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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:
experimental study
Adequacy of study:
key study
Study period:
19-Mar-2003 to 11-Apr-2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to
Guideline:
other: Japanese Substance Control Law (JSCL) Test Guideline III.1 Gene Mutation Test with bacteria
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
semi-solid (amorphous): gel
Remarks:
migrated information: paste
Details on test material:
Name or number of compound: Genamin LAP 100 D
Formula of compound: R-NH-CH2-CH2-CH2-NH2 (R = Lauryl)
Chemical name: Laurylpropylendiamin
CAS-Register number: 5538-95-4
Molecular weight: 246 - 260 g/mol
Batch number: DEGE908461
Purity: approx. 100 %
Appearance: white wax
Storage conditions: at approximately 20 °C in a fume cupboard
Solubility in water: < 1 g/L
Solubility: solution in ethanol
Stability in the solvent: confirmed over 4 hours in ethanol

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: rfa uvrB (TA98 and TA100 also R)
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
S9 rat liver induced with Aroclor 1254
Test concentrations with justification for top dose:
(0.16), 0.5, 1.6, 5, 16, 50, 160, 500, 1600 and 5000 µg/plate
Vehicle / solvent:
vehicle: ethanol
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: sodium-azide, 9-aminoacridine, 2-nitrofluorene, 4-nitroquinoline-N-oxide and 2-aminoanthracene
Details on test system and experimental conditions:
TEST GROUPS
first plate incorporation test (10 % S9 rat liver homogenate):
a: with metabolic activation: 50, 160, 500, 1600 and 5000 µg/plate
b: without metabolic activation: 50, 160, 500, 1600 and 5000 µg/plate
second plate incorporation test (10 % S9 rat liver homogenate):
a: with metabolic activation: 0.5, 1.6, 5, 16, 50 and 160 µg/plate (TA 100, TA 1535, WP2uvrA)
0.16, 0.5, 1.6, 5, 16 and 50 µg/plate (TA 1537, TA 98)
b: without metabolic activation: 0.16, 0.5, 1.6, 5, 16, 50 µg/plate
third plate incorporation test (30 % S9 rat liver homogenate):
a: with metabolic activation: 0.5, 1.6, 5, 16, 50 and 160 µg/plate
b: without metabolic activation: 0.5, 1.6, 5, 16, 50 and 160 µg/plate

Control groups : negative controls:
a: untreated controls
b: solvent controls (0 µg/plate)
positive controls:
a: without metabolic activation: sodium-azide for strain TA 100 and TA 1535
9-aminoacridine for strain TA 1537
2-nitrofluorene for strain TA 98
4-nitroquinoline-N-oxide for strain WP2uvrA
b: with metabolic activation: 2-aminoanthracene for all strains

Formulation of test compound: dissolved in ethanol at appropriate concentrations immediately before use

Formulation of reference compounds: sodium-azide dissolved in deionized water final concentrations: 1.0 µg/plate for strain TA 1535
2.0 µg/plate for strain TA 100
9-aminoacridine dissolved in DMSO final concentration: 50.0 µg/plate for strain TA 1537
2-nitrofluorene dissolved in DMSO final concentration: 2.5 µg/plate for strain TA 98
4-nitroquinoline-N-oxide dissolved in DMSO final concentrations: 2.0 µg/plate (plate inc.) for strain WP2uvrA
2-aminoanthracene dissolved in DMSO final concentrations:
1.5 µg/plate for strains TA 98, TA 100, TA 1535 and TA 1537 (10 % S9-mix)
20.0 µg/plate (plate inc.) for strain WP2uvrA (10 % S9-mix)
1.5 µg/plate for strains TA 100, TA 98 (30 % S9-mix)
10.0 µg/plate for strains TA 1535 and TA 1537 (30 % S9-mix)
30.0 µg/plate for strain WP2uvrA (30 % S9-mix)
The frozen stock solutions of each compound were diluted progressively up to the final concentration on the day of treatment.

Source of bacteria: stock cultures in the bank of "Genetic Toxicology", Aventis Pharma Germany, ProTox; prepared from the original bacterial strains

TEST ORGANISM
Salmonella typhimurium strains: TA 98 hisD3052 rfa uvrB +R,
TA 100 hisG46 rfa uvrB +R,
TA 1535 hisG46 rfa uvrB,
TA 1537 hisC3076 rfa uvrB
Escherichia coli: WP2uvrA pKM101

Experimental conditions in vitro: approx. 37 °C in an incubator

PREPARATION AND STORAGE OF A LIVER HOMOGENATE FRACTION (S9)
The S9 fraction of Spraque Dawley rat liver induced with Aroclor 1254 was obtained by Molecular Toxicology, Inc., 1.57 Industrial Park Dr. Boone, NC 28607, (828) 264-9099. The protein content for every batch was guaranteed by a Quality Control & Production Certificate by the supplier. Also for every batch of S9 an independent validation was performed in the laboratory with a minimum of two different mutagens, e.g. 2-aminoanthracene and benzo(a)pyrene, to confirm metabolic activation by microsomal enzymes.

PREPARATION OF S9-MIX
Sufficient S9 fraction was thawed at room temperature immediately before each test. One volume of S9 fraction (batch no. Moltox 1455 for two plate incorporation tests, protein concentration 39.3 g/l) was mixed with 9 volumes of the S9 cofactor solution (10%, which was kept on ice until used), respectively three volumes of S9 fraction (batch no. Moltox 1455) was mixed with 7 volumes of the S9 cofactor solution (30%). This preparation is termed S9-mix. The concentrations of the different compounds in the S9-mix were:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
4 mM NADP
100 mM phosphate buffer pH 7.4

BACTERIA
The strains of Salmonella typhimurium were obtained from Professor B.N. Ames, University of California, U.S.A. The strain E. colt' was obtained from the E.coli Genetic Stock Center, Yale University, New Haven, U.S.A.
Bacteria were grown overnight in nutrient broth (25 g Oxoid Nutrient Broth No. 2 /liter) at approx. 37 °C. The amount of bacteria in the cell suspension was checked by nephelometry. Inoculation was performed with stock cultures, which had been stored in liquide nitrogen. Each new stock of the different bacterial strains was checked with regard to the respective biotin and histidine requirements, membrane permeability, ampicillin resistance, tetracyclin resistance, crystal violet sensitivity, UV resistance and response to diagnostic mutagens. All criteria for a valid assay were fulfilled.

ASSAY PROCEDURE
Each test was performed in both the presence and absence of S9-mix using all bacterial tester strains and a range of concentrations of the test substance. Positive and negative controls as well as solvent controls were included in each test. Triplicate plates were used.
The highest concentration in the first mutation experiment was 50 mg/ml of the test substance in the chosen solvent, which provided a final concentration of 5000 µg/plate. Further dilutions of 1600, 500, 160 and 50 µg/plate were also used. Dose levels used in the second respectively third experiment were based on findings, including toxicity, in the first respectively second experiment. Toxicity was assessed after microscopic thinning of the bacterial lawn andlor reduction (at least halving) of the number of spontaneously occurring mutants compared to the corresponding solvent control value.
In both tests top agar was prepared which, for the Salmonella strains, contained 100 ml agar (0.6 % (w/v) agar, 0.5 % (w/v) NaCl) with 10 ml of a 0.5 mM histidine-biotin solution. For E. coli histidine was replaced by tryptophan (2.5 ml, 2.0 mM).
The following ingredients were added (in the following order) to 2 ml of molten top agar at approx. 48 °C:
0.5 ml S9-mix (if required) or buffer
0.1 ml of an overnight nutrient broth culture of the bacterial tester strain
0.1 ml test compound solution (dissolved in ethanol)

After mixing, the liquid was poured into a petri dish containing a 25 ml layer of minimal agar (1.5 % (w/v) agar, Vogel-Bonner E medium with 2 % (wlv) glucose). After incubation for approximately 48 hours at approx. 37 °C in the dark, colonies (trp+ revertants) were counted by hand or by a suitable automatic colony counter. The counter was calibrated for each test by reading a test pattern plate to verify the manufacturer's requirements for sensitivity.
Evaluation criteria:
CRITERIA FOR A VALID ASSAY
The assay is considered valid if the following criteria are met:
- the solvent control data are within the laboratory's normal control range for the spontaneous mutant frequency
- the positive controls induce increases in the mutation frequency which are significant and within the laboratory's normal range

CRITERIA FOR A POSITIVE RESPONSE
A test compound is classified as mutagenic if it has either of the following effects:
a) it produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
b) it induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test compound at complete bacterial background lawn.
If the test substance does not achieve either of the above criteria, it is considered to show no evidence of mutagenic activity in this system.

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 / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
SOLUBILITY AND TOXICITY
Genamin LAP 100 D was in ethanol and a stock solution of 50 mg/ml was prepared for the highest concentration, which provided a final concentration of 5000 µg/plate. Further dilutions of 1600, 500, 160, 50, 16, 5, 1.6, 0.5, 0.16 µg/plate were used in the different experiments.
Visible precipitation of Genamin LAP 100 D on the plates was observed at 5000 µg/plate.
In the first plate incorporation test with 10 % S9-mix toxicity in bacteria was observed with metabolic activation in a dose range of 50 to 5000 µg/plate with the test strains TA 1537 and TA 98. In the other test strains toxicity in bacteria was observed with metabolic activation in a dose range of 160 to 5000 µg/plate. In the absence of metabolic activation the test compound proved to be toxic to all bacterial strains at concentrations of 50 µg/plate and above. As less than 5 doses were evaluable with all strains due to toxicity in bacteria a second test with lower doses was performed.

In the second plate incorporation test with 10 % S9-mix toxicity in bacteria was observed with metabolic activation at a dose level of 50 µg/plate with test strains TA 1535, TA 1537 and TA 98 and at a dose level of 160 µg/plate with TA 100 and WP2uvrA. In the absence of metabolic activation Genamin LAP 100 D proved to be toxic to bacterial strains TA 100, TA 1537 and TA 98 at concentrations of 5 µg/plate and above. With tester strain TA 1535 toxicity was observed in a dose range of 16 to 50 µg/plate and with the strain WP2uvrA at a concentration of 50 µg/plate.

In the plate incorporation test with 30 % S9-mix with metabolic activation toxicity in bacteria was observed at a concentration of 160 µg/plate with all Salmonella strains. In the absence of metabolic activation the test compound proved to be toxic to bacterial strain TA 100 at a concentration of 16 µg/plate and above. With the other tester strains toxicity was observed in a dose range of 50 to 160 µg/plate.
Thinning of bacterial lawns and/or a reduction in the number of colonies (at least halving) was observed at these dose levels.

MUTAGENICITY
In the independent mutation tests Genamin LAP 100 D was tested for mutagenicity with the same concentrations. The number of colonies per plate with each strain as well as mean values of 3 plates were given.
Genamin LAP 100 D did not cause a significant increase in the number of revertant colonies at any dose level with any of the tester strains either in the absence or in the presence of S9-mix in each mutation test. No dose-dependent effect was obtained.
All positive controls produced significant increases in the number of revertant colonies. Thus, the sensitivity of the assay and the efficacy of the exogenous metabolic activation system were demonstrated.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Genamin LAP 100 D was not found to be mutagenic in the bacterial reverse mutation test.
Executive summary:

N-Dodecyl-1,3-diaminepropanewas tested for mutagenicity with the strains TA 100, TA 1535, TA 1537 and TA 98 ofSalmonella typhimuriumand withEscherichia coliWP2uvrA.

Three independent mutagenicity studies were conducted (two plate incorporation tests with 10% S9-mix and one plate incorporation test with 30 % S9-mix), each in the absence and in the presence of a metabolizing system derived from a rat liver homogenate.

For all studies, N-Dodecyl-1,3-diaminepropane was dissolved in ethanol, and each bacterial strain was exposed to 5 dose levels in the first plate incorporation test with 10% S9-mix, to 6 dose levels in the second plate incorporation test with 10% S9-mix and also to 6 dose levels in the plate incorporation test with 30 % S9-mix.

Other common vehicles (deionized water, DMSO) were not appropriate due to insolubility of the test compound. As ethanol had to be chosen as vehicle, preincubation was not possible because of vehicle toxicity and therefore, a modified plate incorporation test with 30% S9-mix was performed as confirmatory assay.

The concentrations used for the first plate incorporation test (10% S9-mix) were 50, 160, 500, 1600 and 5000 µg/plate. Because of toxicity in bacteria with the concentrations used in the first experiment, dose levels from 0.16 to 50 µg/plate were chosen in the absence of metabolic activation for all tester strains in the second test. In the presence of metabolic activation dose ranges were variable in the second test to account for varying susceptibilities to toxic effects of the bacterial strains: low dose levels ranged from 0.16 to 50 µg/plate (TA 1537, TA98), and high dose levels ranged from 0.5 to 160 µg/plate (TA 100, TA 1535 and WP2uvrA).

For the plate incorporation test with 30% S9-mix dose levels from 0.5 to 160 µg/plate with and without metabolic activation were chosen with all strains.

Visible precipitation of N-Dodecyl-1,3-diaminepropane on the plates was observed at 5000 µg/plate.

The number of revertant colonies of the solvent controls in individual strains were slightly out of the historical control data range with ethanol. However the existing control data pool with ethanol is limited as this solvent is used rarely. When compared to the historical control data pool with DMSO all values are in the normal range indicating that the validity of the study is not influenced.

 

Toxicity was assessed after microscopic thinning of the bacterial lawn and / or reduction of the number of mutants compared to the corresponding solvent control (at least halving).

In the first plate incorporation test with 10% S9-mix, toxicity in bacteria was observed with metabolic activation in a dose range of 50 to 5000 µg/plate with the test strains TA 1537 and TA 98. In the other test strains toxicity was observed with metabolic activation in a dose range of 160 to 5000 µg/plate. In the absence of metabolic activation the test compound proved to be toxic to all bacterial strains at concentrations of 50 µg/plate and above. As less than 5 doses were evaluable with all strains due to toxicity in bacteria a second test with lower doses was performed.

In the second plate incorporation test with 10 % S9-mix toxicity in bacteria was observed with metabolic activation at a dose level of 50 µg/plate with test strains TA 1535, TA 1537 and TA 98 and at a dose level of 160 µg/plate with TA 100 and WP2uvrA. In the absence of metabolic activation N-Dodecyl-1,3-diaminepropane proved to be toxic to bacterial strains TA 100, TA 1537 and TA 98 at concentrations of 5 µg/plate and above. With tester strain TA 1535 toxicity was observed in a dose range of 16 to 50 µg/plate and with the strain WP2uvrA at a concentration of 50 µg/plate.

In the plate incorporation test with 30% S9-mix with metabolic activation toxicity in bacteria was observed at a concentration of 160 µ/plate with allSalmonella strains.In the absence of metabolic activation the test compound proved to be toxic to bacterial strain TA 100 at a concentration of 16 µg/plate and above. With the other tester strains toxicity in bacteria was observed in a dose range of 50 to 160 µg/plate.

 

In the presence and in the absence of the metabolic activation systemN-Dodecyl-1,3-diaminepropanedid not result in relevant increases in the number of revertants in any of the bacterial strains.

Summarizing, it can be stated thatN-Dodecyl-1,3-diaminepropane is not mutagenicin this bacterial mutation test at any dose level either in the absence or presence of an exogenous metabolic activation system.