Registration Dossier

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Key value for chemical safety assessment

Genetic toxicity in vitro

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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
S. typhimurium TA 1537
Species / strain / cell type:
S. typhimurium TA 97
Species / strain / cell type:
S. typhimurium TA 98
Metabolic activation system:
S9
Test concentrations with justification for top dose:
concentrations for all test strains: 100, 333, 1000, 3333, 10000 μg/plate
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 2-Aminoantracene, 4-nitro-o-phenyl- enediamine
Details on test system and experimental conditions:
S9 in the S9 mixture (metabolic activation enzymes and cofactors from Aroclor 1254-induced male Sprague-Dawley rat or Syrian hamster liver).
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Concentration denotes the the percentage of S9 in the S9 mixture (metabolic activation enzymes and cofactors from Aroclor 1254-induced male Sprague-Dawley rat or Syrian hamster liver) that was added to cultures.

Strain: TA100

S9 Activation

S9 Species

Concentration

No Activation

N/A

N/A

30% RLI

Rat

30%

30% HLI

Hamster

30%

10% RLI

Rat

10%

10% HLI

Hamster

10%

Strain: TA1535

S9 Activation

S9 Species

Concentration

No Activation

N/A

N/A

30% RLI

Rat

30%

30% HLI

Hamster

30%

10% RLI

Rat

10%

10% HLI

Hamster

10%

Strain: TA97

S9 Activation

S9 Species

Concentration

No Activation

N/A

N/A

30% RLI

Rat

30%

30% HLI

Hamster

30%

10% RLI

Rat

10%

10% HLI

Hamster

10%

Strain: TA98

S9 Activation

S9 Species

Concentration

No Activation

N/A

N/A

30% RLI

Rat

30%

30% HLI

Hamster

30%

10% RLI

Rat

10%

10% HLI

Hamster

10%

Conclusions:
Under the study conditions, the test substance is not mutagenic in the Salmonella typhimurium strains TA 97a, TA 98, TA 100 and TA 1535 in absence and presence of metabolic activation.
Executive summary:

A study was conducted to determine the mutagenic potential of the test substance according to Bacterial Reverse Mutation Test. The test substance was examined using four strains of Salmonella typhimurium (TA 97a, TA 98, TA 100 and TA 1535). The test was performed in the presence and absence of S9-mix (Sprague-Dawley rat or Syrian hamster liver S9-mix induced by Aroclor 1254). 5 concentrations were used for all test strains: 100, 333, 1000, 3333 and 10000 μg/plate. Positive and negative controls were included in the study. No significant increase of the number of revertant colonies could be observed at any of the treatment concentrations. Under the study conditions, the test substance was not mutagenic in the Salmonella typhimurium strains TA 97a, TA 98, TA1 00 and TA 1535 in absence and presence of metabolic activation (NTP, 1985).

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
W-B1
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Experiment I
without metabolic activation: 39.8, 49.8, 60, and 75 μg/ml
with metabolic activation: 400, 450, and 500 μg/ml
Experiment II:
without metabolic activation: 24.9, 30.1, and 35 μg/ml
with metabolic activation: 4100, 5000, and 6000 μg/ml
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Trial #:1   Activation: No Activation    Date: 03/01/1984    Harvest Time: 10.5 hour(s)   Trial Call: Negative   
  Dose Total Total Aberrations Complex Aberrations Simple Aberrations Other Abs
µg/mL Cells Examined No. of Abs % Cells No. of Abs % Cells No. of Abs % Cells No. of Abs % Cells
    Abs Per With Abs. Per With Abs. Per With Abs. Per With
      Cell Abs   Cell Abs   Cell Abs   Cell Abs
Vehicle Control: Negative (Not Specified) 0          100 3 0.03 3 2 0.02 2 1 0.01 1 0 0 0
Dimethyl Sulfoxide 0          100 0 0 0 0 0 0 0 0 0 0 0 0
Test Chemical: test sub. 39.8        100 1 0.01 1 0 0 0 1 0.01 1 0 0 0
  49.8        100 1 0.01 1 1 0.01 1 0 0 0 0 0 0
  60          100 2 0.02 2 2 0.02 2 0 0 0 0 0 0
  75          0 0 0 0 0 0 0 0 0 0 0 0 0
Positive Control: Mitomycin-C  0.2        100 16 0.16 11 5 0.05 5 9 0.09 8 2 0.02 1
Mitomycin-C  0.5        25 7 0.28 28 5 0.2 20 2 0.08 8 0 0 0
Trend: 1.337 1.804 -0.453  
Probability: 0.091 0.036 0.675  
Trial #:1_S9   Activation: Induced Rat Liver S9    Date: 03/01/1984    Harvest Time: 10.5 hour(s)   Trial Call: Test Failure   
  Dose Total Total Aberrations Complex Aberrations Simple Aberrations Other Abs
µg/mL Cells Examined No. of Abs % Cells No. of Abs % Cells No. of Abs % Cells No. of Abs % Cells
    Abs Per With Abs. Per With Abs. Per With Abs. Per With
      Cell Abs   Cell Abs   Cell Abs   Cell Abs
Vehicle Control: Dimethyl Sulfoxide 0          100 5 0.05 4 1 0.01 1 4 0.04 4 0 0 0
Test Chemical: test sub. 1000          100 3 0.03 3 1 0.01 1 2 0.02 2 0 0 0
Positive Control: Cyclophosphamide 37.5        25 16 0.64 40 3 0.12 12 13 0.52 36 0 0 0
Trend: 0 0 0  
Probability: 0 0 0  
Trial #:2   Activation: No Activation    Date: 01/26/1984    Harvest Time: 10.5 hour(s)   Trial Call: Negative   
  Dose Total Total Aberrations Complex Aberrations Simple Aberrations Other Abs
µg/mL Cells Examined No. of Abs % Cells No. of Abs % Cells No. of Abs % Cells No. of Abs % Cells
    Abs Per With Abs. Per With Abs. Per With Abs. Per With
      Cell Abs   Cell Abs   Cell Abs   Cell Abs
Vehicle Control: Negative (Not Specified) 0          100 5 0.05 5 2 0.02 2 2 0.02 2 1 0.01 1
Dimethyl Sulfoxide 0          100 4 0.04 4 2 0.02 2 2 0.02 2 0 0 0
Test Chemical: test sub. 24.9        100 3 0.03 3 2 0.02 2 1 0.01 1 0 0 0
  30.1        100 5 0.05 5 2 0.02 2 3 0.03 3 0 0 0
  35          100 4 0.04 4 0 0 0 4 0.04 4 0 0 0
Positive Control: Mitomycin-C  0.25       100 19 0.19 15 8 0.08 7 11 0.11 8 0 0 0
Mitomycin-C  0.5        50 17 0.34 24 7 0.14 12 10 0.2 18 0 0 0
Trend: 0.251 -1.07 1.148  
Probability: 0.401 0.858 0.125  
Trial #:2_S9   Activation: Induced Rat Liver S9    Date: 01/11/1984    Harvest Time: 10.5 hour(s)   Trial Call: Negative   
  Dose Total Total Aberrations Complex Aberrations Simple Aberrations Other Abs
µg/mL Cells Examined No. of Abs % Cells No. of Abs % Cells No. of Abs % Cells No. of Abs % Cells
    Abs Per With Abs. Per With Abs. Per With Abs. Per With
      Cell Abs   Cell Abs   Cell Abs   Cell Abs
Vehicle Control: Negative (Not Specified) 0          100 3 0.03 2 0 0 0 3 0.03 2 0 0 0
Dimethyl Sulfoxide 0          100 3 0.03 2 0 0 0 3 0.03 2 0 0 0
Test Chemical: test sub. 400          100 3 0.03 3 0 0 0 3 0.03 3 0 0 0
  450          100 2 0.02 2 0 0 0 2 0.02 2 0 0 0
  500          100 7 0.07 3 0 0 0 7 0.07 3 0 0 0
Positive Control: Cyclophosphamide 25          50 16 0.32 22 6 0.12 10 10 0.2 16 0 0 0
Cyclophosphamide 50          100 12 0.12 12 5 0.05 5 7 0.07 7 0 0 0
Trend: 0.277 0 0.277  
Probability: 0.391 0 0.391  
Trial #:3_S9   Activation: Induced Rat Liver S9    Date: 03/22/1984    Harvest Time: 10.5 hour(s)   Trial Call: Negative   
  Dose Total Total Aberrations Complex Aberrations Simple Aberrations Other Abs
µg/mL Cells Examined No. of Abs % Cells No. of Abs % Cells No. of Abs % Cells No. of Abs % Cells
    Abs Per With Abs. Per With Abs. Per With Abs. Per With
      Cell Abs   Cell Abs   Cell Abs   Cell Abs
Vehicle Control: Negative (Not Specified) 0          100 1 0.01 1 0 0 0 1 0.01 1 0 0 0
Dimethyl Sulfoxide 0          100 2 0.02 2 0 0 0 2 0.02 2 0 0 0
Test Chemical: test sub. 4100          100 0 0 0 0 0 0 0 0 0 0 0 0
  5000          100 2 0.02 2 1 0.01 1 1 0.01 1 0 0 0
  6000          100 2 0.02 1 0 0 0 2 0.02 1 0 0 0
Positive Control: Cyclophosphamide 5          100 8 0.08 8 0 0 0 8 0.08 8 0 0 0
Cyclophosphamide 37.5        25 9 0.36 28 3 0.12 8 5 0.2 20 1 0.04 4
Trend: -0.188 0.474 -0.448  
Probability: 0.574 0.318 0.673  
Conclusions:
Under the study conditions, the test substance was negative for genotoxicity in a chromosome aberration assay.
Executive summary:

A study was conducted to investigate the mutagenic potential of the test substance in the chromosomal aberration assay. In the first trial, test substance concentrations of 39.8, 49.8, 60, and 75 μg/mL were tested without metabolic activation, and doses of 400, 450, and 500 μg/mL were tested with metabolic activation. In the second trial, concentrations of 24.9, 30.1, and 35 μg/mL of the test substance were tested without metabolic activation, and concentrations of 4100, 5000,and 6000 μg/mL were tested with metabolic activation. Vehicle (DMSO) and appropriate positive controls were used. Under the study conditions, the test substance was negative for genotoxicity in a chromosome aberration assay (NTP, 1984).

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
not specified
Type of assay:
sister chromatid exchange assay in mammalian cells
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Additional strain / cell type characteristics:
other: W-B1
Metabolic activation:
with and without
Test concentrations with justification for top dose:
Experiment I:
without metabolic activation: 5, 7, 10, and 50 μg/ml; with metabolic activation: 30, 100, 300, and 1000 μg/ml
Experiment II:
without metabolic activation: 15.1, 19.95, 25.2, and 30.2 μg/ml; with metabolic activation: 202, 298.2, 396, and 497 μg/ml;
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
Cell harvest and fixation:
Two to three hours after addition of colcemid, cells were collected by mitotic shake-off and treated for up to 3 min at room temperature with hypotomic KCl (75 mM). Cells were then washed twice with fixative (3:1, methanol: glacial acetic acid, v/v), dropped onto slides, and ar-dried).
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
other: weakly positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Trial #: 1    Activation: No Activation    Date: 12/13/1983    Trial Call: Negative   
  Dose Number Number Total SCE / SCE / Hours in % Increase
µg/mL Cells Chromosomes Number Chromosome Cell BRDU Over Solvent
  Examined Examined SCEs       Control
Vehicle Control Dimethyl Sulfoxide 0          50 1026 468 0.456 9.36 25.3 0
Test Chemical test sub. 5          50 1003 484 0.483 9.68 25.3 5.79
  7          50 1036 482 0.465 9.64 25.3 1.997
  10          50 1005 479 0.477 9.58 25.3 4.489
  50          0 0 0 0 0 0 N/A
Positive Control Mitomycin-C  0.0015     50 1021 714 0.699 14.28 25.3 53.311
Mitomycin-C  0.01       5 104 244 2.346 48.8 25.3 414.349
Trend: 0.587        
Probability: 0.279        
Trial #: 2    Activation: No Activation    Date: 01/11/1984    Trial Call: Negative   
  Dose Number Number Total SCE / SCE / Hours in % Increase
µg/mL Cells Chromosomes Number Chromosome Cell BRDU Over Solvent
  Examined Examined SCEs       Control
Vehicle Control Dimethyl Sulfoxide 0          50 1027 469 0.457 9.38 25.8 0
Test Chemical test sub. 15.1        50 1029 410 0.398 8.2 25.8 -12.75
  19.95       50 1029 487 0.473 9.74 32.8 3.636
  25.2        50 1035 504 0.487 10.08 32.8 6.632
  30.2        0 0 0 0 0 0 N/A
Positive Control Mitomycin-C  0.0015     50 1037 662 0.638 13.24 25.8 39.79
Mitomycin-C  0.01       5 102 264 2.588 52.8 25.8 466.763
Trend: 1.848        
Probability: 0.032        
Trial #: 1_S9    Activation: Induced Rat Liver S9    Date: 12/06/1983    Trial Call: Weakly Positive   
  Dose Number Number Total SCE / SCE / Hours in % Increase
µg/mL Cells Chromosomes Number Chromosome Cell BRDU Over Solvent
  Examined Examined SCEs       Control
Vehicle Control Dimethyl Sulfoxide 0          50 1025 494 0.482 9.88 25.3 0
Test Chemical test sub. 30          50 1017 531 0.522 10.62 25.3 8.335
  100          50 1024 561 0.548 11.22 25.3 13.674
  300          50 1012 655 0.647 13.1 34.1 34.294
  1000          0 0 0 0 0 0 N/A
Positive Control Cyclophosphamide  0.4        50 1012 734 0.725 14.68 25.3 50.492
Cyclophosphamide 2          5 101 204 2.02 40.8 25.3 319.088
Trend: 4.986        
Probability: 0        
Trial #: 2_S9    Activation: Induced Rat Liver S9    Date: 12/13/1983    Trial Call: Weakly Positive   
  Dose Number Number Total SCE / SCE / Hours in % Increase
µg/mL Cells Chromosomes Number Chromosome Cell BRDU Over Solvent
  Examined Examined SCEs       Control
Vehicle Control Dimethyl Sulfoxide 0          50 1012 538 0.532 10.76 25.3 0
Test Chemical test sub. 202          50 1024 618 0.604 12.36 25.3 13.524
  298.2        50 1021 610 0.597 12.2 25.3 12.383
  396          50 1017 730 0.718 14.6 25.3 35.021
  497          0 0 0 0 0 0 N/A
Positive Control Cyclophosphamide  0.4        50 1018 865 0.85 17.3 25.3 59.833
Cyclophosphamide 2          5 104 226 2.173 45.2 25.3 308.765
Trend: 4.896        
Probability: 0        
Conclusions:
Under the study conditions, the test substance was weakly positive in a sister chromatid exchange assay in the presence of metabolic activation.
Executive summary:

A study was conducted to determine the genotoxic potential in mammalian cells according to a sister chromatid exchange assay. Two trials were performed. In the first trial, concentrations of 5, 7, 10, and 50 μg/mL of the test substance were tested without metabolic activation, and doses of 30, 100, 300, and 1000 μg/mL were tested with metabolic activation. In the second trial, concentrations of 15.1, 19.95, 25.2, and 30.2 μg/mL of the test substance were tested without metabolic activation, and concentrations of 202, 298.2, 396, and 497 μg/mL were tested with metabolic activation. Vehicle (DMSO) and appropriate positive controls were used. Under the study conditions, the test substance was weakly positive in a sister chromatid exchange assay in the presence of metabolic activation (NTP, 1984).

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

Genetic toxicity in vivo

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: gene mutation
Remarks:
mouse Mutatect tumor model
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: RA study
Justification for type of information:
Refer to the section 13 for details on the read across justification.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
other: mouse Mutatect tumor model
Deviations:
not applicable
GLP compliance:
not specified
Type of assay:
other: mouse Mutatect tumor model
Species:
mouse
Strain:
other: C57BL/6 mice
Sex:
not specified
Details on test animals and environmental conditions:
The mouse Mutatect tumor model is a transplantable fibrosarcoma developed as an experimental paradigm to study the contributions of tumor-infiltrating leukocytes and the reactive nitrogen oxide species they produce on genetic instability. These subcutaneous tumors become infiltrated with leukocytes, predominantly neutrophils, which express inducible nitric oxide synthase, the principal source of reactive nitrogen oxide species. The number of neutrophils is strongly associated with the mutation frequency at the hypoxanthine phosphoribosyltransferase (Hprt) locus in the tumor cells. When injected into mice, utatect cell lines engineered to express human interleukin 8 (IL-8) produce tumors with high levels of neutrophil infiltration and correspondingly high Hprt gene mutation frequencies. The high-frequency loss of IL-8 transgenecontaining cells that occurs in these tumors may be due to a combination of generalized genotoxicity and selective cytotoxicity against IL-8 –secreting tumor cells by neutrophil-derived reactive nitrogen oxide species. Additional evidence of an increase in reactive nitrogen oxide species in these tumors is the presence of protein nitrotyrosine.

Mutatect TM-28 cells, a clone that expresses human IL-8, were injected subcutaneously into 6- to 8-week-old C57BL/6 mice (typically 9 per group; Charles River Laboratories, Quebec, Canada). Tumors were harvested when they reached 1 cm in size (typically at 2.5 to 3 weeks after injection). Dietary vitamin E supplements (0, 25, 50, or 100 mg/kg body weight per day) were added to the tocopherol-stripped rodent pellets that were fed to the mice from 7 days before injection of tumor cells until the mice were killed and their tumors harvested (i.e., a total of 3.5 to 4 weeks). Dietary supplementation with tocopherol had no observable effects on tumor volumes or on mouse behavior or survival (data not shown). The cellular fractions of the tumors were analyzed for Hprt gene mutation frequencies and myeloperoxidase (MPO) activity.
Route of administration:
oral: feed
Details on exposure:
Experiments:
I - Hprt gene mutation frequencies - increasing doses of the test substance on Hprt gene mutation frequency in cells isolated from Mutatect TM-28 tumors

II - effects of dietary tocopherol on the number of neutrophils in tumors – measurement of the activity of MPO, a neutrophil-specific marker in single-cell and stromal fractions of Mutatect TM-28 tumors
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
25 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Control animals:
yes
Statistics:
Two tailed nonparametric tests were used for all statistical analyses. Nonparametric Kruskal–Wallis tests were used to compare three or more unpaired groups. Where P values were less than .05, we used Dunn’s multiple comparison post hoc test to compare values between two groups. The Analyse-it (version 1.65; www.analyse-it.com) was used to calculate 95% confidence intervals (CIs) and Graphpad Prism (version 3; Graphpad Software).
Key result
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
not specified
Additional information on results:
Results:
I - Hprt gene mutation frequencies:

- In two experiments, the Hprt gene mutation frequency decreased with increasing test substance dose.

- Nonparametric two-factor (experiment [exp, random], dose [D, fixed]) Model III analysis of variance revealed a statistically significant difference in the overall Hprt gene mutation frequency between the two experiments (Scheirer–Ray–Hare Hexp = 12.2, df = 1, P<.001).

II - effects of dietary tocopherol on the number of neutrophils in tumors:

- There was a statistically significant decrease in MPO activity in the single-cell fraction with increasing dose of the test substance in two experiments. A statistically significant decrease in MPO activity was observed in the single-cell fraction of tumors from mice fed 50 or 100 mg/kg test substance as compared with mice fed 0 mg/kg test substance (Kruskal–Wallis pooled over experiment = 35.1, df = 3, P<.001).
- The MPO activity in stromal fractions was unaffected by the test substance at doses up to 100 mg/kg

Table 1.

Experiment I

group 1:

 

dose

No. mice

Hprt gene

mutation frequency

Difference (95% CI)

1503 (1095 to 2158) 

0 (referent) 

25 

1577 (0 to 2422) 

75 (−1296 to 752) 

 

50 

26 (0.8 to 1011) 

−1477 (1700 to 1661) 

 

100 

14 (4 to 79) 

−1488 (1273 to 1662) 

 

group 2:

 

dose

No. mice

Hprt gene

mutation frequency

Difference (95% CI)

65 (7 to 129) 

0 (referent) 

25 

14 (0 to 102) 

−51 (1 to 92) 

50 

10 (3 to 35) 

−55 (15 to 94) 

100 

5 (2 to 25) 

−60 (24 to 101) 

 

Experiment II

 

group 1:

Dose

Myeloperoxidase activity

Difference (95% CI)

0

271 (197 to 535) 

0 (referent) 

25

115 (22 to 235) 

−156 (55 to 321) 

50

19 (4 to 105) 

−252 (177 to 428) 

100

7 (0.7 to 12) 

−264 (194 to 428) 

 

group 2:

dose

 

 

Myeloperoxidase activity

Difference (95% CI)

0

 

 

249 (6 to 305) 

0 (referent) 

25

 

 

180 (67 to 225) 

−69 (12 to 189) 

50

 

 

12 (2 to 68) 

−237 (150 to 286) 

100

 

 

13 (7 to 124) 

−236 (116 to 281) 

 Same number of mices was used as in above experiment I

Conclusions:
Based on the results of the read across study,the test substance (both 50 mg and 100 mg) statistically significantly decreased mutation frequency and MPO activity. The test substance was therefore negative for genotoxicity in a Mutatect tumor model
Executive summary:

A study was conducted to determine the potential of the read across substance to induce Hprt gene mutation frequency in mouse Mutatect tumor model. The Hprt gene mutation frequency is associated with the number of tumor-infiltrating neutrophils. An indirect measure of neutrophil number is expressed as number of 6-thioguanine–resistant colonies per 10E+5 clonable tumor cells, IL-8 transgene loss, and myeloperoxidase activity. Mutatect TM-28 cells, a clone that expresses human IL-8, were injected subcutaneously into 6- to 8-week-old C57BL/6 mice. Dietary vitamin E supplements (0, 25, 50, or 100 mg/kg bw/d) were added to the tocopherol-stripped rodent pellets that were fed to the mice from 7 days before injection of tumor cells until the mice were killed and their tumors harvested (i.e., a total of 3.5 to 4 weeks when they reached 1 cm in size). The cellular fractions of the tumors were analyzed for Hprt gene mutation frequencies and myeloperoxidase (MPO) activity. The test substance was found to significantly decrease the mutation frequency and MPO activity. Under the study conditions, the test substance was therefore negative for genotoxicity in a Mutatect tumor model (Soo, 2004).

Endpoint:
in vivo mammalian somatic cell study: gene mutation
Remarks:
mouse Mutatect tumor model
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: RA study
Justification for type of information:
Refer to the section 13 for details on the read across justification.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
other: mouse Mutatect tumor model
Deviations:
not applicable
GLP compliance:
not specified
Type of assay:
other: mouse Mutatect tumor model
Specific details on test material used for the study:
source: Novartis, Mississauga, ON, Canada
Species:
mouse
Strain:
other: C57BL/6 mice
Sex:
not specified
Route of administration:
oral: feed
Vehicle:
Dietary test substance was administered by diluting it in soy oil and adding the liquid to dry standard rodent chow (Charles River Laboratories). Animals were isolated, one per cage, to ensure that the pellet was consumed. Control animals were treated similarly.
Details on exposure:
Control animals: 0.45 IU of test substance per day from their standard chow (90 IU test sub./kg chow).
Test substance-supplemented animals received an additional 2 IU of per mouse per day (400 IU test sub./kg chow); they were estimated to have also received 0.50 mg of gamma-tocopherol and 0.25 mg omega-tocopherol from the 20 uL of soybean oil used as a vehicle.
Dose / conc.:
90 other: IU test sub./kg chow
Remarks:
control animals
Dose / conc.:
400 other: IU test sub./kg chow
Remarks:
test substance-supplemented animals
Control animals:
yes
Details of tissue and slide preparation:
Cells and culture conditions:

Mutatect cells were cultured in nonselective medium (Dulbecco’s modified Eagle medium with 10% fetal calf serum), HAT medium (supplemented with 100 M hypoxanthine, 0.4 M aminopterin, and 15 M thymidine), HT medium (without aminopterin), or 6-TG medium (supplemented with 50 M 6-thioguanine), as described previously. Before use, Mutatect cells were grown in HAT medium for 7 days to kill any pre-existing mutants, and then they were transferred to HT medium for 2 days to allow recovery from HAT treatment.

Mutatect tumor formation, detection of mutants, and myeloperoxidase measurement:

Tumors from Mutatect MN-11, MT-6, and TM-28 cells were established by subcutaneous injection of cells into the flanks of C57BL/6 female mice, 8–10 weeks of age. Tumors were excised when they reached 1 cm in size (2–3 weeks, depending on the cell line); single-cell suspensions were prepared by gentle mechanical dispersion of the tumor fragments with a plastic syringe. Tumor cells were established in culture for 2–4 days, and the frequency of mutations arising in vivo was then estimated from the number of colonies capable of growth in 6-TG medium, as described earlier. Mutation frequency is expressed as the number of 6-thioguanine-resistant colonies per 1+E5 clonable tumor cells. The same tumor cell suspension was also used for measurement of myeloperoxidase activity, a marker of neutrophil infiltration.

Vitamin E supplements to the diet:

Test substance supplements were started 7 days before tumor cell injection and were continued until the animals were killed (i.e., a total of 3–4 weeks, depending on the cell line). Control animals received 0.45 IU of test substance per day from their standard chow (90 IU test sub./kg chow).

Test substance-supplemented animals received an additional 2 IU of per mouse per day (400 IU test sub./kg chow); they were estimated to have also received 0.50 mg of gamma-tocopherol and 0.25 mg omega-tocopherol from the 20 uL of soybean oil used as a vehicle. Control animals received neither vehicle nor supplementary tocopherol. Experiments were carried out according to guidelines of the Canadian Council on Animal Care. The test substance determinations were carried out with the use of a HPLC method.

Induction of mutations by glyceryl trinitrate and by molsidomine in MN-11 tumor-bearing animals:

On day 12 after injection of MN-11 cells, mice received an intraperitoneal injection of either injectable glyceryl trinitrate or molsidomine. Control mice were given an injection of phosphate-buffered saline. Two days after treatment, tumors were recovered and established in culture as described above. Cells were cultured for 8 days to allow expression of the mutant phenotype before challenge with 6-thioguanine.
Statistics:
Nonparametric tests were used for the statistical analyses. Two groups were compared by use of the Mann–Whitney U test. Correlation between two variables was determined with the use of the Spearman rank coefficient. All P values shown are two-tailed. A P value of <.05 was considered to be statistically significant, and a P value of <.01 was considered to be highly statistically significant. Confidence intervals (CIs) of median differences were calculated with the use of Analyse-it Version 1.5. Other statistical calculations were done with the use of GraphPad Prism Version 3 (GraphPad Software).
Key result
Sex:
not specified
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
not specified
Additional information on results:
Effect of the test substance on spontaneous mutation frequency in MN-11 tumors:
- Supplementation of standard chow (which contains 90 IU test sub./kg chow) with 400 IU test sub./kg chow for 3 weeks reduced the median mutation frequency by 24.9% (P = 0.01).

Decrease in myeloperoxidase and mutation frequency by the test substance in TM-28 tumors:
- In two separate experiments, test substance reduced the median mutation frequency by 68.9% (P = 0.0019) and 84.1% (P = 0.011) and myeloperoxidase levels by 75.3% (P = 0.0002) and 75.5% (P = 0.026), respectively

- Dietary test substance afforded strong protection against both spontaneously arising and nitric oxideinduced mutations. Two separate protective mechanisms by the test substance may be operating: scavenging of a nitric oxide-related genotoxic species and altering the infiltration of neutrophils into tumors.
Conclusions:
Based on the results from the read across study, the supplementation of standard chow with the test substance for 3 weeks reduced the median mutation frequency by 24.9% (P = 0.01) in MN-11 tumors. Additionally in two separate experiments, test substance reduced the median mutation frequency in TM-28 tumors by 68.9% (P = 0.0019) and 84.1% (P = 0.011) and myeloperoxidase levels by 75.3% (P = 0.0002) and 75.5% (P = 0.026), respectively
Executive summary:

A study was conducted to determine the potential of the read across substance to induce Hprt gene mutation frequency in mouse Mutatect tumor model. In this study the mutatect cells were grown in mice as subcutaneous tumors for 2–3 weeks and then the tumor cells were recovered, and 6 -thioguanine-resistant (i.e., hprt mutant) colonies were scored. Myeloperoxidase activity was used as a measure of neutrophil infiltration in TM-28 tumors.Based on the results from the read across study, the test substance was found to reduce the median mutation frequency by 24.9% (P = 0.01) in MN-11 tumors. Additionally in two separate experiments, test substance reduced the median mutation frequency in TM-28 tumors by 68.9% (P = 0.0019) and 84.1% (P = 0.011) and myeloperoxidase levels by 75.3% (P = 0.0002) and 75.5% (P = 0.026), respectively. Under the study conditions, the test substance was therefore negative for genotoxicity(Sandhu, 2000).

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

Additional information

Genetic toxicity in vitro

Study 1:

A study was conducted to determine the mutagenic potential of the test substance according to Bacterial Reverse Mutation Test. The test substance was examined using four strains of Salmonella typhimurium (TA 97a, TA 98, TA 100 and TA 1535). The test was performed in the presence and absence of S9-mix (Sprague-Dawley rat or Syrian hamster liver S9-mix induced by Aroclor 1254). 5 concentrations were used for all test strains: 100, 333, 1000, 3333 and 10000 μg/plate. Positive and negative controls were included in the study. No significant increase of the number of revertant colonies could be observed at any of the treatment concentrations. Under the study conditions, the test substance was not mutagenic in the Salmonella typhimurium strains TA 97a, TA 98, TA1 00 and TA 1535 in absence and presence of metabolic activation (NTP, 1985).

Study 2:

A study was conducted to determine the genotoxic potential in mammalian cells according to a sister chromatid exchange assay. Two trials were performed. In the first trial, concentrations of 5, 7, 10, and 50 μg/mL of the test substance were tested without metabolic activation, and doses of 30, 100, 300, and 1000 μg/mL were tested with metabolic activation. In the second trial, concentrations of 15.1, 19.95, 25.2, and 30.2 μg/mL of the test substance were tested without metabolic activation, and concentrations of 202, 298.2, 396, and 497 μg/mL were tested with metabolic activation. Vehicle (DMSO) and appropriate positive controls were used. Under the study conditions, the test substance was weakly positive in a sister chromatid exchange assay in the presence of metabolic activation (NTP, 1984).

Study 3:

A study was conducted to investigate the mutagenic potential of the test substance in the chromosomal aberration assay. In the first trial, test substance concentrations of 39.8, 49.8, 60, and 75 μg/mL were tested without metabolic activation, and doses of 400, 450, and 500 μg/mL were tested with metabolic activation. In the second trial, concentrations of 24.9, 30.1, and 35 μg/mL of the test substance were tested without metabolic activation, and concentrations of 4100, 5000,and 6000 μg/mL were tested with metabolic activation. Vehicle (DMSO) and appropriate positive controls were used. Under the study conditions, the test substance was negative for genotoxicity in a chromosome aberration assay (NTP, 1984).

Genetic toxicity in vivo

Study 1:

A study was conducted to determine the potential of the read across substance to induce Hprt gene mutation frequency in mouse Mutatect tumor model. The Hprt gene mutation frequency is associated with the number of tumor-infiltrating neutrophils. An indirect measure of neutrophil number is expressed as number of 6-thioguanine–resistant colonies per 10E+5 clonable tumor cells, IL-8 transgene loss, and myeloperoxidase activity. Mutatect TM-28 cells, a clone that expresses human IL-8, were injected subcutaneously into 6- to 8-week-old C57BL/6 mice. Dietary vitamin E supplements (0, 25, 50, or 100 mg/kg bw/d) were added to the tocopherol-stripped rodent pellets that were fed to the mice from 7 days before injection of tumor cells until the mice were killed and their tumors harvested (i.e., a total of 3.5 to 4 weeks when they reached 1 cm in size). The cellular fractions of the tumors were analyzed for Hprt gene mutation frequencies and myeloperoxidase (MPO) activity. The test substance was found to significantly decrease the mutation frequency and MPO activity. Under the study conditions, the test substance was therefore negative for genotoxicity in a Mutatect tumor model (Soo, 2004).

Study 2:

A study was conducted to determine the potential of the read across substance to induce Hprt gene mutation frequency in mouse Mutatect tumor model. In this study the mutatect cells were grown in mice as subcutaneous tumors for 2–3 weeks and then the tumor cells were recovered, and 6 -thioguanine-resistant (i.e., hprt mutant) colonies were scored. Myeloperoxidase activity was used as a measure of neutrophil infiltration in TM-28 tumors. Based on the results from the read across study, the test substance was found to reduce the median mutation frequency by 24.9% (P = 0.01) in MN-11 tumors. Additionally in two separate experiments, test substance reduced the median mutation frequency in TM-28 tumors by 68.9% (P = 0.0019) and 84.1% (P = 0.011) and myeloperoxidase levels by 75.3% (P = 0.0002) and 75.5% (P = 0.026), respectively. Under the study conditions, the test substance was therefore negative for genotoxicity (Sandhu, 2000).

Justification for classification or non-classification

Based on the results of in vitro and in vivo genotoxicity studies, the test substance does not require classification for genotoxicity according to EU CLP (EC 1272/2008) criteria.