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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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:
: from 18-MAR-1986 to 13-MAY-1986
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: No indication of purity of test substance (no analytical certificate).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1986
Report Date:
1986

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
EU Method B.13/14 (Mutagenicity - reverse mutation test using bacteria) 19.9.84
Deviations:
not specified
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Species / strain
Species / strain / cell type:
other: TA 98, TA 100, TA 1535, TA 1537, TA 1538
Metabolic activation:
with and without
Metabolic activation system:
S9 (see table in other information)
Test concentrations with justification for top dose:
0.05; 0.1; 0.5; 1; 2 µl/plate
Vehicle / solvent:
- VEHICLE: DMSO (0.1 ml/ml)
> Justification for choice of solvent/vehicle: the solvent was chosen because of its solubility properties and its relative non-toxicity to bacteria
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see details on test system
Details on test system and experimental conditions:
- VEHICLE: DMSO (0.1 ml/ml)
> Justification for choice of solvent/vehicle: the solvent was chosen because of its solubility properties and its relative non-toxicity to bacteria
> Vehicle controls tested: no
- NEGATIVE CONTROL(S): DMSO (100 µl/plate)
- POSITIVE CONTROL(S)
2-nitrofluorene in TA 98 and TA 1538 (0.001 µl/plate) without metabolic activation
9-aminoacridine in TA 1537 (0.05 µl/plate) without metabolic activation
ethylmethanesulphonate in TA 1535 (10 µl/plate) without metabolic activation
methylmethanesulfonate in TA 100 (0.1 µl/plate) without metabolic activation
Amino-2 anthracene: TA 98, TA 100, TA 1535, TA 1537, TA 1538 (0.002 µl/plate) with metabolic activation
>Justification for choice of positive control(s): Standard mutagen products.

DETAILS ON TEST SYSTEM AND CONDITIONS:
*Preliminary experiment: served to identify toxicity. The strain TA 100 was exposed without activation metabolic system to following concentrations: 0.5, 1, 5, 10, 50 and 100 µl/plate

*First experiment: 3 replicates of S. Typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538 with and without metabolic activation system were exposed to 0.05, 0.1, 0.5, 1 or 2 µl/plate of test substance. A preparation of 2.5 ml of agar +0.1 ml of bacteria culture and 0.5 ml of S9 mix (if required) was prepared extemporaly and added to 100 ml of substance in DMSO at adapted concentration. This mixture was agitated and spread out over a layer of Vogel and Bonner medium. The plates were incubated at 37°C for 48h.
Reading of results was done with electronic meter of colonies.

*Second experiment: same conditions than first experiment.
For both experiments, positive and negative controls for each strain tested with and without S9 mix were done.
Sterility controls were done on product solutions tested, DMSO and S9 mix.
Genetic strain control with and without metabolic activation system and without test substance were done.

- METHOD OF APPLICATION: in agar (plate incorporation)
- DURATION:
> Exposure duration: 48 hours
- Expression time (cells in growth medium): 16 hours at 37°C in nutritive culture to obtain plate phase (around 2.10E9 bacteria/ml)
- NUMBER OF REPLICATIONS: 3
OTHER: Electronic count.
Evaluation criteria:
Results were considered positive by the authors when the revertant numbers were equal or more than 2 fold the negative control. Quantitative method of mutagen power was applied by the statistic method of least square to evaluate mutagen power.
Statistics:
no quantitative evaluation by linear regression (method of least square). Mutagen power was expressed in reverse/µl of product.

Results and discussion

Test resultsopen allclose all
Species / strain:
other: TA100, TA1535
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
2 µl/plate for both strains
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: TA98, TA1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 2 µl/plate for TA 98 and TA 1538 with S9, and 1 µl/plate for TA 1538 without S9.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 2 µl/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In a preliminary study, the strain TA 100 was exposed without activation metabolic system to following concentrations: 0.5, 1, 5, 10, 50 and
100 µl/plate.
The concentration of 5 µl/plate was toxic, and concentrations for the main study were determined as follow: 0.05, 0.1, 0.5, 1 and 2 µl/plate.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table of results: Number of revertants per plate

 

 Strain

 

S9-mix

Concentration (µl/plate)

 

 

 

0

0.05

0.1

0.5

1

2

Positive controls

 TA 98

1st study

-

 27/32/27

23/28/42

28/18/28 

28/29/33 

36/50/

36 

6/3/3

216/144

 

 

+

36/27/34

32/28/41

38/40/37

51/41/64

52/62/

47

 27/27/27 T

1841/1796

 

2ndstudy

-

41/19/23

34/29/28

37/24/32

31/40/36

37/31/

33

7/11/11

264/259

 

 

+

34/38/25

37/34/32

41/37/41

38/41/43

67/62/

56

3/12/6T

1520/1449

 TA 100

1st study

-

86/86/81

180/143/ 176

222/208/

215

427/518/

497

870/772/

809

182/45/0 T

302/316

 

 

+

100/124/

130

155/178/ 171

237/211/

250

472/567/

541

895/909/ 941

68/6/96 T

2441/3076

 

2ndstudy

-

104/87/77

130/114/ 130

170/178/

200

409/413/

394

551/507/ 483

111/192/

25 T

343/338

 

 

+

98/95/100

148/133/ 140

189/160/

184

422/485/

456

607/594/ 568

268/298/

289 T

1233/1135

 TA 1535

1st study

-

6/12/11

21/32/40

41/52/65

122/155/

147

179/182/ 169

15/9/2T

5989/6781

 

 

+

7/11/11

34/54/41

54/49/67

195/182/

158

224/222/ 204

122/157/

112 T

158/149

 

2ndstudy

-

7/12/14

27/40/33

69/46/59

100/130/

139

158/130/ 142

25/29/18 T

6275/6611

 

 

+

12/15/15

25/25/25

46/41/24

143/138/

127

143/174/ 160

156/176/174

136/176

 TA 1537

1st study

-

6/5/6

10/3/6

6/5/12

7/18/11

9/14/18

1/2/1T

614/577

 

 

+

5/6/5

6/6/7

9/5/9

20/11/12

25/16/10

3/1/0T

183/152

 

2ndstudy

-

10/5/6

3/6/5

7/6/3

12/14/9

16/18/11

2/0/0 T

620/549

 

 

+

2/3/11

3/1/10

10/2/2

9/14/10

11/12/10

0/2/3 T

179/187

 TA 1538

1st study

-

27/25/23

27/24/19

18/23/24

33/18/28

15/18/12 T

1/1/0T

167/158

 

 

+

36/40/16

38/40/37

42/41/33

24/31/31

36/24/34

19/18/19 T

1069/883

 

2ndstudy

-

19/15/15

18/15/21

9/12/14

19/27/15

12/7/16

0/3/0 T

182/262

 

 

+

20/19/25

18/27/24

20/27/24

27/19/20

25/28/21

2/6/5T

713/656

T: cytotoxicity

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive with and without metabolic activation

- INTERPRETATION OF RESULTS:
Regarding results and applying actual criteria evaluation (the test item is considered as mutagen if an increase in revertants number exceeding the
threshold of twice for strains TA 98, TA 100, and thrice for strains TA 1535, TA 1537, TA 1538 as compared with solvent control), the test item
presented mutagenic activity with and without S9 mix in Salmonella Typhimurium TA 100 and TA 1535 (base substitution mutagen). A weak positive response in Salmonella Typhimurium TA 1537 (frameshift mutagen) and negative response for strains TA 98 and TA 1538 (frameshift mutagen)
were observed.
Reproductibility between first, second experiment and positive control results in accordance with positive criteria, validated the study.
Executive summary:

In a reverse gene mutation assay in bacteria (Hazleton, 1986), strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 of S. typhimurium were exposed to paratrifluoromethylaniline at concentrations of0.05,0.1, 0.5, 1 or 2 µl/plate in the presence and absence of mammalian metabolic activation.
Paratrifluoromethylaniline was tested up to cytotoxic concentrations. The positive controls induced the appropriate responses in the corresponding strains.

In this study, the test item presented mutagenic activity with and without S9 -mix in Salmonella typhimurium TA100 and TA1535. A weak positive response in TA1537 and negative response for TA98 and TA1538.

Therefore, p-TFMA is considered as mutagenic for Salmonella typhimurium under the test conditions.
 
This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.