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Toxicological information

Genetic toxicity: in vivo

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Administrative data

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.
Cross-reference
Reason / purpose:
read-across source
Reference
Endpoint:
in vivo mammalian somatic cell study: gene mutation
Remarks:
mouse Mutatect tumor model
Type of information:
other: publication
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
secondary literature
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:
Under the study conditions, 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 test 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 10+E5 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 typically 9 per group. Dietary vitamin E supplements (0, 25, 50, or 100 mg/kg bw 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 when they reached 1 cm in size). The cellular fractions of the tumors were analyzed for Hprt gene mutation frequencies and myeloperoxidase (MPO) activity. Under the study conditions, 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 (Soo, 2004).

Data source

Reference
Reference Type:
publication
Title:
Dose-dependent effects of dietary alpha- and gamma-Tocopherols on genetic instability in mouse mutatect tumors
Author:
Soo C. C., Haqqani A. S., Hidiroglou N., Swanson J.E., Parker R. S., Birnboim H.C.
Year:
2004
Bibliographic source:
Journal of the National Cancer Institute, Vol. 96, No. 10
Report Date:
2004

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: mouse Mutatect tumor model
Deviations:
not applicable
GLP compliance:
not specified
Type of assay:
other: mouse Mutatect tumor model

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified

Test animals

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.

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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).

Results and discussion

Test results
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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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).