13-Docosenamide, N-[3-(dimethylamino)propyl]-, (13Z)-

Genetic toxicity: in vitro

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

Study period:

From 5 April 2005 to 19 December 2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was performed similarly to the OECD guideline 471 and is in compliance with the Japanese GLP. The test was performed on a very similar structure (saturated chain instead of an insaturated chain).

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Minister of Labor Standard based on the industrial safety and health Law Article 57-2 Paragraph 1 (Notification No. 77 of JMOL on September 1, 1988) and Notification on partial revision of the standard (Notification No. 67 of JMOL on June 2, 1997)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Remarks:

Minister of Labor Standard based on the industrial safety and health Law Article 34-3 Paragraph 2 (Notification No. 76 of JMOL on September 1, 1988) and Notification on partial revision of the standard (Notification No. 13 of JMOL on March 29, 2000)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Docosanamide,N-[3-(dimethylamino)propyl]-

IUPAC Name:

Docosanamide,N-[3-(dimethylamino)propyl]-

Constituent 2

Reference substance name:

N-[3-(dimethylamino)propyl]docosanamide

EC Number:

262-134-8

EC Name:

N-[3-(dimethylamino)propyl]docosanamide

Cas Number:

60270-33-9

Molecular formula:

C27H56N2O

Test material form:

other: white granular solid

Details on test material:

- Name of test material (as cited in study report):N-[3-(dimethylamino)propyl]docosanamide, other name: AMIDET APA-22
- Stability under test conditions: stable in room temperature
- Storage condition of test material: in room temperature, shield from light

Method

Target gene:

Histidine gene for S. typhimurium, tryphtophan locus for E. coli WP2 uvrA

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction prepared from rat liver homogenates that had been treated with phenobarbital and 5,6-benzoflavone. S9 fraction purchased from Kikkoman Corporation (Japan). Cofactor for S9 mix was purchased from Roche Diagnostics K.K. (lot 732).

Test concentrations with justification for top dose:

Preliminary test: 5; 20; 78; 313; 1250 and 5000 µg/plate
Main test: 39, 78, 156, 313, 625, 1250, 2500 and 5000 µg/plate in the absence of metabolic activation
313, 625, 1250, 2500 and 5000 µg/plate in the presence of metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: tetrahydrofuran
- Justification for choice of solvent/vehicle: In a solubility test, distilled water and three different organic solvents of dimethylsulfoxide (DMSO), acetone and tetrahydrofuran, were used. The concentration of the test susbstance when distilled water and DMSO were used was set at 50mg/mL, and the concentration was set at 100 mg/mL for acetone and 250 mg/mL for tetrahydrofuran. The test substance dissolved in tetrahydrofuran by the treatment of sonic waves. The test substance suspended in acetone, but uniform dispersion was not obtained though treatment by supersonic waves was done. On the other hand, the response of the test substance such as exothermic reaction was not recognized in any solvents. Therefore, the test substance was thought stable in these solvents. But the test substance could not disperse in distilled water and DMSO though the treatment by supersonic waves was done.
From these results, tetrahydrofuran was used as the solvent by the reason that it could obtain solution, and the test substance is judged stable. It was confirmed previously that the solvent did not cause bad influence to growth of the bacterial strains and metabolism of drug-metabolizing enzymes.

Controls

Untreated negative controls:

yes

Remarks:

sterility test with: S9 mix, 0.1M Phosphate buffer, nutrient broth No.2 and the test substance solution of the maximum concentration.

Negative solvent / vehicle controls:

yes

Remarks:

tetrahydrofuran

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

benzo(a)pyrene

other: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation (with and without metabolic activation)

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 hours

SELECTION AGENT (mutation assays): not applicable

NUMBER OF REPLICATIONS: 2 plates/dose, 2 independant experiments (named Preliminary and main study)

NUMBER OF CELLS EVALUATED: not applicable

DETERMINATION OF CYTOTOXICITY
- Method: toxic effect was examined with a stereoscopic microscope.

Evaluation criteria:

Negative and positive control data were compared to the laboratory historical control data (mean +/- 2SD).

Acceptance criteria:
when the experiment satisfies the following criteria, it was judged that the test result was obtained from the test of the appropriate procedure.
1) The growth of the contaminant in the test materials, such as Nutrient broth, the test substance solution of the highest concentration, S9 Mix, sodium phosphate buffer are not detected from the result of the sterility test.
2) the negative control values satisfy the acceptance criteria
3) the positive control values satisfy acceptance criteria. And the number of the revertant colonies of the positive control increases two-fold or more compared with the negative control.

Judgment of the test results:
the test substance was judged positive for mutagenic activity when clear dose-related increase in the number of the revertant colonies and two-fold or more increase in the number of revertant colonies compared with the negative control were observed with reappearance.

Statistics:

no statistics analysis was used for evaluation of the test results.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

See details in Tables 7.6.1/1 to 7.6.1/4

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In TA1535, TA1537, TA98 and TA100 in the absence of metabolic activation, see below for more details and in Tables 7.6.1/1 to 7.6.1/4.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: not applicabe
- Precipitation: in the preliminary test, the precipitation of the test substance was detected at the dose of 313 µg/plate or more in both the presence and absence of metabolic activation, but it did not have a bad influence on the observation of the revertant colonies. In the main test, precipitation of the test substance was detected in the absence of metabolic activation at the dose of 156 µg/plate or more and in the presence of metabolic activation at the dose of 313 µg/plate or more.
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES: the preliminary reverse mutation test was performed to determine the most favorable dose levels of the test substance in the reverse mutation test at each of the bacterial strain. Cytotoxicity was observed in S. typhimurium TA98; TA100, TA1535 and TA1537 in the absence of metabolic activation at the dose of 1250 µg/plate or more. Based on the foregoing results, the maximum dose of the main reverse mutation test was set at 1250 µg/plate and a total of six different dose levels with factor 2 from the maximum dose were employed. The maximum dose levels of the main test of the other bacterial strains were set at 5000 µg/plate and a total of five different dose levels with factor 2 from the maximum dose was employed as toxic effect of the test substance was not detected.

COMPARISON WITH HISTORICAL CONTROL DATA: the numbers of the revertant colonies of the negative control and the positive control were within the range of the standard value of the historical data in the laboratory. Furthemore, all positive controls give an two-fold increase or more in the number of the revertant colonies compared with the negaive control with all bacterial strains. The test can be considered therefore as valid.

ADDITIONAL INFORMATION ON CYTOTOXICITY: the test substance showed toxic effects in the strains TA1535 and TA1537 in the absence of the metabolic activation at the dose of 625 µg/plate or more. Similar toxic effects were observed in the strains TA98 and TA100 in the absence of metabolic activation at the dose of 1250 µg/plate or more.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 7.6.1/1:Number of revertants per plate in the absence of metabolic activation in the preliminary test

Test item Concentration (µg/plate)	TA 1535	TA1537	TA 98	TA 100	WP2 uvrA
	Mean$	Mean$	Mean$	Mean$	Mean$
0	10	9	25	129	22
5	8	8	22	132	23
20	10	7	22	134	27
78	9	10	30	130	24
313	8#	9#	26#	121#	20#
1250	6*#	3*#	7*#	56*#	21#
5000	5*#	3*#	11*#	62*#	21#
Positive control**	482	385	507	602	123

$: Mean of duplicate

*: Toxic effect of the test substance was observed

# : Precipitation was observed

**Mutagens positive controls:

- NaN₃(0.5 µg/plate) in TA1535 strain

- 9AA (80 µg/plate) in TA1537 strain

- AF-2 (0.01 µg/plate) in TA 100 and WP2 uvrA strains

- AF-2 (0.1 µg/plate) in TA 98 strain

 

 

Table 7.6.1/12: Number of revertants per plate in the presence of metabolic activation (S9 mix) in the preliminary test 

Test item Concentration (µg/plate)	TA 1535	TA1537	TA 98	TA 100	WP2 uvrA
	Mean$	Mean$	Mean$	Mean$	Mean$
0	11	10	31	142	25
5	9	9	34	141	26
20	9	9	35	153	25
78	9	7	32	145	20
313	10#	8#	30#	145#	25#
1250	8#	10#	30#	140#	21#
5000	10#	9#	30#	126#	20#
Positive control**	240	85	384	1068	941

$: Mean of duplicate

# : Precipitation was observed

**Mutagens positive controls:

- 2AA (2 µg/plate) in TA1535 strain

- 2AA (10 µg/plate) in WP2 uvrA strain

-B[a]P (5 µg/plate) in TA100; TA98 and TA1537 strains

 

Table 7.6.1/3: Number of revertants per plate in the absence of metabolic activation in the main test

Test item Concentration (µg/plate)	TA 1535	TA1537	TA 98	TA 100	WP2 uvrA
	Mean$	Mean$	Mean$	Mean$	Mean$
0	9	7	29	132	26
39	7	7	23	124	-
78	8	8	29	117	-
156	8#	7#	28#	137#	-
313	8#	7#	29#	116#	22#
625	7*#	6*#	27#	102#	26#
1250	2*#	2*#	22*#	71*#	25#
2500	-	-	-	-	23#
5000	-	-	-	-	23#
Positive control**	484	430	554	465	127

$: Mean of duplicate

*: Toxic effect of the test substance was observed

# : Precipitation was observed

**Mutagens positive controls:

- NaN₃(0.5 µg/plate) in TA1535 strain

- 9AA (80 µg/plate) in TA1537 strain

- AF-2 (0.01 µg/plate) in TA 100 and WP2 uvrA strains

- AF-2 (0.1 µg/plate) in TA 98 strain

 

Table 7.6.1/4: Number of revertants per plate in the presence of metabolic activation (S9 mix) in the main test

 

Test item Concentration (µg/plate)	TA 1535	TA1537	TA 98	TA 100	WP2 uvrA
	Mean$	Mean$	Mean$	Mean$	Mean$
0	9	7	28	139	25
313	10#	8#	32#	144#	25#
625	9#	8#	36#	142#	26#
1250	9#	9#	34#	146#	21#
2500	8#	7#	32#	137#	25#
5000	9#	7#	29#	140#	25#
Positive control**	220	91	334	1044	898

$: Mean of duplicate

# : Précipitation was observed

**Mutagens positive controls:

- 2AA (2 µg/plate) in TA1535 strain

- 2AA (10 µg/plate) in WP2 uvrA strain

- B[a]P (5µg/plate) in TA100, TA98 and TA1537 strains

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the test conditions, N-[3-(dimethylamino)propyl]docosanamide is not mutagenic in S. typhimurium and E. coli in the absence and presence of metabolic activation.

Executive summary:

In a reverse gene mutation assay in bacteria, performed similarly to the OECD No.471 guideline and in compliance with the Japanese GLP, N-[3-(dimethylamino)propyl]docosanamide (named also as Amidet APA-22) (purity of 99.1%) diluted in tetrahydrofuran was tested inS. typhimuriumTA1535, TA1537, TA100, TA98 strains and E. coli WP2 uvrA strain in the presence and the absence of mammalian metabolic activation (S9) using the preincubation method. Five known mutagens (Sodium azide (NaN3); 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2); 9-aminoacridine (9AA), 2-aminoanthracene (2AA) and benzoapyrene (B[a]P)) were used to check the sensitivity of the test system. They gave appropriate response, therefore the test was considered as valid.

In the preliminary assay the test item was used in the presence and absence of metabolic activation at the following concentrations: 0; 5; 20; 78; 313; 1250 and 5000 µg/plate (duplicate). Cytotoxicity was observed in S. typhimurium TA98; TA100, TA1535 and TA1537 in the absence of metabolic activation at the dose of 1250 µg/plate or more. The maximum dose of the main reverse mutation test was thus set at 1250 µg/plate and a total of six different dose levels with factor 2 from the maximum dose were employed. The maximum dose levels of the main test of the other bacterial strains were set at 5000 µg/plate and a total of five different dose levels with factor 2 from the maximum dose was employed as toxic effect of the test substance was not detected.

In the main assay the test item was used at the following concentration: 38; 78; 156; 313; 625 and 1250 in S. typhimurium TA100, TA1535, TA98 and TA1537 in the absence of metabolic activation; 313; 625; 1250; 2500 and 5000 µg/plate in E. coli WP2 uvrA and 313; 625; 1250; 2500 and 5000 µg/plate in all strains in the presence of metabolic activation (duplicate). The test substance showed toxic effects in the strains S. typhimurium TA1535 and TA1537 in the absence of the metabolic activation at the dose of 625 µg/plate or more. Similar toxic effects were observed in the strains TA98 and TA100 in the absence of metabolic activation at the dose of 1250 µg/plate or more.

A precipitation of the test substance was observed in both experiments but it did not have a bad influence on the observation of the revertant colonies.

In both experiments, there was no increase of the number of revertant colonies compared to the vehicle control. Therefore, under the test conditions, N-[3-(dimethylamino)propyl]docosanamide did not show any mutagenic activity in the bacterial reverse mutation test using Salmonella typhimurium and E. coli. This study is considered as acceptable as it satisfied the criteria of the OECD Guideline No. 471.