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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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: (as the data is used in a read-across approach, a maximal reliability score of 2 was attributed).
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:

- Lot/batch No.: FAL 07/25
- Expiration date of the lot/batch: 2009
- Storage condition of test material: at room temperature (preference between 2°C and 10°C)

Method

Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain characteristics:
DNA polymerase A deficient
Metabolic activation:
with and without
Metabolic activation system:
S9 and S9 Mix addition
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 and S9 mix addition
Test concentrations with justification for top dose:
Assay 1 and assay 2 : for strain TA1535, TA1537, TA98, TA100 and TA102, doses in µg/plate are 0, 50, 150, 500, 1500 and 3000.
Vehicle:
The test item LAB 3822 was dissolved in dimethylsulfoxyde (DMSO)
Controlsopen allclose all
Negative controls:
no
Solvent controls:
yes
Remarks:
without metabolic activation
True negative controls:
not specified
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
other: Sodium azide ; 9-amino-acridine ; 2-nitro fluorene ; mitomycin C
Remarks:
Without metabolic activation:sterility of the air media (agar only)
Negative controls:
no
Solvent controls:
yes
Remarks:
with metabolic activation
True negative controls:
not specified
Positive controls:
yes
Remarks:
with metabolic activation
Positive control substance:
other: 2-anthramine ; benzo(a)pyrene
Remarks:
Assay for the control of the media sterility are performed like in the mutagenicity assay without metabolic activation.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Remarks on result:
other:
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Remarks on result:
other:
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Under these experimental conditions, the test item LAB3822 (Batch FAL 07/25) induced no mutagenic activity in any of the five Salmonella typhimurium tested in presence as well as in absence of metabolic activation.
The determination of LAB3822 in treatment solutions was performed using a validated analytical method. The dosing results are thus considered as reliable. The results obtained for the concentration assay of LAB3822 in treatment solutions used in the Ames test FSP-IPL 070707, were within the range 80-120% of the theoretical values, except for 4 samples, with 67.6 to 123.5% of recovery between theoretical and nominal values. Nevertheless, this was considered only as a slight deviation and did not affect either the integrity or the validity of the current study. It is noteworthy that the blanks were below the limit of quantification, as expected. Finally, LAB3822 is considered as stable in DMSO solutions for 8 months, when stored at –80°C.
Executive summary:

BACTERIAL MUTAGENICITY TEST ON SALMONELLA TYPHIMURIUM HIS- (5 STRAINS) USING B.N. AMES'S TECHNIQUE

THIS STUDY WAS CARRIED OUT IN COMPLIANCE WITH GOOD LABORATORY PRACTICE REGULATIONS

METHODS

Strains used : TA 1535, TA1537, TA98, TA100, TA102

Preliminary test of toxic activity : carried out on 5 strains – Incubation period: 48 hours

Sterility test : absence of contamination

Mutagenicity test : carried out both with and without metabolic activation using hepatic microsomes from rat livers induced by Aroclor 1254 – Incubation period: 48h

Number of assays : 2 (the second assay with metabolic activation was performed according to pre-incubation method)

Solvent : DMSO

Limiting factor for maximum dose : maximum dose according to OECD guideline

Doses used in main test : expressed as μg/plate pure LAB3822

CYTOTOXICITY ASSAY

 Cytotoxicity assay     Doses in µg/plate        TA1535  TA1537        TA98    TA100      TA102
 T  P  T  P  T  P  T  P  T  P
LAB 3822 WITHOUT S9 -mix                  0  -  -  -  -  -  -  -  -  -
 50  -  -  -  -  -  -  -  -  -  -
 150  -  -  -  -  -  -  -  -  -  -
 500  -  -  -  -  -  -  -  -  -  -
 1500  -  -  -  -  -  -  -  -  -  -
 5000  -  -  -  -  -  -  -  -  -
 Top Dose in 1st mutagenicity assay  -     3000     5000     5000     5000     3000
LAB 3822 WITH S9 - mix                 0  -  -  -  -  -  -  -  -  -  -
 50  -  -  -  -  -  -  -  -  -  -
 150  -  -  -  -  -  -  -  -  -  -
 500  -  -  -  -  -  -  -  -  -  -
 1500  -  -  -  -  -  -  -  -  -  -
 5000  -  -  -  -  -  -  -  -  -  -
  Top Dose in 1st mutagenicity assay  -     5000     5000     5000     5000     3000

T = Toxicity (- non toxic; + slightly toxic; ++ moderately toxic; +++ strongly toxic; N no bacterial growth)

P = Precipitate (- absence; + slight precipitate; ++ moderate precipitate; +++ important precipitate hindering scoring)

The test item LAB3822 induced no toxicity either with or without metabolic activation, whatever the dose tested. However, the highest concentration of 5000 μg/plate induced a decrease in the number of reverstants in strain TA 102, in presence and in absence of metabolic activation, and in strain TA 1535 in absence of metabolic activation.

The maximum dose retained for the first mutagenicity assay was 5000 μg/plate with and without metabolic activation in all strains tested, except in strain TA1535, without metabolic activation, and strain TA102, with and without metabolic activation, where the top dose retained was of 3000 μg/plate.

 Assay 1        TA1535     TA1537     TA98     TA100     TA102  
 Doses µg/plate  Induction ratio(a)  Doses µg/plate    Induction ratio(a)   Doses µg/plate   Induction ratio(a)   Doses µg/plate   Induction ratio(a)   Doses µg/plate  Induction ratio(a)   
LAB 3822 WITHOUT S9 -mix                  0  -  0  -  0  -  0  -  0  -  
 50  0.8  50  1.0  50  1.0  50  0.9  50  1.0  
 150  0.7  150  1.2  150  0.7  150  1.0  150  1.2  
 500  0.7  500  1.2  500  0.5  500  0.9 500   1.0  
 1500  0.7  1500  1.2  1500  0.4  1500  0.7  1500  0.8  
 3000  0.6  3000  0.9  3000  0.4  3000  0.2  3000  0.5  
                                
LAB 3822 WITH S9 -mix                  0  -  0  -  0  -  0  -  0  -
 50  1.7  50  2.1  50 0.9   50 1.3   50 0.7 
 150  1.3  150 1.1   150 0.9   150 1.4  150  0.8 
 500  1.6  500 1.1   500 1.3   500 1.4   500 0.7 
 1500  1.0  1500 1.6   1500 1.2   1500 1.3   1500 0.6 
 3000  1.2  3000 0.6   3000 1.1   3000 1.3   3000 0.2 

(a) Induction Ratio = number of revertants in the treated/number of revertants in the control

 Assay 2      TA1535     TA1537     TA98     TA100     TA102  
 Doses µg/plate  Induction ratio(a)  Doses µg/plate    Induction ratio(a)   Doses µg/plate   Induction ratio(a)   Doses µg/plate   Induction ratio(a)   Doses µg/plate  Induction ratio(a)   
LAB 3822 WITHOUT S9 -mix                  0  -  0  -  0  -  0  -  0  -  
 50  0.9  50 0.9  50 0.8  50  0.9  50  1.0  
 150  0.8  150  1.6  150  0.7  150 0.9  150 1.0  
 500  0.7  500  0.6  500 0.9  500 0.8 500   1.0  
 1500  0.5  1500 0.4  1500  0.6  1500  0.9  1500  0.9  
 3000  0.6  3000  0.2  3000  0.5  3000  0.7  3000  0.7  
                                
LAB 3822 WITH S9 -mix                  0  -  0  -  0  -  0  -  0  -
 50  1.1  50  1.1  50 0.7   50 1.0   50 0.7 
 150  0.9  150 1.3   150 0.9   150 0.8  150  0.9 
 500  0.8  500 1.1   500 0.7   500 0.9   500 0.6 
 1500  1.1  1500 0.6   1500 0.5   1500 0.8   1500 0.6 
 3000  0.5  3000 0.8   3000 0.6   3000 0.6   3000 0.2 

(a) Induction Ratio = number of revertants in the treated/number of revertants in the control

CONCLUSION

Under these experimental conditions, the test item LAB3822 (Batch FAL 07/25) provided by ROQUETTE induced no mutagenic activity in any of the five Salmonella typhimurium tested in presence as well as in absence of metabolic activation.

The determination of LAB3822 in treatment solutions was performed using a validated analytical method. The dosing results are thus considered as reliable. The results obtained for the concentration assay of LAB3822 in treatment solutions used in the Ames test FSP-IPL 070707, were within the range 80-120% of the theoretical values, except for 4 samples, with 67.6 to 123.5% of recovery between theoretical and nominal values. Nevertheless, this was considered only as a slight deviation and did not affect either the integrity or the validity of the current study. It is noteworthy that the blanks were below the limit of quantification, as expected. Finally, LAB3822 is considered as stable in DMSO solutions for 8 months, when stored at –80°C.