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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:
30 March 2017 to 13 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: 'Standards for Mutagenicity Tests using Microorganisms' (Notification No. 77, September 1, 1988 & partial revision: Notification No. 67, June 2, 1997, Ministry of Labour, Japan and Notification No. 120, December 25, 2000, Ministry of Labour, Japan)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: "JAPAN: "Guidelines for Toxicity Testing of New Chemical Substances"
Version / remarks:
(Notification No. 7 of 0331 Pharmaceutical and Food safety Bureau, MInistry of Health, Labour and Welfare, No. 5 of the Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry, and No. 110331009 of Environmental Policy Bureau, Ministry of the Environment, Japan, 31 Macrh 2011)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
3-[ethyl[4-[(4-nitrophenyl)azo]phenyl]amino]propiononitrile
EC Number:
250-654-8
EC Name:
3-[ethyl[4-[(4-nitrophenyl)azo]phenyl]amino]propiononitrile
Cas Number:
31482-56-1
Molecular formula:
C17H17N5O2
IUPAC Name:
3-[ethyl[4-[(4-nitrophenyl)azo]phenyl]amino]propiononitrile
Test material form:
solid: particulate/powder

Method

Target gene:
Salmonella typhimurium
TA1537 (frame shift mutations)
TA98 (frame shift mutations)
TA1535 (base-pair mutations)
TA100 (base-pair mutations)

Escherichia coli
WP2 uvrA (base-pair substitution)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Preliminary test - 1.2, 4.9, 20, 78, 313, 1250 and 5000 µg/plate.

In the preliminary test, the growth inhibition by the test material was not observed in any strains either with or without metabolic activation. An increase in the number of revertant colonies more than twice that of the negative control was observed in S. typhimurium TA strains without metabolic activation and all strains with metabolic activation. Test material precipitate was observed at 5000 µg/plate both with and without metabolic activation.

5000 µg/plate was selected as the highest dose in the main tests for S. typhimurium TA 1535 without metabolic activation and E. coli WP2 uvraA both with and without metabolic activation. This dose was diluted four times (using a common ratio of 2) to provide a total of five doses. 5000 µg/plate was selected as the highest dose for S. typhimurium TA100 and TA98 without metabolic activation and diluted 8 times (using a common ratio of 2) to provide a total of 9 doses. 5000 µg/plate was also selected for S. typhimurium TA1537 without metabolic activation and S. typhimurium TA100, TA1535 and TA98 with metabolic activation and diluted 10 times (using a common ration of 2) to provide a total of 11 doses.

Doses tested are detailed in the results section.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: N,N-dimethylformamide (DMF)
- Justification for choice of solvent/vehicle: the test material could be suspended in DMF at 100 mg/mL and dissolved at 50 mg/mL.
Controls
Untreated negative controls:
yes
Remarks:
DMF
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
furylfuramide
other: 2-aminoanthracene and 2-methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine·2HCl
Details on test system and experimental conditions:
TEST PROCEDURES
For the tests without metabolic activation, 0.5 mL of 0.1 M Na-phosphate buffer (pH 7.4) and 0.1 mL of each fresh bacterial culture were added to each tube containing 0.05 mL of the test solution or the negative control solution. For the tests with metabolic activation, 0.5 mL of the S9 mix was added to each tube instead of the buffer. The mixture was pre-incubated in a water bath at 37°C for 20 minutes while shaking horizontally and then 2 mL of top agar was added to the mixture and the contents of each tube were poured over the surface of the minimal glucose agar plate. The same test with 0.1 mL of the positive control solution was also performed.

One minimal glucose agar plate was used for each dose level in the preliminary test and three minimal glucose agar plates were used for each dose level in the two main tests which were performed at the same doses.

For the sterility test, 0.05 mL of the test solution of the maximum concentration and 0.5 mL of the S9 mix were put into each tube, 2 mL of top agar was then added to the tube and the contents of each tube were poured over the surface of the minimal glucose agar plates.

As top agar, the 0.5 mM biotin / 0.5 mM L-histidine solution and the 0.05 mM L-tryptophan solution were added to the soft agar solution (0.6% agar and 0.5% NaCl) by volume of 1/10 for the S. typhimurium TA strains and the E. coli strain, respectively.

All plates were incubated at 37°C for 48 hours and the number of revertant colonies was counted. Afterwards, growth inhibition of the test strains was checked using a stereoscopic microscope.

COUNTING PROCEDURES
The number of revertant colonies was counted visually due to the precipitate of the test material on the plates. The revertant colonies of the positive controls were counted with a colony counter.
Evaluation criteria:
In the main tests, if the number of revertant colonies on the test plates increased significantly in comparison with that on the control plates (based on twice as many as the negative control), and dose-response and reproducibility were also observed, the test material was to be judged positive. The results at each concentration were demonstrated with the mean and standard deviation.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In the main tests, a dose-dependent increase in the number of revertant colonies, more than twice that of the negative control, and reproducibility were observed in S. typhimurium TA100, TA98 and TA1537 without metabolic activation and all strains with metabolic activation. An increase in the number of revertant colonies was observed in S. typhimurium TA5135 without metabolic activation and was more than twice that of the negative control in the first main test.

The revertant colonies of the positive controls showed an increase of more than twice that of the negative controls and they were within the appropriate limits of the historical control data indicating that the study was valid.

The test material was considered to be positive for mutagenicity.

The growth inhibition of the test strains by the test material was not observed. Test material precipitate on plates was observed at doses of 2500 µg/plate and higher both with and without metabolic activation.

No growth of the bacteria was observed in the sterility test.

Any other information on results incl. tables

Preliminary test results

   Number of revertant colonies/plate            
 With or without S9 mix  Dose (µg/plate)  TA100  TA1535  WP2 uvr A  TA98  TA1537
 -S9 mix  Solvent control  107  8  22  30  8
   1.2  109  6  23  24  6
   4.9  118  8  29  38  13
   20  137  9  26  28  23
   78  190  11  29  63  39
   313  245  13  20  139  90
   1250  315  13  21  313  156
   5000*  444  20  18  715  247
 +S9 mix  Solvent control  135  16  34  35  19
   1.2  115  16  29  43  15
   4.9  155  13  28  53  14
   20  258  44  21  278  23
   78  295  45  31  371  66
   313  386  43  49  631  210
   1250  856  38  108  1321  1000
   5000*  1405  64  172  1913  1127
 Positive control without S9 mix  Name   (Dose, µg/plate)  AF-2 (0.01)  NaN3  (0.5)  AF-2 (0.01)  AF-2 (0.1)  ICR-191 (1.0)
   Number of colonies/plate  526 464 171  509 1176 
  Name (Dose, µg/plate)  B[α]P (5.0)   2AA (2.0)  2AA (10.0)  B[α]P (5.0)  B[α]P (5.0)
   Number of colonies/plate  779  340  409  226  57

AF-2: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide

NaN3: sodium azide

ICR-191: 2-methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylaminoacridine · 2HCl

B[α]P: Benzo [α] pyrene

2AA: 2-aminoanthracene

* the precipitate of the test material on the plates was observed

NT = not tested

First main test results

   Number of revertant colonies/plate            
 With or without S9 mix  Dose (µg/plate)  TA100  TA1535  WP2 uvr A  TA98  TA1537
 -S9 mix  Solvent control  111 ± 5.0  13 ± 2.0  29 ± 2.5  34 ± 2.3  8 ± 2.0
   4.9  NT  NT  NT  NT  11 ± 1.2
   10  NT  NT  NT  NT  16 ± 3.5
   20  124 ± 17.8  NT  NT  50 ± 9.0  20 ± 3.0
   39  142 ± 8.1  NT  NT  82 ± 9.3  36 ± 6.9
   78  162 ± 5.1  NT  NT  113 ± 2.9  49 ± 5.1
   156  199 ± 21.3  NT  NT  172 ± 21.7  76 ± 6.1
   313  237 ± 25.4  17 ± 0.6  23 ± 4.6  249 ± 18.0  96 ± 11.0
   625  297 ± 10.2  20 ± 2.0  26 ± 1.5  299 ± 12.1  138 ± 8.7
   1250  321 ± 3.5  24 ± 2.5  24 ± 1.5  442 ± 22.5  172 ± 8.9
   2500*  394 ± 26.4  20 ± 1.7  27 ± 3.5  699 ± 25.8  260 ± 28.6
   5000*  523 ± 18.8  27 ± 1.0  25 ± 2.3  1008 ± 28.8  318 ± 16.8
 Positive control without S9 mix  Name  AF-2  NaN3  AF-2  AF-2  ICR-191
   Dose (µg/plate)  0.01  0.5  0.01  0.1  1.0
   Number of colonies/plate  537 ± 12.9  426 ± 17.0  136 ± 10.4  536 ± 16.6  1295 ± 73.5
 +S9 mix  Solvent control  124 ± 10.2  14 ± 2.9  34 ± 4.0  40 ± 3.1  17 ± 2.1
   4.9  142 ± 9.0  14 ± 2.5  NT  58 ± 1.7  NT
   10  211 ± 6.2  20 ± 3.0  NT  178 ± 24.3  NT
   20  287 ± 14.4  38 ± 4.6  NT  313 ± 23.1  26 ± 2.1
   39  331 ± 29.3  48 ± 4.9  NT  399 ± 8.7  35 ± 3.6
   78  315 ± 18.9  50 ± 3.5  NT  421 ± 21.7  64 ± 3.2
   156  388 ± 13.0  46 ± 5.7  NT  578 ± 41.9  118 ± 10.0
   313  452 ± 8.2  47 ± 4.6  46 ± 5.6  699 ± 19.1  229 ± 10.1
   625  656 ± 41.1  37 ± 3.6  79 ± 5.0  1191 ± 120.4  465 ± 34.2
   1250  983 ± 22.9  47 ± 10.4  130 ± 12.2  1521 ± 74.6  847 ± 72.1
   2500*  1465 ± 53.9  53 ± 6.0  167 ± 4.0  1861 ± 28.0  1217 ± 65.7
   5000*  1591 ± 61.3  55 ± 2.6  192 ± 4.2  2149 ± 134.0  1221 ± 72.2
 Positive control with S9 mix  Name  B[α]P  2AA  2AA  B[α]P  B[α]P
   Dose (µg/plate)  5.0  2.0  10.0  5.0  5.0
   Number of colonies/plate  878 ± 64.1  318 ± 18.1  416 ± 44.1  237 ± 7.8  72 ± 8.7

AF-2: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide

NaN3: sodium azide

ICR-191: 2-methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylaminoacridine·2HCl

B[α]P: Benzo [α] pyrene

2AA: 2-aminoanthracene

* the precipitate of the test material on the plates was observed

NT = not tested

Second main test results

     Number of revertant colonies/plate        
 With or without S9 mix  Dose (µg/plate)  TA100  TA5135  WP2 uvr A  TA98  TA1537
 -S9 mix  Solvent control  96 ± 13.5  14 ± 0.0  22 ± 2.3  24 ± 3.2  8 ± 1.5
   4.9  NT  NT  NT  NT  13 ± 3.2
   10  NT  NT  NT  NT  18 ± 2.1
   20  115 ± 5.0  NT  NT  43 ± 3.1  22 ± 2.0
   39  125 ± 7.2  NT  NT  60 ± 2.1  28 ± 2.6
   78  145 ± 8.4  NT  NT  83 ± 11.1  41 ± 3.1
   156  176 ± 15.4  NT  NT  114 ± 9.8  83 ± 7.4
   313  230 ± 14.8  13 ± 2.1  22 ± 2.1  167 ± 28.6  93 ± 14.7
   625  252 ± 17.1  13 ± 2.3  20 ± 0.6  246 ± 22.2  123 ± 16.0
   1250  268 ± 24.9  18 ± 3.2  21 ± 1.7  331 ± 5.5  181 ± 23.6
   2500*  340 ± 22.1  20 ± 4.5  26 ± 2.6  468 ± 10.7  216 ± 15.3
   5000*  438 ± 22.0  22 ± 2.1  25 ± 1.2  731 ± 26.5  307 ± 23.2
 Positive control without S9 mix  Name  AF-2  NaN3  AF-2  AF-2  ICR-191
   Dose (µg/plate)  0.01  0.5  0.01  0.1  1.0
   Number of colonies/plate  531 ± 17.1  425 ± 9.6  128 ± 19.8  516 ± 4.6  1408 ± 241.3
 +S9 mix  Solvent control  118 ± 11.0  13 ± 1.0  3.7 ± 3.1  39 ± 2.3  14 ± 0.6
   4.9  123 ± 15.3  17 ± 1.2  NT  67 ± 9.5  NT
   10  185 ± 1.2  23 ± 4.9  NT  191 ± 7.8  NT
   20  253 ± 22.2  32 ± 3.0  NT  263 ± 25.0  25 ± 3.6
   39  248 ± 9.2  46 ± 4.2  NT  301 ± 18.3  38 ± 5.7
   78  274 ± 17.4  41 ± 5.1  NT  357 ± 48.7  62 ± 8.0
   156  294 ± 9.0  40 ± 5.5  NT  502 ± 34.6  116 ± 3.2
   313  391 ± 13.5  44 ± 6.6  51 ± 4.2  678 ± 34.0  236 ± 14.2
   625  501 ± 3.6  39 ± 2.3  76 ± 2.3  905 ± 16.9  517 ± 28.1
   1250  849 ± 37.3  51 ± 4.0  110 ± 0.6  1360 ± 175  886 ± 54.3
   2500*  1211 ± 45.4  54 ± 3.1  185 ± 9.7  1587 ± 47.9  1077 ± 27.1
   5000*  1420 ± 2.0  61 ± 9.0  212 ± 21.5  2160 ± 160.5  1101 ± 26.4
 Positive control with S9 mix  Name  B[α]P  2AA  2AA  B[α]P  B[α]P
   Dose (µg/plate)  5.0  2.0  10.0  5.0  5.0
   Number of colonies/plate  787 ± 51.4  288 ± 24.9  338 ± 41.6  189 ± 13.0  60 ± 6.7

AF-2: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide

NaN3: sodium azide

ICR-191: 2-methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylaminoacridine·2HCl

B[α]P: Benzo [α] pyrene

2AA: 2-aminoanthracene

* the precipitate of the test material on the plates was observed

NT = not tested

Applicant's summary and conclusion

Conclusions:
Under the conditions of the study, the test material was determined to be mutagenic in the bacterial reverse mutation assay.
Executive summary:

The mutagenicity potential of the test material was assessed with Salmonella typhimurium TA100, TA1535, TA98, TA1537 and Escherichia coli WPA uvrA following the standardised guidelines OECD 471, the Japanese Standards for Mutagenicity Tests using Microorganisms, and the Japanese Guidelines for Toxicity Testing of New Chemical Substances. The study was conducted under GLP conditions.

In the two main tests, a dose-dependent increase in the number of revertant colonies, more than twice that of the negative control, and reproducibility was observed in S. typhimurium TA100, TA98 and TA5137 without metabolic activation and in all strains with metabolic activation. It is concluded that the test material is mutagenic to bacteria under the study conditions.