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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was published in 1983 and included in this review in 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Unnamed
Year:
1991
Reference Type:
publication
Title:
Salmonella mutagenicity test results for 250 chemicals
Author:
Steve Haworth et al
Year:
1983
Bibliographic source:
Environmental mutagenisis supplement, 1, 1983, 3-142

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Testing was performed as reported by Ames et at! (1975) with modifications described in Haworth et al, (1983).
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Batch number 0708501022
Description Red crystalline solid
See certificate of analysis:
Purity 99.8%
Storage requirements Store under a nitrogen atmosphere in a cool, dry, well-ventilated area away from flammable materials and sources of heat or flame.
Expiration date September 1, 2012
Specific details on test material used for the study:
Substance name: TITANOCENE DICHLORIDE
CAS NO.1271-19-8
Titanocene dichloride was sent to the laboratory as a coded aliquot from Radian Corporation, Austin, TX.

Method

Target gene:
histidine locus
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
metabolic activation enzymes and cofactors from Aroclor 1254-induced male Sprague-Dawley rat or Syrian hamster liver
Test concentrations with justification for top dose:
High dose was limited by toxicity or solubility
0.0, 33.3, 100.0, 333.3, 1000.0 and 3,333.3 µg/plate
Vehicle / solvent:
dimethylsulfoxide
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
dimethylsulfoxide
True negative controls:
no
Positive controls:
yes
Remarks:
for all strains
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
dimethylsulfoxide
True negative controls:
no
Positive controls:
yes
Remarks:
for strain TA98
Positive control substance:
other: 4-nitro-o-phenylenediamine
Remarks:
without S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
dimethylsulfoxide
True negative controls:
no
Positive controls:
yes
Remarks:
for strains TA100 and TA1535
Positive control substance:
sodium azide
Remarks:
without S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
dimethylsulfoxide
True negative controls:
no
Positive controls:
yes
Remarks:
for strain TA1537
Positive control substance:
9-aminoacridine
Remarks:
without S9-mix
Details on test system and experimental conditions:
Testing was performed as reported by Ames et al. (1975) with modifications as listed below and described in greater detail in Haworth et al, (1983). The test chemical was incubated with the Salmonella typhimurium tester strain (TA98,TA100, TA1535, TA1537) either in buffer or S9 mix for 20 minutes at 37ºC prior to the addition of soft agar supplemented with e-histidine and d-biotin, and subsequent plating on minimal glucose agar plates. Incubation was continued for an additional 48 hours.
In this assay, each test consisted of triplicate plates of concurrent positive and negative controls and of at least 5 doses of the test chemical. High dose was limited by toxicity. All positive assays were repeated under the conditions that elicited the positive response.
Evaluation criteria:
A positive response was defined as a reproducible, dose-related increase in histidine-independent (revertant) colonies in any one strain/activation combination. An equivocal response was defined as an increase in revertants that was not dose-related, not reproducible, or of insufficient magnitude to support a determination of mutagenicity. A negative response was obtained when no increase in revertant colonies was observed following chemical treatment.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
slight toxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
slight toxicity in absence of S9-mix
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
slight toxicity in absence of S9-mix
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
slight toxicity in absence of S9-mix
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Any other information on results incl. tables

Titanocene dichloride was tested at concentrations of up to 3,333 µg/plate for the induction of gene mutations in Salmonella typhimurium strains TA100, TA1535, TA1537, and TA98 both in the presence and in the absence of Aroclor 1254-induced male Sprague-Dawley rat or Syrian hamster liver S9. A positive response was observed only for the base-substitution strain TAl00 tested in the absence of S9; all other strain/activation combinations yielded negative results.

Mutagenicity of Titanocene Dichloride in Salmonella typhimurium (a) 

 

Revertants/plate (b)

Strain

Dose (µg/plate)

-S9

+10% hamster S9

+10% rat S9

Trial 1

Trial 2

TA100

0.0

91 ± 0.9

83 ± 7.6

104 ± 2.1

82 ± 5.1

 

33.3

166 ± 9.7

102 ± 0.0

110 ± 9.2

93 ± 2.5

 

100.0

178 ± 11.0

210 ± 8.5

91 ± 12.3

94 ± 4.6

 

333.3

193 ± 9.3

271 ± 4.3

90 ± 8.4

89 ± 5.6

 

1000.0

184(c) ± 20.9

153(c) ± 13.8

101 ± 10.0

99 ± 7.2

 

3333.3

85(c) ± 11.6

64(c) ± 4.4

100 ± 13.4

93 ± 6.3

Trial summary

Positive

Positive

Negative

Negative

Positive control (d)

352 ± 16.0

486 ± 21.5

1482 ± 54.3

338 ± 9.4

 

TA1535

0.0

11 ± 2.0

 

5 ± 0.3

7 ± 1.5

 

33.3

18 ± 0.7

 

5 ± 0.9

7 ± 1.2

 

100.0

13 ± 3.5

 

6 ± 2.0

7 ± 0.0

 

333.3

13 ± 1.5

 

7 ± 0.7

12 ± 2.3

 

1000.0

14(c) ± 3.2

 

7 ± 0.6

15 ± 1.0

 

3333.3

4(c) ± 2.6

 

14 ± 5.0

16 ± 5.2

Trial summary

Negative

 

Negative

Negative

Positive control (d)

285 ± 7.8

 

404 ± 18.0

211 ± 12.4

 

TA1537

0.0

7 ± 0.7

 

7 ± 1.0

18 ± 0.3

 

33.3

6 ± 0.7

 

5 ± 0.6

14 ± 2.3

 

100.0

6 ± 1.8

 

6 ± 1.5

18 ± 1.2

 

333.3

7 ± 1.2

 

4 ± 0.6

19 ± 1.0

 

1000.0

7(c) ± 1.5

 

5 ± 0.6

16 ± 2.0

 

3333.3

5(c) ± 0.7

 

4 ± 1.5

13 ± 1.9

Trial summary

Negative

 

Negative

Negative

Positive control (d)

312 ± 24.1

 

466 ± 35.1

83 ± 6.9

 

TA98

0.0

24 ± 4.2

 

33 ± 4.1

36 ± 3.1

 

33.3

33 ± 4.6

 

27 ± 3.5

30 ± 1.2

 

100.0

38 ± 1.5

 

25 ± 5.4

24 ± 2.0

 

333.3

30 ± 1.2

 

32 ± 2.6

26 ± 1.2

 

1000.0

29(c) ± 5.6

 

29 ± 1.9

28 ± 5.4

 

3333.3

11(c) ± 3.2

 

26 ± 3.6

21 ± 4.1

Trial summary

Equivocal

 

Negative

Negative

Positive control (d)

529 ± 11.8

 

1222 ± 59.4

153 ± 12.2

(a) Study performed at SRI International. Cells and the test chemical or solvent (dimethylsulfoxide) were incubated in the absence of exogenous metabolic activation or with Aroclor 1254-induced S9 from male Syrian hamster liver or male Sprague-Dawley rat liver. High dose was limited by toxicity or solubility, but did not exceed 10 mg/plate; 0 µg/plate dose is the solvent control.

(b) Revertants are presented as mean ± the standard error from 3 plates.

(c) Slight toxicity

(d) 2-aminoanthracene was used for all strains in the presence of S9. In the absence of metabolic activation, 4-nitro-o-phenylenediamine was tested on TA98, sodium azide was tested on TAlOO and TA1535, and 9-aminoacridine was tested on TA1537.

Applicant's summary and conclusion

Conclusions:
Titanocene dichloride was mutagenic in Salmonella typhimurium strain TA100 in the absence of exogenous metabolic activation (S9); it was not mutagenic in TA100 with S9, nor was it mutagenic in TA1535, TA1537, or TA98 with or without S9.
Executive summary:

Titanocene dichloride, tested at concentrations of 33 to 3,333 µg/plate, induced gene mutations in the S. typhimurium base pair substitution strain TA100 in the absence of S9, but was negative in several frameshift strains, with and without S9 activation