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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:
2004-06-14 to 2005-01-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 21 July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Succinimide
EC Number:
204-635-6
EC Name:
Succinimide
Cas Number:
123-56-8
Molecular formula:
C4H5NO2
IUPAC Name:
pyrrolidine-2,5-dione
Test material form:
solid: flakes
Details on test material:
- Name of test material (as cited in study report): "Succinimide"
- Chemical name: 2,5-Diketopyrrolidine
- Physical state: White scales
- Lot/batch No.: SSGS 029
- Solubility in water: 330 g/L at 20 °C
- Melting range: 123- 125 °C
- Boiling point: 287 °C
- Stability under conditions of storage: Stable.
- Expiration date of the lot/batch: 30 June 2005
- Storage condition of test material: In the dark, may be used under light.
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance solution for the highest concentration group was prepared by dissolving 500 mg of "SUCCINIMIDE" in 10 mL of deionised water. The solutions for the lower concentrations were prepared by subsequent dilution of one volume of the higher concentrated solution with two volumes of deionised water. The solvent, deionised water, was also used for the negative control group.
The test substance was dissolved and diluted immediately before the start of the Ames test. The time between the preparation and the administration to the last petri dish was less than 3 hours.

The solutions for the lower concentrations were prepared by subsequent dilution of one
volume of the higher concentrated solution with two volumes of deionised water. The solvent, deionised water, was also used for the negative control group.
The test substance was dissolved and diluted immediately before the start of the Ames test. The time between the preparation and the administration to the last petri dish was less than 3 hours.

The solutions for the lower concentrations were prepared by subsequent dilution of one
volume of the higher concentrated solution with two volumes of deionised water. The solvent, deionised water, was also used for the negative control group.
The test substance was dissolved and diluted immediately before the start of the Ames test. The time between the preparation and the administration to the last petri dish was less than 3 hours.

Method

Target gene:
Histidine
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535
Additional strain / cell type characteristics:
other:
Remarks:
see Table 1 in box "Any other information on materials & methods incl. tables"
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
other:
Remarks:
see Table 1 in box "Any other information on materials & methods incl. tables"
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
other:
Remarks:
see Table 1 in box "Any other information on materials & methods incl. tables"
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
other:
Remarks:
see Table 1 in box "Any other information on materials & methods incl. tables"
Species / strain / cell type:
S. typhimurium, other: TA97a
Additional strain / cell type characteristics:
other:
Remarks:
see Table 1 in box "Any other information on materials & methods incl. tables"
Metabolic activation:
with and without
Metabolic activation system:
S9-MixType and composition of metabolic activation system:
- source of S9: microsomal fraction of homogenised livers of female Sprague Dawley rats treated once with 500 mg/kg of Aroclor 1254
- method of preparation of S9 mix: The livers of the animals were removed and homogenised in cold 0.15 mol/L KCI. Three mL of homogenate were obtained per gram of liver. Then the homogenate was centrifuged for 10 minutes at 9000 x g. The supernatant contained the microsomes. Small portions of the microsomes were stored in liquid nitrogen. Immediately before use they were thawed and mixed with the cofactor solution.
- concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): The metabolic activity of the microsomes was verified by the positive control substances of each study.
Test concentrations with justification for top dose:
5000, 1667, 556, 185, 62 µg/plate
Vehicle / solvent:
Deionised water was used as solvent for the test substance and for the negative control group.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-Nitro-o-phenylene-diamine
Remarks:
for TA97a, 10 µg, without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
for TA98, 2 µg, without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
for TA100 (2 µg) and TA1535 (1µg), without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: t-Butyl-hydroperoxide
Remarks:
for TA102, 50 µg, without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
for TA97a, 10 µg, with S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2- Aminoanthracene
Remarks:
for TA98 (1 µg), TA100 (2 µg) and TA1535 (2 µg), with S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 1,8-Dihydroxy-anthraquinone
Remarks:
for TA102, 50 µg, with S9
Details on test system and experimental conditions:
The exposure was performed according to the "Plate Incorporation Assay", in which bacteria, test substance (and microsomes) are in contact on the plate without preceding incubation in liquid state. The number of viable cells in the overnight-cuture is in the range of 200 000 000 cells per mL.
For each sample the following solutions were combined:
• 0.1 mL of the overnight culture of the bacteria,
• 0.5 mL of S9-mix (or phosphate buffered saline for samples without metabolic activation),
• 0.1 mL of the appropriate test- or reference substance solution and
• 2 mL of top agar.
The combined solutions were mixed and spread over a plate with minimal agar (9 cm diameter). After the top agar had solidified, the plates were incubated at 37 °C until the colonies were visible (2 days).

Bacterial strains of S. typhimurium (TA97a, TA98, TA100, TA102 and TA1535) were obtained from Prof. Bruce N. Ames, Berkely, California. The bacteria were stored in small portions in a solutions of 6 % DMSO in PBS in liquid nitrogen.

Counting of colonies: The plates of the strains with a low spontaneous revertant rate, i.e. TA98 and TA1535 were counted visually by marking the colonies with a felt tipped pen. The plates of the other strains were photographed with a video camera and the picture files were scanned for colonies by a computer program.

The results of the first experiment were verified by a second, independent experiment.

Triplicate repetitions were run for each dose group in each of the two separate experiments that were conducted, for the control groups six-fold repetitions were run.

Positive controls:
Strain TA97a: 4-NOPD 10 µg (without S9), DMBA 10 µg (with S9)
Strain TA98: 2-NF 2 µg (without S9), 2-AA 1 µg (with S9)
Strain TA100: Sodium-azide 2 µg (without S9), 2-AA 2ug (with S9)
Strain TA102: t-BHPO 50 µg (without S9), DHA 50 µg (with S9)
Strain TA1535: Sodium-azide 1 µg (without S9), 2-AA 2 µg (with S9)

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate):
- Number of independent experiments: 3

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 2 x 10^8 cells per mL.
For each sample the following solutions were combined:
• 0.1 mL of the overnight culture of the bacteria,
• 0.5 mL of S9-mix (or phosphate buffered saline for samples without metabolic activation),
• 0.1 mL of the appropriate test- or reference substance solution and
• 2 mL of top agar.
The combined solutions were mixed and spread over a plate with minimal agar (9 cm
diameter).

TREATMENT AND HARVEST SCHEDULE:
- After the top agar had solidified, the plates were incubated at 37 °C until the colonies were visible (2 days).

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method:
The following signs of toxicity, if present, were recorded:
• A reduced bacterial background lawn (mottled instead of homogeneous).
• Microcolonies of bacteria instead of a homogeneous background lawn.
• No background lawn.
• Clearly reduced numbers of revertant colonies.
- Any supplementary information relevant to cytotoxicity:

METHODS FOR MEASUREMENTS OF GENOTOXICIY:
Counting of colonies:
The plates with less than about 50 revertant colonies, i.e. the plates of TA98 and TA1535 with the exception of the positive controls, were counted visually by marking the colonies with a felt tipped pen. The other plates were photographed with a video camera and the picture files were scanned for colonies by a computer program.
Rationale for test conditions:
The concentrations for the first experiment were set according to a preliminary toxicity test: 50 mg of the test substance was dissolved in 1 mL of deionised water and was mixed with phosphate buffer, bacteria (TA100) and top-agar, as described in 3.5 and spread over a plate with minimal agar. The plate was incubated at 37 °C for 2 days and the growth of the bacterial background and the density of revertant colonies was determined.
The test substance was not toxic up to 5000 µg/petri dish. It was therefore decided to use 5000 µg/plate as the highest concentration which is the limit concentration according to the guidelines. Each of the other 4 concentrations was 1/3 of the preceding one. The number and intervals of the concentrations are in accordance with the guidelines (5 concentrations with intervals of about 10).
Evaluation criteria:
Means and standard deviations were calculated for the number of mutants in every concentration group.
The criteria for a positive result were:
A reproducible increase of the number of revertants to more than the following threshold values for at least one of the concentrations:
• For the strains with a low spontaneous revertant rate i.e. TA98 and TA1535: 250 % of the amount of the spontaneous revertants.
• For the strains with a high spontaneous revertant rate i.e. TA97a, TA100 and TA102: 167 % of the amount of the spontaneous revertants.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA97a
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
PRECIPITATION:
No precipitation of the test substance was seen in any of the concentration groups.

RANGE-FINDING/SCREENING STUDIES:
In a preliminary test no toxicity was seen up to 5000 µg/plate.

COMPARISON WITH HISTORICAL CONTROL DATA:
The numbers of spontaneous revertants were comparable with the historic control data for the negative controls. See also Table 2 in box "Any other information on results incl. tables."

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No toxicity was seen up to 5000 µg/plate.

Any other information on results incl. tables

Table 2: Historical control data

 

without metabolisation 

with metabolisation (S9-mix) 

strain: 

substance, 

concentration 
(µg/plate) 

revertants/plate 

 mean SD min/max 

 

substance, 

concentration 
(µg/plate) 

revertants/plate 

 mean SD min/max 

 

TA97a 
 

Control  

 88  20 55 / 179 

Control 

 116  19       83 / 192 

4-NoPD, 10 µg 

 363  126 198 / 865 

DMBA, 10 µg 

 368  121 176 / 844 

TA98 
 

Control 

 9.4  2.1         5 / 16 

Control 

 13.3  2.6           9 / 21 

2-NF, 2 µg 

 197  48 101 / 307 

2-AA, 1 µg 

 462  180 174 / 1062 

TA100 
 

Control 

 74.1  16.6 40 / 128 

Control 

 87.0  12.6       58 / 118 

Na-Azide, 2 µg 

 479  114 246 / 738 

2-AA, 2 µg 

 1620  423 633 / 2324 

TA102 
 

Control 

 152  32 69 / 240 

Control 

 219  38 120 / 320 

tBHPO, 50 µg 

 483  151 186 / 855 

DHA, 50 µg 

 706  193 246 / 1248 

TA1535 
 

Control 

 12.9  5.3         7 / 31 

Control 

 11.8  4.7          6 / 32 

Na-azide, 1 µg 

 269  106 94 / 587 

2-AA, 2 µg 

 152  70       43 / 374 

DHA = 1,8-Dihydroxy-anthraquinone

tBHPO = tert.-Butyl-hydroperoxide

4-NoPD = 4-Nitro-o-phenylene-diamine

DMBA = 7,12-Dimethylbenz[a]anthracene

2-NF = 2 -Nitro-fluorene

2-AA = 2-Aminoanthracene

Applicant's summary and conclusion

Conclusions:
Succinimide is not mutagenic in the Ames test with the strains of Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 up to 5000 µg per plate.
Executive summary:

In a reverse gene mutation assay in bacteria conducted according to OECD guideline 471, strains of TA97a, TA98, TA100, TA102 and TA1535 of S. typhimurium were exposed to succinimide dissolved in deionised water (500 mg succinimide dissolved in 10 mL water) at concentrations of 5000, 1667, 556, 185 and 62 μg/plate in the presence and absence of mammalian metabolic activation. The exposure was performed according to the Plate Incorporation Assay.

Succinimide was tested up to the limit concentration of 500 µg/petri dish. In a preliminary toxicity test, the test substance was not toxic up to this concentration.

The positive controls induced the appropriate responses in the corresponding strains.There wasno evidenceof induced mutant colonies over background.

 

This study is classified asacceptable.  This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

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