Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
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:
Test article name : Emulsamine AT-O
Chemical name : alkylamidoamine
Batch number: 6514
Purity: > 95%
Origin: Feuchy Plant
Storage condition of test material : at room temperature, protected from light
Physical state: brown liquid

Method

Target gene:
HIS-locus
Species / strain
Species / strain / cell type:
other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
The bacterial strains obtained from Professor B.N. Ames (University of California, Berkeley - U.S.A.) are kept in the laboratory and preserved in liquid nitrogen. The strains are regularly checked for their various characteristics :
· Histidine requirements
· Sensitivity to the reference mutagens
· Loss of cell-wall lipopolysaccharides : rfa mutations (sensitivity to crystal violet) for strains
Salmonella typhimurium
· uvr B- mutation (sensitivity to uvB), apart from strain TA102
· Presence of pKM 101 plasmid (ampicillin resistance) from strains TA98, TA100 and TA102
· Presence of pAQ1 plasmid (tetracyclin resistance) from strain TA102 only
Additional strain / cell type characteristics:
other: Each strain derived from Salmonella typhimurium LT 2 contains one mutation in the histidine operon, resulting in a requirement for histidine. Please see materials and methods for more information
Metabolic activation:
with and without
Metabolic activation system:
S9 metabolic activation system
Test concentrations with justification for top dose:
Due to its insolubility in water, the test compound was dissolved in DMSO at a maximum concentration of 50 mg/ml. After that, this solution was used at 100 μl/plate giving a final concentration of 5000 μg/plate as recommended by OECD procedures. Successive dilutions were prepared with DMSO and used at 100 μl/plate.

Vehicle / solvent:
The vehicle was dimethylsulfoxide (DMSO) for chromatography MERCK batch # k30049278211
Controlsopen allclose all
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
The vehicle control was tested both with and without S9 mix
True negative controls:
not specified
Positive controls:
yes
Remarks:
Dissolved in dimethylsulfoxide to check the sensitivity of the test system
Positive control substance:
other: sodium azide 1 µg/plate
Remarks:
Without S9 mix. Used on the TA 1535 and TA 100 strains
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
The vehicle control was tested both with and without S9 mix
True negative controls:
not specified
Positive controls:
yes
Remarks:
Dissolved in dimethylsulfoxide to check the sensitivity of the test system
Positive control substance:
other: 9-aminoacridine 50 µg/plate
Remarks:
without S9 mix. Used on the TA 1537 strain.
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
The vehicle control was tested both with and without S9 mix
True negative controls:
not specified
Positive controls:
yes
Remarks:
Dissolved in dimethylsulfoxide to check the sensitivity of the test system
Positive control substance:
other: 2-nitrofluorene 2 µg/plate
Remarks:
without S9 mix. Used on the TA 98 strain.
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
The vehicle control was tested both with and without S9 mix
True negative controls:
not specified
Positive controls:
yes
Remarks:
Dissolved in dimethylsulfoxide to check the sensitivity of the test system
Positive control substance:
other: mitomycin C 0.125 µg/plate
Remarks:
without S9 mix. Used on the TA 102 strain.
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
The vehicle control was tested both with and without S9 mix
True negative controls:
not specified
Positive controls:
yes
Remarks:
Dissolved in dimethylsulfoxide to check the sensitivity of the test system
Positive control substance:
other: other:2-Anthramine
Remarks:
with S9 mix. 2 µg/plate was used without pre-incubation and 1 µg/plate was used with pre-incubation for TA 1535, TA 1537, TA 100 and TA 98 strains.
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
The vehicle control was tested both with and without S9 mix
True negative controls:
not specified
Positive controls:
yes
Remarks:
Dissolved in dimethylsulfoxide to check the sensitivity of the test system
Positive control substance:
other: benzo(a)pyrene 2 µg/plate
Remarks:
with S9 mix. Used for the TA 102 strain
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Without metabolic activation
The following technique is performed for each one of the strains used in the test : 0.1 ml of the
test substance at the relevant concentration and 0.1 ml of a bacterial suspension from a culture
agitated overnight at 37°C are successively added to 2 ml of top agar to which 10 % of
0.5 mM biotin histidine solution, maintained in a state of superfusion at 45°C, has been added.
The content of each tube is agitated, then spread out in a Petri plate containing 20 ml of minimum
agar. Three plates are used per treatment. The plates are then incubated at 37°C for 48 h
approximately. Finally, colonies of revertants are counted for each plate.
At the same time, solvent controls (0.1 ml solvent/plate ) are performed under the same
conditions but using 6 plates per solvent. Appropriate positive
reference controls are also performed.
At each assay, 3 Petri plates, each containing 20 ml of minimal agar receive 2 ml of top agar
only and are incubated under the same conditions of assay for the control of media sterility: no
colony must be observed after 48 hours at 37°C.
With metabolic activation
The method with metabolic activation is the same as the one described above, except that
immediately before spreading in the plates, 0.5 ml of the S9-mix metabolic activation system is added in soft agar.
Solvent controls, positive controls and assay for the control of media sterility are performed like in
the mutagenicity assay without metabolic activation.
Furthermore, the sterility control of the S9-mix is performed.

NUMBER OF REPLICATIONS:
Without metabolic activation
The test is subsequently repeated in an independent assay. The same method is used, but the test doses may be modified, depending on the results obtained during the first test.
With metabolic activation
According to the results obtained in the first assay in the presence of metabolic activation, the second assay can either be performed using a standard plate-incorporation technique, or be extended by use of the pre-incubation modification of the assay. If a positive response was observed in the first assay, the same standard plate-incorporation technique is used in a repeat assay with the confirmatory purpose. But the test doses may be modified, depending on the results obtained during the first test. If a negative response was observed in the first assay, the method used in a second assay is the pre-incubation test.
The use of the standard plate-incorporation protocol with S9-mix is known to be sub-optimal in the detection of a number of bacterial promutagens.
On a technical point of view, this method is the same as the one using agar plate incorporation with, however, the following modifications:
The following solutions are added in this order: the test compound solution (50 μl if an organic solvent is used and 100 μl if aqueous solvent), S9-mix (500 μl) and at the end, the bacterial strain to be tested (100 μl). The mixture is preincubated with stirring at 37°C for 60 minutes prior to adding soft agar and spreading out in a Petri plate.


In order to choose the range of doses for the test, the cytotoxic activity of the test compound is determined. The cytotoxicity assay is carried out in all the test strains to be tested under the same conditions than the mutagenicity test but using only one plate per dose instead of 3 plates per dose. The plates are incubated for approximatively 48-72 hours at 37°C, and the revertants are counted. Cytotoxicity is checked by microscopic examination of the background lawn and is noted as follows :
- : non -toxic
+ : slightly toxic
++ : moderately toxic
+++ : strongly toxic
N : no bacterial growth
In this assay, the possible presence of a precipitate is also noted according to a scoring :

Generally 5 concentrations are tested.
· For non-toxic and very soluble compounds, the top concentration used is generally 5000 μg/plate (100 μl/plate for extracts)
· For non-toxic and slightly soluble compounds, the top concentration used is the lowest dose provoking a slight precipitate when added to the top agar
· For toxic compounds, irrespective of the solubility, the top concentration used is the dose provoking a moderate thinning of the lawn and/or a reduction in the number of revertants close to 75 % in comparison with the controls. Four other lower doses, distributed by geometrical (half-log) ratio, are also used.

Evaluation criteria:
CRITERIA FOR DETERMINING THE MUTAGENIC EFFECT
Given that the procedures followed in order to evaluate mutagenic activity are semi-quantitative, the criteria used to determine a positive effect must of course be subjective and based mainly on experience. However, under our experimental conditions, and when the validity criteria are reached, the following decision criteria may be used.
Criteria based on biological significance
. Strains TA 1535, TA 1537
A product causing a positive response proportional to the dose for at least 3 concentrations with, for the highest increase, a value greater than or equal to 3 times the value for the solvent control, is considered positive in the assay.
. Strains TA 98, TA 100, TA102
A product causing a positive response proportional to the dose for at least 3 concentrations with, for the highest increase, a value greater than or equal to 2 times the value for the solvent control, is considered positive in the assay.
Criteria based on statistical significance
In parallel, data are analysed by means of Dunnett's method allowing the comparison of the mean value for each dose to the mean value for the corresponding
solvent control.
Reproducibility
If a product causes a positive response during a single assay and that result cannot be reproduced in at least 2 independent assays, the initial positive result may be considered as not significant.
All these criteria are not absolute, but they, however, help in coming to a decision which can be conclusive in the majority of the cases.

Results and discussion

Test results
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
from 50 µg/plate without metabolic activation, from 150 µg/plate with metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The results of the test for toxic activity showed that the compound demonstrated a strong toxic effect that prevented bacterial growth from 50 to 5000 μg/plate without metabolic activation, and from 150 to 5000 μg/plate with metabolic activation. The maximum dose chosen for the mutagenicity assay was 15 μg/plate for all strains tested without metabolic activation, and 50 μg/plate for strains TA1535, TA100 and TA102, and 100 μg/plate for strains TA1537, TA98 in the assay with metabolic activation.

With and without metabolic activation, no biologically significant increase in the number of revertants was noted
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Doses used in main test : express as µg/ plate pure test substance

Without S9 mix

Strain

TA1535

TA1537

TA98

TA100

TA102

Assay

1

2

1

2

1

2

1

2

1

2

Doses

µg / plate

0

0

0

0

0

0

0

0

0

0

0.15

0.15

0.15

0.15

0.15

0.15

0.15

0.15

0.15

0.15

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

1.5

1.5

1.5

1.5

1.5

1.5

1.5

1.5

1.5

1.5

5

5

5

5

5

5

5

5

5

5

15

15

15

15

15

15

15

15

15

15

Without S9 mix

Strain

TA1535

TA1537

TA98

TA100

TA102

Assay

1

2*

1

2*

1

2*

1

2*

1

2*

Doses

µg / plate

0

0

0

0

0

0

0

0

0

0

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

1.5

1.5

1.5

1.5

1.5

1.5

1.5

1.5

1.5

1.5

5

5

5

5

5

5

5

5

5

5

15

15

15

15

15

15

15

15

15

15

50

50

50

50

50

50

50

50

50

50

100

100

*with pre-incubation

The test substance induced no mutagenic activity in the five Salmonella typhimurium strains tested.

Applicant's summary and conclusion

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

The test substance induced no mutagenic activity in the five Salmonella typhimurium strains tested.
Executive summary:

The objective of this study was to evaluate the potential of the test item N-[3-(dimethylamino)propyl]-C18 unsaturated-alkylamide (unsaturated C18) to induce reverse mutation inSalmonella typhimurium.

The study was performed according to the international guidelines (OECD 471 and Commission Directive No. B13/14) and in compliance with the principles of Good Laboratory Practice.

Methods

A preliminary toxicity test was performed to define the dose-levels of Cocoamidopropyldimethylamine to be used for the mutagenicity study. The test item was then tested in three experiments, with and/or without a metabolic activation system, the S9 mix, prepared from a liver post‑mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254.

All experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C).

Five strains of bacteriaSalmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to at least five dose-levels of the test item (three plates/dose‑level). After 48 hours of incubation at 37°C, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

The test item was dissolved in dimethylsulfoxide (DMSO).

The dose-levels of the positive controls were as follows:

without S9 mix

- 1 µg/plate of sodium azide (NaN3): TA 1535 and TA 100 strains,

- 50 µg/plate of 9-Aminoacridine (9AA): TA 1537 strain,

- 2 µg/plate of 2-Nitrofluorene (2NF): TA 98 strain,

- 0.125 µg/plate of Mitomycin C (MMC): TA 102 strain.

     

with S9 mix

- 2 µg/plate (without pre-incubation) or 1µg/plate (with pre-incubation) of 2-Anthramine (2AM): TA 1535, TA 1537, TA 98 and TA 100 strains,

- 2 µg/plate of Benzo(a)pyrene (BAP): TA 102 strain,

 

Results

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

Since the test item was toxic in the preliminary test, the choice of the highest dose-level was based on the level of toxicity, according to the criteria specified in the international guidelines.

Experiments without S9 mix

The selected treatment-levels were:

- 0, 0.5, 1.5, 5, 15 and 50 µg/plate for all the strains in the two experiments,

 

No precipitate was observed in the Petri plates when scoring the revertants at any dose-levels.

A moderate to strong toxicity was noted at dose-levels 50 µg/plate in all strains.

The test item did not induce any increase in the number of revertants, which could be considered as biologically relevant, in any of the five strains.

Experiments with S9 mix

The selected treatment-levels were:

- 0, 0.15, 0.5, 1.5, 5 and 15 µg/plate for all the strains in the two experiments,

 

No precipitate was observed in the Petri plates when scoring the revertants at any dose-levels.

A moderate to strong toxicity was noted at dose-levels15 µg/plate in all strains

The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains.

Conclusion

Under our experimental conditions, the test item N-[3-(dimethylamino)propyl]-C18 unsaturated-alkylamide (unsaturated C18) did not show any mutagenic activity in the bacterial reverse mutation test withSalmonella typhimurium.

Under the conditions of this study, the test substance was not considered to be mutagenic and as such, does not require classification according to Regulation EC No. 1272/2008 and Directive 67/548/EEC.