Based on results obtained in a preliminary toxicity trial, dose-levels selected were 500, 1000 and 2000 mg/kg bodyweight for male and female animals. Hsd:ICR (CD-I) mice were dosed once by gavage with the vehicle alone (carboxymethylcellulose 0.5%), the test material in the vehicle at the selected dose-levels or with the positive control substance Mitomycin-C. Each group consisted of five male and five female animals with the exception of the control and high-dose group, which included an additional five animals of each sex per group. Five animals per sex from each group were sacrificed at the 24 hour sampling time. The additional animals were sacrificed at the 48 hour sampling time. Bone-marrow smear slides were made and stained with May-Gruenwald and Giemsa stains. At least 2000 polychromatic cells per animal were examined for the presence of micronuclei. The slides were coded prior to scoring. The results obtained at each sampling time were subjected to statistical analysis using a modified chi-squared test. Following treatment with the test substance, no statistically significant increase in the incidence of micronucleated PCE's over the control value was observed at any dose level, at any sampling time. Following treatment with the positive control Mitomycin-C, statistically significant increases in the incidence of micronucleated PCE's over the control values were observed, indicating the correct functioning of the test system. No clinical signs or depression of bone marrow erythropoietic cell division, were observed at any dose-level, at any sampling time. It is concluded that, under the reported experimental conditions, the test item administered by oral gavage at the selected dose-levels to male and female mice, does not induce micronuclei in the polychromatic erythrocytes.

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

Additional information

Potential mutagenicity of 6,6',6''-(1,3,5-Triazine-2,4,6-triyltriimino)trihexanoic acid was tested:

-      Twice according to OECD 471 with negative results

-      Once according to OECD 487 with negative result

-      Once according to OECD 473 with positive result

-      Once according to OECD 476 with negative result

-      Once according to OECD 474 with negative result

All tests together enable thorough assessment for gene mutations and effects on chromosome structure and number.

6,6',6''-(1,3,5-Triazine-2,4,6-triyltriimino)trihexanoic acid has no potential to cause chromosomal damage in vivo.

According to Annexes VII, VIII, IX & X no further tests are required.

 

It can be concluded that 6,6',6''-(1,3,5-Triazine-2,4,6-triyltriimino)trihexanoic acid is not genotoxic.

Justification for classification or non-classification

All tests together enable thorough assessment for gene mutations and effects on chromosome structure and number.

6,6',6''-(1,3,5-Triazine-2,4,6-triyltriimino)trihexanoic acid has no potential to cause chromosomal damage in vivo.

It can be concluded that 6,6',6''-(1,3,5-Triazine-2,4,6-triyltriimino)trihexanoic acid is not genotoxic.