Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1996
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
Study predates the OECD guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guideline
Qualifier:
no guideline followed
Version / remarks:
Study predates the OECD guideline 471. Although the protocol used seems to be similar to the OECD guideline, the lack of information concerning the test conditions does not allow to conclude that they are formally similar.
Deviations from OECD 471:Only 4 strains tested / duplicate plating / no follow-up experiment.
Principles of method if other than guideline:
Mutagenicity in the Salmonella typhimurium plate incorporation assay (Reverse mutation assay):

The test substance was evaluated for mutagenicity in Salmonella typhimurium strains TA100, TA1535, TA97a and TA98 with and without an exogenous metabolic activation system (S9). The maximum concentration tested was 5000 µg/plate. Concentrations of 2500, 1000, 500, 100, 50 and 10 µg/plate were also tested in comparison to a negative (solvent) control. The negative and the diluent for the test substance was ethyl alcohol.
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
See confidential details on test material

Method

Target gene:
Not specified.
Species / strain
Species / strain / cell type:
S. typhimurium, other: TA100, TA1535, TA97a and TA98
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
The maximum concentration tested was 5000 µg/plate.
Concentrations of 2500, 1000, 500, 100, 50 and 10 µg/plate were also tested.
Vehicle / solvent:
ethyl alcohol
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
ethyl alcohol
Positive controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2AA)
Remarks:
With metabolic activation
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without metabolic activation
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Without metabolic activation
Positive controls:
yes
Positive control substance:
other: ICR-191
Remarks:
Without metabolic activation
Details on test system and experimental conditions:
Not specified.
No details provided in the report.
Rationale for test conditions:
No information on the test conditions
Evaluation criteria:
According to the test method, the test substance was classified as positive when:
1. The average number of revertants in any strain at any test substance concentration studied was at least two times greater than the average number of revertants in the concurrent negative control.
AND
2. There was a positive dose response relationship in that same strain.

The test substance was classified as negative when:
1. Ther were no test concentrations with an average number of revertants at least two times greater than the average number of revertants in the negative control.
OR
2. There was a positive dose-response relationship.

A test substance was classified as equivocal when the test substance was not clearly negative yet did not meet the criteria for a positive response.
Statistics:
For each strain, the average of revertants and the standard deviation (S.D.) at each concentration with and without S9 activation were calculated.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Background lawn was noticeably thinner and/or size of microcolonies was slightly larger than control at 50 microgram/plate both in presence and absence of S9-mix. More evident signs of cytotoxicity were observed at higher concentrations.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Background lawn was noticeably thinner and/or size of microcolonies was slightly larger than control at 500 microgram/plate (S9mix -) and 100 (S9mix +). More evident signs of cytotoxicity were observed at higher concentrations.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Background lawn was noticeably thinner and/or size of microcolonies was slightly larger than control at 50 microgram/plate both in presence and absence of S9-mix. More evident signs of cytotoxicity were observed at higher concentrations.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 97
Remarks:
TA97a strain
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Background lawn was noticeably thinner and/or size of microcolonies was slightly larger than control at 100 microgram/plate both in presence and absence of S9-mix. More evident signs of cytotoxicity were observed at higher concentrations.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
According to the test method, acceptability criteria:
An individual trial must have included at least five test concentrations, a negative controm, and a positive indicator for each selected tester strain. Data acceptability criteria were as follows:
- A single data point may have been rejected if contamination or excessive toxicity was seen on a treatment plate. A single data point may also have been rejected if excessive precipitate on the plate prevented accurate colony counting.
- A negative control data point may have been rejected if it fell outside the acceptable spontaneous mutation range.
- A concentration level was rejected if there were less than two data points at the treatment level or if the data point values were judged by the study director to be too divergent.
- A trial for the affected strain was rejected if the negative control was rejected or if there was no evidence of mutagenic activity on any positive indicator plate.

Only those trials which met the criteria of acceptability were included in the report.

Any other information on results incl. tables

Table 1. Mutagentic activity of the test item in strain TA100

      Without Metabolic Activation   With Metabolic Activation    
 Concentration (µg/plate)  Revertant average (SD)  Observations  Concentration (µg/plate)  Revertant average (SD)  Observations
0.0  94 (6)  T0, P0  0.0  125 (4)  T0, P0 
10.0  88 (4)  T0, P0  10.0  106 (4)  T0, P0 
50.0  78 (4)  T1, P0  50.0  92 (6)  T1, P0 
100.0  80 (3)  T1, P0  100.0  102 (1)  T1, P0 
500.0  87 (2)  T1, P0  500.0  110 (6)  T2, P0 
1000.0  70 (0)  T3, P0  1000.0  100 (12)  T1, P0 
2500.0  61 (1)  T3, P0  2500.0  94 (5)  T3, P1 
5000.0  75 (1)  T3, P0  5000.0  90 (1)  T3, P1 

 Positive control:

NAAZ

   

Positive control:

2AA 

   
2 µg/plate   610 (23) 1 µg/plate  2341 (306) 

Table 2. Mutagentic activity of the test item in strain TA1535

      Without Metabolic Activation   With Metabolic Activation    
 Concentration (µg/plate)  Revertant average (SD)  Observations  Concentration (µg/plate)  Revertant average (SD)  Observations
0.0  20 (1)  T0, P0  0.0  25 (1)  T0, P0 
10.0  18 (2)  T0, P0  10.0  22 (1)  T0, P0 
50.0  21 (1)  T0, P0  50.0  20 (4)  T0, P0 
100.0  19 (8)  T0, P0  100.0  18 (1)  T1, P0 
500.0  15 (1)  T1, P0  500.0  19 (2)  T1, P0 
1000.0  15 (1)  T2, P0  1000.0  15 (1)  T2, P0 
2500.0  20 (1)  T2, P0  2500.0  9 (1)  T2, P0 
5000.0  17 (1)  T3, P0  5000.0  7 (2)  T2, P1 

 Positive control:

NAAZ

   

Positive control:

2AA 

   
2 µg/plate  547 (4)  2 µg/plate  536 (23) 

Table 3. Mutagentic activity of the test item in strain TA98

      Without Metabolic Activation   With Metabolic Activation    
 Concentration (µg/plate)  Revertant average (SD)  Observations  Concentration (µg/plate)  Revertant average (SD)  Observations
0.0  34 (2)  T0, P0  0.0  21 (1)  T0, P0 
10.0  23 (1)  T0, P0  10.0  28 (2)  T0, P0 
50.0  24 (2)  T1, P0  50.0  28 (2)  T1, P0 
100.0  23 (1)  T1, P0  100.0  20 (1)  T1, P0 
500.0  19 (2)  T1, P0  500.0  26 (2)  T1, P0 
1000.0  18 (2)  T2, P0  1000.0  30 (1)  T2, P0 
2500.0  6 (1)  T3, P0  2500.0  17 (2)  T3, P0 
5000.0  9 (1)  T3, P0  5000.0  18 (1)  T3, P0 

 Positive control:

2NF

   

Positive control:

2AA 

   
25 µg/plate  1710 (31)  2 µg/plate  2048 (214) 

Table 4. Mutagentic activity of the test item in strain TA97a

      Without Metabolic Activation   With Metabolic Activation    
 Concentration (µg/plate)  Revertant average (SD)  Observations  Concentration (µg/plate)  Revertant average (SD)  Observations
0.0  89 (3)  T0, P0  0.0  98 (3)  T0, P0 
10.0  108 (0)  T0, P0  10.0  84 (7)  T0, P0 
50.0  65 (1)  T0, P0  50.0  69 (10)  T0, P0 
100.0  48 (6)  T1, P0  100.0  78 (2)  T1, P0 
500.0  63 (9)  T2, P0  500.0  85 (6)  T1, P0 
1000.0  71 (0)  T2, P0  1000.0  92 (1)  T2, P0 
2500.0  72 (4)  T2, P0  2500.0  105 (5)  T2, P0 
5000.0  55 (1)  T2, P0  5000.0  67 (5)  T3, P0 

 Positive control:

ICR-191

   

Positive control:

2AA 

   
2 µg/plate  2326 (45)  1 µg/plate  1491 (1) 

Abbreviations used in the tables:

Evidence for test substance toxicity to the bacteria was documented by recording the appearance of the plates and background lawn using the following key:

- T0: Background lawn appeared normal.

- T1: Background lawn was noticeably thinner and/or size of micrcolonies was slightly larger than controls.

- T2: Background lawn was markedly thinner and/or size of micrcolonies was markedly larger than controls.

- T3: Background lawns were severely reduced and/or micrcolonies were greatly enlarged relative to controls.

- T4: Background lawn was absent and/or microcolonies could be seen readily by the unaided eye.

- T5: Colony formation was reduced or absent relative to controls.

Formation of a precipitate by the test material was documented using the following key:

- P0: No evidence of precipitate was detected.

- P1: Microscopic precipitate was present.

- P2: Marked precipitate was present but did not interfere with automated colony counting.

- P3: Marked precipitate was present and required the plate to be counted by hand.

- P4: Heavy precipitate prevented accurate colony counting and/or obscured the background lawn.

- N: The absence of any noteworthy observations.

- R: Plate rejected.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, no evidence of mutagenic activity was detected.
Executive summary:

The assessment of the mutagenic activity of the test substance was carried out using the Salmonella typhimurium reverse mutation assay (plate incorporation method).

The test substance was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA100, TA1535, TA97a and TA98). The test was performed in the absence and presence of S9 -mix.

Under the conditions of this study Fluorosulfonic Adduct did not induce a significant dose-related increase in the number of revertant (His+ ) colonies in each of the four tester strains (TA1535, TA97a, TA98 and TA100) both in the absence and presence of S9-metabolic activation.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.