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Toxicological information

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 experimental phase of this study was performed between 15 October 2003 and
03 November 2003.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report date:
2003

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
484-440-2
EC Name:
-
Cas Number:
502157-74-6
Molecular formula:
C84H68O12S3P2
IUPAC Name:
4-(4-hydroxybenzenesulfonyl)phenol bis(tetraphenylphosphanium) bis(4-(4-hydroxybenzenesulfonyl)benzen-1-olate)
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Sponsor's identification SBCAT-03
Description White powder
Batch number UK030622B
Date received 09 October 2003
Storage conditions Room temperature in the dark

Data relating to the identity, purity and stability of the test material are the responsibility of the
Sponsor.

The test material was accurately weighed and approximate half-log dilutions prepared in dimethyl
formamide by mixing on a vortex mixer on the day of each experiment. Dimethyl formamide is
considered an acceptable vehicle for use in this test system. Analysis for concentration,
homogeneity and stability of the test material formulations is not a requirement of the test
guidelines and was, therefore, not determined. Prior to use, the solvent was dried using molecular
sieves (sodium alumino-silicate) ie 2 mm pellets with a nominal pore diameter of 4 x 10-4
microns.

Method

Target gene:
Histidine auxotrophs of Salmonella typhimurium
Tryptophan auxotrophs of Escherichia coli
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 was prepared in-house on 19 July 2003 from the livers of male Sprague-Dawley rats weighing
~ 250g. These had each orally received three consecutive daily doses of
phenobarbitone/β-naphthoflavone (80/100 mg per kg per day) prior to S9 preparation on Day 4.
Before use, each batch of S9 was assayed for its ability to metabolise appropriate indirect
mutagens used in the Ames Test. The S9 was stored at -196°C.

The S9-mix was prepared immediately before use using sterilised co-factors and maintained on
ice for the duration of the test.
S9 5.0ml
1.65 M KCl/0.4 M MgCh 1.0 ml
0.1 M Glucose-6-phosphate 2.5ml
0.lMNADPH 2.0ml
0.lMNADH 2.0 ml
0.2 M Sodium phosphate buffer (pH 7.4) 25.0 ml
Sterile distilled water 12.5 ml
A 0.5 ml aliquot of S9-mix and 2 ml of molten, trace histidine or tryptophan supplemented, top
agar was overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of
the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.
Top agar was prepared using 0.6% Difeo Bacto agar and 0.5% sodium chloride with 5 ml of
1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 ml of top
agar. Vogel-Bonner Minimal agar plates were purchased from ILS Ltd.
Test concentrations with justification for top dose:
In order to select appropriate dose levels for use in the main test, a preliminary assay was carried
out to determine the toxicity of the test material. The concentrations tested were 0, 0.15, 0.5, 1.5,
5, 15, 50, 150, 500, 1500 and 5000 μg/plate. The assay was performed by mixing 0.1 ml of
bacterial culture (TAlO0 or WP2uvrA"), 0.1 ml of test material formulation, 0.5 ml of S9-mix or
phosphate buffer and 2 ml of molten, trace histidine or tryptophan supplemented, top agar and
overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 ml/plate). Ten concentrations of
the test material and a vehicle control (dimethyl formamide) were tested. In addition, 0.1 ml of
the maximum concentration of the test material and 2 ml of molten, trace histidine or tryptophan
supplemented, top agar was overlaid onto a sterile Nutrient agar plate in order to assess the
sterility of the test material. After approximately 48 hours incubation at 37°C the plates were
assessed for numbers of revertant colonies using a Domino colony counter and examined for
effects on the growth of the bacterial background lawn.
Vehicle / solvent:
The test material was insoluble in sterile distilled water, acetone, and ethanol at 50mg/ml (the most concentrated stock solution) but was soluble in dimethyl formamide at the same concentration in solubility checks performed in house. Dimethyl formamide was, therefore, selected as the vehicle of choice. The test material was poorly soluble in dimethyl sulphoxide and was, therefore, not selected.
The test material was accurately weighed and approximate half-log dilutions prepared in dimethyl
formamide by mixing on a vortex mixer on the day of each experiment. Dimethyl formamide is
considered an acceptable vehicle for use in this test system (Maron D et al (1981) Mutation Research, 88, 343-350).
Analysis for concentration, homogeneity and stability of the test material formulations is not a requirement of the test guidelines and was, therefore, not determined. Prior to use, the solvent was dried using molecular sieves (sodium alumino-silicate) ie 2 mm pellets with a nominal pore diameter of 4 x 10-4 microns.
Vehicle and positive controls were used in parallel with the test material. A solvent treatment
group was used as the vehicle control and the positive control materials were as follows:

N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG): 2 μg/plate for WP2uvrA-, 3 μg/plate for TA100
and 5 μg/plate for TA1535

9-Aminoacridine (9AA): 80 μg/plate for TA1537

4-Nitroquinoline-1-oxide ( 4NQO): 0.2 μg/plate for TA98

In addition, 2-Aminoanthracene (2AA) and Benzo(a)pyrene (BP), which are non-mutagenic in the
absence of metabolising enzymes, were used in the series of plates with S9-mix at the following
concentrations:

2AA at 1 μg/plate for TA100
2AA at 2 μg/plate for TA1535 and TA1537
2AA at 10 μg/plate for WP2uvrABP
at 5 μg/plate for T A98
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
Details on test system and experimental conditions:
Seven concentrations of the test material (5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were
assayed in triplicate against each tester strain, using the direct plate incorporation method.
Additional dose levels were included to allow for test material induced toxicity, ensuring that a
minimum of four non-toxic doses were achieved.
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes
followed by 2.0 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of the
test material formulation, vehicle or positive control and either 0.5 ml of S9-mix or phosphate
buffer. The contents of each test tube were mixed and equally distributed onto the surface of
Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in
triplicate, for each bacterial strain and for each concentration of test material both with and
without S9-mix.
All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant
colonies assessed using a Domino colony counter.
Mutation Test - Experiment 2 (Main Test)
The second experiment was performed using methodology as described for the range-finding test, using fresh bacterial cultures, test material and control solutions. The test material dose range was the same as the range-finding test (5 to 5000 μg/plate ).
Rationale for test conditions:
In accordance with test guidelines
Evaluation criteria:
The test material may be considered positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and statistically (Dunnett's
method of linear regression significant increase in the revertant count in at least one strain of
bacteria.
Statistics:
Dunnett's method of linear regression

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
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 determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
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 determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Preliminary Toxicity Test
The test material was initially toxic at 500 μg/plate to the strains of bacteria used (TAlO0 and
WP2uvrA). The test material formulation and S9-mix used in this experiment were both shown
to be effectively sterile.
The number of revertant colonies for the toxicity assay were: see "Any other information on results"

Prior to use, the master strains were checked for characteristics, viability and spontaneous
reversion rate (all were found to be satisfactory). These data are not given in the report. The
S9-mix used in both experiments of the main test was shown to be sterile.
Results for the negative controls (spontaneous mutation rates) are presented in "Any other information on results" and were
considered to be acceptable. These data are for concurrent untreated control plates performed on
the same day as the Mutation Test.
The individual plate counts, the mean number of revertant colonies and the standard deviations for the test material, vehicle and positive controls both with and without metabolic activation, are
presented in "Any other information on results" with the results also expressed graphically in "Illustrations".
Information regarding the equipment and methods used in these experiments as required by the
Japanese Ministry of Economy, Trade and Industry and Japanese Ministry of Health, Labour and
Welfare are presented in "Any other information on materials".
A history profile of vehicle and positive control values is presented in "Any other information on materials".

The test material caused a visible reduction in the growth of the bacterial background lawn to all
of the tester strains, both in the presence and absence of S9, from 1500 μgiplate. The test material was tested up to the maximum recommended dose level of 5000 μgiplate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence
of S9-mix.
No significant increases in the frequency of revertant colonies were recorded for any of the
bacterial strains, at any dose level either with or without metabolic activation.
All of the positive control chemicals used in the test induced marked increases in the frequency of
revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial
strains.

Any other information on results incl. tables

The number of revertant colonies for the toxicity assay were:




















































































With (+) or without (-) S9-mix



Strain



Dose (µg/plate)



0



0.15



0.5



1.5



5



15



50



150



500



1500



5000



-



TA100



101



87



93



81



98



72



98



88



63*



0*



0*



+



TA100



121



85



93



95



90



101



73



75



69



0*



0*



-



WP2urvA-



26



23



23



31



20



24



19



24



21



0*



0*



+



WP2urvA-



25



43



25



26



32



22



25



24



25



9*



0*



*- Partial or complete absence of bacterial background lawn


 


Spontaneous Mutation Rates (Concurrent Negative Controls)


Range-finding Test


 









































Number of revertants (mean number of colonies per plate)



Base-pair substitution type



Frameshift type



TA100



TA1535



WP2uvrA-



TA98



TA1537



118



36



22



21



5



120                      (124)



34                (33)



35                  (26)



18               (20)



5                 (7)



134



30



22



20



12



 


Main Test


 









































Number of revertants (mean number of colonies per plate)



Base-pair substitution type



Frameshift type



TA100



TA1535



WP2uvrA-



TA98



TA1537



96



33



30



C



21



119                (103)



37                  (34)



25                    (29)



19                 (17)



12                  (15)



93



33



32



15



11



C=Contaminated


 


Test Results: Range-Finding Test- Without Metabolic Activation
























































































































































































































































Test Period



From: 27 October 2003



To: 30 October 2003



With or without


 S9 mix



Test substance concentration (µg/plate)



Number of revertants (mean number of colonies per plate)



Base-pair substitution type



Frameshift type



TA100



TA1535



WP2uvrA-



TA98



TA1537



-



0



83



33



15



25



6



95           (89)



23          (28)



22        (18)



18          (22)



20         (11)



90              6.0#



28           5.0



18         3.5



22           3.5



6            8.1



-



5



67



33    



22



25



9



85           (84)



34           (32)



28        (22)



24           (29)



10          (9)



101          17.0



30            2.1



15        6.5



38           7.8



9           0.6



-



15



96



33



11



16



4     



116        (107)



33          (34)



26         (19)



20            (20)



9            (6)



108           10.1



36           1.7



21         7.6



24            4.0



5           2.6



-



50



89



27



27



21



6



121        (107)



27         (29)



20         (23)



26           (27)



12           (9)



111           16.4



33          3.5



21          3.8



34          6.6



8             3.1



-



150



106



33



12



16



16



109         (113)



28         (32)



24        (18)



19           (15)



8            (11)



123           9.1



36          4.0



18         6.0



11           4.0



8           4.6



-



500



83



30



17



18



8



62            (78)



23         (27)



20         (15)



18         (23)



8             (8)



88           13.8



29          3.8



8           6.2



33          8.7



7           0.6



-



1500



0*



0*



8*



8*



0*



0*            (0)



0*         (0)



5*        (15)



5*          (6)



0*          (0)



0*            0.0



0*         0.0



4*        6.4



4*        2.1



0*          0.0



-



5000



0*



0*



0*



0*



0*



0*           (0)



0*         (0)



0*         (0)



0*          (0)



0*          (0)



0*           0.0



0*         0.0



0*        0.0



0*          0.0



0*          0.0



Positive controls


 


S9 mix


 


-


 


 



Name


Concentration (µg/plate)


 


No. colonies per plate


 



ENNG



ENNG



ENNG



4NQO



9AA



3



5



2



0.2



80



460



247



608



103



730



453          (515)



249        (239)



624          (694)



125          (137)



732         (730)



631          100.8



222        15.0



851          135.9



183           41.3



727            2.5



ENNG = N-ethyl-N'-nitro-N-nitrosoguanidine


4NQO = 4-Nitroquinoline-1-oxide


9AA = 9-Aminoacridine


* = Partial or complete absence of bacterial background lawn


# = Standard deviation


 


Test Results: Range-Finding Test- With Metabolic Activation
























































































































































































































































Test Period



From: 27 October 2003



To: 30 October 2003



With or without


 S9 mix



Test substance concentration (µg/plate)



Number of revertants (mean number of colonies per plate)



Base-pair substitution type



Frameshift type



TA100



TA1535



WP2uvrA-



TA98



TA1537



+



0



117



12



24



22



16



114          (107)



8             (15)



27           (28)



20            (21)



11              (14)



89            15.4#



25           8.9



34            5.1



21             1.0



15               2.6



+



5



106



12



34



27



10



101          (107)



12           (11)



22           (29)



27            (30)



10              (11)



113            6.0



8              2.3



32            6.4



36            5.2



12               1.2



+



15



112



16



34



22



9



90            (97)



15           (14)



22          (30)



35           (29)



15               (14)



90             12.7



12           2.1



35           7.2



30           6.6



18               4.6



+



50



98



11



23     



35



9



98            (98)



13           (12)



17          (26)



23            (27)



8                 (10)



98             0.0



11            1.2



39          11.4



22            7.2



13                2.6



+



150



95



23



15



28



7



115         (105)



15           (19)



28          (23)



27             (27)



9                  (9)



104          10.0



19            4.0



26           7.0



25              1.5



10                1.5



+



500



82



10



21



35



15



85            (89)



18           (12)



17           (20)



25            (27)



11                (16)



100          9.6



7             5.7



23           3.1



20            7.6



22                 5.6



+



1500



0*



0*



18*



12*        



0*



0*           (0)



0*           (0)



10*        (12)



13*          (11)



0*               (0)



0*           0.0



0*           0.0



8*         5.3



9*            2.1



0*              0.0



+



5000



0*



0*



0*



0*         



0*



0*          (0)



0*         (0)



0*           (0)



0*            (0)



0*               (0)



0*           0.0



0*          0.0



0*          0.0



0*            0.0



0*              0.0



Positive controls


 


S9 mix


 


+


 


 



Name


Concentration (µg/plate)


 


No. colonies per plate


 



2AA



2AA



2AA



BP



2AA



1



2



10



5



2



1876



459



983



188



351



2035



360



948



188



361



1850



421



765



211



512



BP = Benzo( a )pyrene


2AA = 2-Aminoanthracene


*  = Partial or complete absence of bacterial background lawn


# = Standard deviation


 


Test Results: Main Test- Without Metabolic Activation
























































































































































































































































Test Period



From: 31 October 2003



To: 03 November 2003



With or without


 S9 mix



Test substance concentration (µg/plate)



Number of revertants (mean number of colonies per plate)



Base-pair substitution type



Frameshift type



TA100



TA1535



WP2uvrA-



TA98



TA1537



-



0



100



43



18



17



21



121          (105)



43            (40)



21           (20)



20           (18)



9            (14)



94             14.2#



34             5.2



20           1.5



18             1.5



11          6.4



-



5



101



38



19



19



17



112         (109)



39            (39)



16           (18)



17            (15)



10          (14)



115            7.4



40             1.0



19            1.7



10             4.7



16           3.8



-



15



93



40



20



15



10



109         (105)



41            (40)



29           (21)



12            (12)



8              (9)



113         10.6



40           0.6



13           8.0



10            2.5



8              1.2



-



50



106



39



16



13



22



105         (108)



38            (36)



15           (18)



23            (15)



19           (17)



113          4.4



30            4.9



24           4.9



9               7.2



9              6.8



-



150



136



32



26



19



8



119          (125)



32            (34)



18           (20) 



16            (15)



10            (9)



119          9.8



39            4.0



17           4.9



9             5.1



10           1.2



-



500



91



25



8



8



4



109          (9.2)



28           (26)



12           (10)



6              (8)



2              (4)



75            17.0



25          1.7



10           2.0



10          2.0



6             2.0



-



1500



0*



0*



3*



6*



0*



0*            (0)



0*             (0)



10*          (6)



6*             (5)



0*             (0)



0*            0.0



0*            0.0



6*           3.5



3*            1.7



0*             0.0



-



5000



0*



0*



0*



0*



0*



0*            (0)



0*             (0)



0*           (0)



0*           (0)



0*              (0)



0*           0.0



0*            0.0



0*           0.0



0*          0.0



0*             0.0



Positive controls


 


S9 mix


 


-


 


 



Name


Concentration (µg/plate)


 


No. colonies per plate


 



ENNG



ENNG



ENNG



4NQO



9AA



3



5



2



0.2



80



467



267



578



113



632



479        (468)



263          (265)



605           (606)



147          (139)



512          (592)



458         10.5



266           2.1



635            28.5



158            23.5



633           69.6



ENNG = N-ethyl-N'-nitro-N-nitrosoguanidine


4NQO = 4-Nitroquinoline-1-oxide


9AA = 9-Aminoacridine


* = Partial or complete absence of bacterial background lawn


# = Standard deviation


 


Test Results: Main Test- With Metabolic Activation


 
























































































































































































































































Test Period



From: 31 October 2003



To: 03 November 2003



With or without


 S9 mix



Test substance concentration (µg/plate)



Number of revertants (mean number of colonies per plate)



Base-pair substitution type



Frameshift type



TA100



TA1535



WP2uvrA-



TA98



TA1537



+



0



112



16



19



37



16



93            (101)



16              (15)



20            (23)



38             (37)



12             (14)



98             9.8#



12               2.3



31              6.7



35             1.5



15              2.1



+



5



103



13



28



35



21



101          (104)



17             (13)



24             (23)



37            (35)



17              (20)



109            4.2



9                4.0



17              5.6



32            2.5



21               2.3



+



15



108



10



22



35



12



107          (111)



19             (16)



16             (20)



36            (38)



12              (12)



119            6.7



20              5.5



23            3.8



43            4.4



13               0.6



+



50



90



13



16



34



12



91             (91)



19            (16)



27             (20)



30            (33)



17              (13)



93            1.5



17             3.1



17             6.1



34            2.3



10                3.6



+



150



104



16



29



35



11



90            (105)



10            (14)



18            (22)



29            (33)



12              (9)



122          16.0



15             3.2



20             5.9



29             3.5



5                 3.8



+



500



100



4



10



29



8



103           (99)



11             (8)



17            (17)



28            (29)



6                (7)



94             4.6



8               3.5



23             6.5



30            1.0



8                1.2



+



1500



0*



0*



7*



19



0*



0*              (0)



0*             (0)



8*            (8)



20             (17)



0*              (0)



0*             0.0



0*             0.0



9*             1.0



12              4.4



0*             0.0



+



5000



0*



0*



0*



0*



0*



0*             (0)  



0*           (0)



0*            (0)



0*             (0)



0*               (0)



0*             0.0



0*           0.0



0*            0.0



0*             0.0



0*              0.0



Positive controls


 


S9 mix


 


+


 


 



Name


Concentration (µg/plate)


 


No. colonies per plate


 



2AA



2AA



2AA



BP



2AA



1



2



10



5



2



1845



283



1051



108



429



2000           (1939)



269             (280)



1204         (1135)



117         (121)



425           (389)



1971              82.4



288                9.8



1151           77.7



139           15.9



314            65.3



BP = Benzo( a )pyrene


2AA = 2-Aminoanthracene


*  = Partial or complete absence of bacterial background lawn


# = Standard deviation


 


COMBINED VEHICLE AND UNTREATED CONTROL VALUES 2001


 




























































































































































Strain


S9-mix



TA100



TA1535



WP2urvA-



TA102



TA98



TA1537



TA1538



WP2uvrA- pKM101



TA97a



TA104



-S9



+S9



-S9



+S9



-S9



+S9



-S9



+S9



-S9



+S9



-S9



+S9



-S9



+S9



-S9



+S9



-S9



+S9



-S9



+S9



Mean



106



116



16



15



22



26



278



305



23



32



11



13



14



nd



144



147



116



131



512



nd



SD



23



23



4



4



5



6



45



39



7



8



4



4



5



nd



61



14



21



24



93



nd



Min



58



62



8



7



11



13



195



201



10



13



2



4



6



nd



74



131



84



114



389



nd



Max



178



178



37



37



41



52



405



421



56



58



23



29



20



nd



292



164



134



134



616



nd



Values



817



658



790



621



656



501



244



157



814



662



793



630



5



nd



10



4



5



2



5



nd



 nd = no data available


 


POSITIVE CONTROL VALUES 2001


 




























































































































































Strain


S9-mix



TA100



TA1535



WP2urvA-



TA102



TA98



TA1537



TA1538



WP2uvrA- pKM101



TA97a



TA104



-S9



+S9



-S9



+S9



-S9



+S9



-S9



+S9



-S9



+S9



-S9



+S9



-S9



+S9



-S9



+S9



-S9



+S9



-S9



+S9



Mean



505



1582



431



336



685



756



849



830



140



303



1415



428



153



501



1946



2102



618



750



825



1445



SD



190



541



412



110



323



243



179



159



43



96



713



146



60



246



794



844



62



142



209



202



Min



259



454



98



113



248



242



461



485



72



136



197



139



113



322



672



956



547



649



644



1224



Max



1829



2738



2039



990



1960



1340



1541



1303



362



679



3616



792



133



413



2790



3100



655



850



1125



1621



Values



165



163



163



161



156



155



92



93



166



164



162



160



4



4



7



7



3



2



4



3



 


 


 


 


 


COMBINED VEHICLE AND UNTREATED CONTROL VALUES 2002


 





















































































































Strain


S9-mix



TA100



TA1535



WP2urvA-



TA102



TA98



TA1537



WP2uvrA- pKM101



-S9



+S9



-S9



+S9



-S9



+S9



-S9



+S9



-S9



+S9



-S9



+S9



-S9



+S9



Mean



91



100



20



17



23



27



322



349



22



35



12



17



169



215



SD



16.6



18.0



5.5



4.3



4.8



5.3



47.7



34.6



5.2



6.8



4



4.7



42.8



50.2



Min



61



68



8



8



10



14



183



255



11



13



4



5



92



131



Max



160



162



38



37



47



45



414



449



44



66



29



38



234



277



Values



939



742



912



718



685



521



329



210



946



749



918



718



25



11



 


POSITIVE CONTROL VALUES 2002


 





















































































































Strain


S9-mix



TA100



TA1535



WP2urvA-



TA102



TA98



TA1537



WP2uvrA- pKM101



-S9



+S9



-S9



+S9



-S9



+S9



-S9



+S9



-S9



+S9



-S9



+S9



-S9



+S9



Mean



460



1880



347



310



621



866



1126



858



136



239



1953



453



1384



2608



SD



118.2



594.3



204.0



119.7



235.5



350.5



289.4



169.5



38.2



803.1



803.1



145.9



333.7



1109.6



Min



235



499



80



91



185



210



686



568



66



486



486



140



960



186



Max



952



3397



1385



810



1295



3406



2456



2009



323



4622



4622



1365



2073



3900



Values



190



190



188



186



169



168



117



118



192



192



188



184



14



14



 


 


SD = Standard deviation


Min = Minimum value


Max = Maximum value

Applicant's summary and conclusion

Conclusions:
The test material was considered to be non-mutagenic under the conditions of this test.
Executive summary:

Introduction.


The method was designed to conform to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method Bl3/14 of Commission Directive 2000/32/EC and the USA, EPA (TSCA) OPPTS harmonised guidelines.



Methods.


Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA· were treated with the test material using the Ames plate incorporation method at seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and was 5 to 5000 μg/plate. The experiment was repeated on a separate day using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test material formulations.
Additional dose levels were included to allow for test material induced toxicity, ensuring that a minimum of four non-toxic dose levels were achieved.
Results. The vehicle ( dimethyl formamide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test material caused a visible reduction in the growth of the bacterial background lawn to all of the tester strains, both in the presence and absence of S9, from 1500 μg/plate.


The test material was tested up to the maximum recommended dose level of 5000 μg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence
of S9-mix.
No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.


Conclusion.


The test material was considered to be non-mutagenic under the conditions of this
test.