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Genetic toxicity: in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Experimental data of read across substances
Justification for type of information:
Weight of evidence approach based on structurally similar chemicals
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
read-across source
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from study report
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
other: "Commission Directive 2000/32/EC, L 1362000, Annex 40", dated May 19,2000.
Principles of method if other than guideline:
The experiments were performed to assess the potential of the test item to induce gene mutations by means of two independent Salmonella typhimurium reverse mutation assays.
Experiment I was performed as a plate incorporation assay. Since a negative result was obtained in this experiment, experiment II was performed as a pre-incubation assay.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
The histidine dependent strains are derived from S. typhimurium strain LT2 through a mutation in the histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation (deletion of the uvrB gene) causes an inactivation of the excision repair system. The latter alteration also includes a deletion in the nitrate reductase and biotin genes. In the strains TA 98, TA 100, and TA 102 the R-factor plasmid pKM 101 carries umu DC analogous genes that are involved in error-prone repair and the ampicillin resistance marker. The strain TA 102 does not contain the uvrB--mutation and is excision repair proficient. Additionally, TA 102 contains the multicopy plasmid pAQ1 carrying the hisG428 mutation (ochre mutation in the hisG gene ) and a tetracycline resistance gene (5).
In summary, the mutations of the TA strains used in this study can be described as follows:
Salmonella typhimurium
Strains Genotype Type of mutations indicated
TA 1537 his C 3076; rfa-; uvrB-: frame shift mutations
TA 98 his D 3052; rfa-; uvrB-;R-factor " "
TA 1535 his G 46; rfa-; uvrB-: base-pair substitutions
TA 102 his G 428; rfa-; uvrB+;R-factor " "
TA 100 his G 46; rfa-; uvrB-;R-factor " "
Regular checking of the properties of the strains regarding the membrane permeability,ampicillin- and tetracycline resistance as well as spontaneous mutation rates is performed in the laboratory of RCC Cytotest Cell Research according to B. Ames et al.and D.Maron and B. Ames.In this way it was ensured that the experimental conditions set down by Ames were fulfilled.
The bacterial strains TA 1535, TA 1537 TA 98, TA 100 and TA 102 were obtained from Trinova Biochem GmbH (35394 Gießen, Germany).
Species / strain / cell type:
S. typhimurium TA 1535
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 1537
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
liver microsomal activation
Test concentrations with justification for top dose:
In the pre-experiment the concentration range of the test item was 3 – 5000 μg/plate. The pre-experiment is reported as part of experiment I. Since the criteria mentioned above were met 5000 μg/plate were chosen as maximal concentration.
The concentration range included two logarithmic decades. The following concentrations were tested:

Pre-Experiment: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment I: 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:
With metabolic activation
Strains: TA 1535, TA 1537, TA 98, TA 100, TA 102 - DMSO

Without metabolic activation
Strains: TA 1535, TA 100 - water deionised
Strains: TA 1537, TA 98 - DMSO
Strain: TA 102 - water deionised
- Justification for choice of solvent/vehicle:The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Remarks:
Concentration: 10 μg/plate
Positive control substance:
sodium azide
Remarks:
For Strains: TA 1535, TA 100 without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Remarks:
Concentration: 10 μg/plate in TA 98, 50 μg/plate in TA 1537
Positive control substance:
other: 4-nitro-o-phenylene-diamine, 4-NOPD
Remarks:
For Strains: TA 1537, TA 98 without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Remarks:
Concentration: 4.0 μL/plate
Positive control substance:
methylmethanesulfonate
Remarks:
For Strain: TA 102 without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Remarks:
Concentration: 2.5 μg/plate (10.0 μg/plate in TA 102)
Positive control substance:
other: 2-aminoanthracene, 2-AA
Remarks:
For Strains: TA 1535, TA 1537, TA 98, TA 100, TA 102 with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

Storage
The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO (MERCK, D-64293 Darmstadt) in liquid nitrogen.

Precultures
From the thawed ampoules of the strains 0.5 mL bacterial suspension was transferred into 250 mL Erlenmeyer flasks containing 20 mL nutrient medium. A solution of 20 μL ampicillin (25 μg/mL) was added to the strains TA 98, TA 100, and TA 102. Additionally 20 μL tetracycline (2 μg/mL) was added to strain TA 102. This nutrient medium contains per litre:
8 g Merck Nutrient Broth (MERCK, D-64293 Darmstadt)
5 g NaCl (MERCK, D-64293 Darmstadt)
The bacterial cultures were incubated in a shaking water bath for 4 hours at 37° C.

Selective Agar
The plates with the minimal agar were obtained from E. Merck, D-64293 Darmstadt.

Overlay Agar
The overlay agar contains per litre:
6.0 g MERCK Agar Agar*
6.0 g NaCl*
10.5 mg L-Histidine x HCl x H2O*
12.2 mg Biotin*
* (MERCK, D-64293 Darmstadt)

Sterilisations were performed at 121° C in an autoclave.

Experimental Performance
For each strain and dose level, including the controls three plates were used.
The following materials were mixed in a test tube and poured onto the selective agar plates:
- 100 μL Test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control),
- 500 μL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
- 100 μL Bacteria suspension (cf. test system, pre-culture of the strains),
- 2000 μL Overlay agar
In the pre-incubation assay 100 μL test solution, 500 μL S9 mix / S9 mix substitution buffer and 100 μL bacterial suspension were mixed in a test tube and incubated at 37°C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45° C) was added to each tube. The mixture was poured on minimal agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 37° C in the dark .
Evaluation criteria:
The Salmonella typhimurium reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce a significant increase in mutant colony frequencies

Evaluation of Results
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100, and TA 102.
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test item C 008 was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102.
The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls, were tested in triplicate. The
test item was tested at the following concentrations:

Pre-Experiment: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment I: 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate

Reduced background growth was observed with and without S9 mix in strains TA 98 and TA 100 in experiment I. In experiment II, reduced background growth was observed with and without S9 mix in all strains used (cf. tables of results).
Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5) were observed at the following concentrations (μg/plate):see below table.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with C 008 at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance with the exception of strain TA 98 in the absence of metabolic activation in experiment I. This strain showed a minor increase in revertant colony numbers at 1000 μg/plate. However, the required threshold of two times the number of the corresponding solvent control only slightly exceeded (indication factor of 2.2). To verify the results of this experiment an independent repeat experiment was performed under identical conditions with strain 98 in the absence of metabolic activation. No increase in the number of revertant colonies occurred in the repeat experiment and the effect observed in the first experiment was judged as biologically irrelevant. The results of the repeat experiment are reported as experiment I A. Also in experiment II strain TA 98 showed a minor increase in the absence of metabolic activation. The number of colonies reached the threshold of twice the number of the corresponding solvent control at concentrations as low as 100 μg/plate. Since the factor is just reached an did not show a dose dependent increase this effect is judged as biological irrelevant.
Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.
The number of colonies exceeded the laboratory´s historical control range slighty in the solvent control of strain TA 102 without metabolic activation in experiment I. Since this
deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies and has no impact on the outcome of the study.
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Remarks on result:
other: The test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Strain

Experiment I and I A

Experiment II

 

without S9 mix

with S9 mix

without S9 mix

with S9 mix

TA 1535

5000

/

/

/

TA 1537

5000

5000

5000

5000

TA 98

5000 (I)

/ (IA)

5000

1000 - 5000

5000

TA 100

2500, 5000

5000

1000 - 5000

5000

TA 102

2500, 5000

5000

2500, 5000

5000

/ no toxic effects observed

HISTORICAL CONTROL DATA

Strain

 

 

without S9 mix

 

 

with S9 mix

 

 

 

Mean

SD

Min

Max

Mean

SD

Min

Max

 

Solvent control

20

6

9

30

18

10

7

39

TA 1535

Negative control

18

5

10

29

18

10

9

38

 

Positive control

3042

756

1003

3618

357

111

172

476

 

Solvent control

11

7

4

29

18

9

6

31

TA1537

Negative control

12

6

5

29

18

6

8

29

 

Positive control

97

21

52

191

141

47

94

380

 

Solvent control

24

9

14

58

37

13

21

57

TA 98

Negative control

26

10

15

52

43

15

17

64

 

Positive control

379

98

137

976

1239

510

229

2566

 

Solvent control

121

29

91

198

149

36

109

281

TA 100

Negative control

141

23

101

189

147

43

103

254

 

Positive control

2089

408

1262

2872

921

346

546

2589

 

Solvent control

338

77

242

430

426

70

332

514

TA 102

Negative control

326

53

242

390

450

60

280

556

 

Positive control

2764

1479

1220

5593

2104

752

872

3052

 

 

 

 

 

 

 

 

 

Mean = mean value of revertants/plate

 

 

 

 

 

 

SD = standard deviation

Min = minimal value/Max = maximal value

Summary of Results Pre-Experiment

 

Metabolic

Test

Dose Level

Revertant Colony Counts (Mean ±SD)

Activation

Group

(µg/plate)

 

 

 

 

 

 

 

TA 98

TA 100

Without

DMSO

 

19

± 10

115 ± 23

Activation

Untreated

 

28

± 6

111 ± 35

 

C 008

3 µg

23

± 4

77

± 7

 

 

10 µg

22

± 7

86

± 5

 

 

33 µg

31

± 6

93

± 10

 

 

100 µg

27

± 6 D

122 ± 26 D

 

 

333 µg

30

± 5 D

95

± 5 D

 

 

1000 µg

42

± 3 D

81

± 17 D

 

 

2500 µg

35

± 5 D

35

± 6 D M R

 

 

5000 µg

7 ± 2 D M R

7 ± 3 D M R

 

4-NOPD

10 µg

259 ± 20

 

 

 

NaN3

10 µg

 

 

1950 ± 318

 

With

DMSO

 

38 ± 4

114

± 10

Activation

Untreated

 

37 ± 5

117

± 5

 

C 008

3 µg

38

± 11

85

± 20

 

 

10 µg

36

± 6

88

± 12

 

 

33 µg

41

± 8

88

± 11

 

 

100 µg

43

± 3 D

109

± 8 D

 

 

333 µg

34

± 4 D R

77

± 22 D

 

 

1000 µg

57

± 7 D M R

78

± 23 D

 

 

2500 µg

60

± 8 D P M

61

± 3 D M R

 

 

5000 µg

12

± 2 D M R

31

± 4 D M R

 

 

2-AA

2.5 µg

1963 ± 143

1710 ± 142

Key to Positive Controls

 

Key to Plate Postfix Codes

NaN3

sodium azide

 

D

Densely coloured plate

2-AA

2-aminoanthracene

 

M

Manual count

4-NOPD

4-nitro-o-phenylene-diamine

R

Reduced background growth

 

 

 

P

Precipitate

Summary of Results Experiment I A

Metabolic

Test

Dose Level

Revertant Colony Counts (Mean ±SD)

Activation

Group

(µg/plate)

 

 

 

 

 

TA 98

Without

Deionised water

 

30

± 9

Activation

Untreated

 

29

± 9

 

C 008

3 µg

26

± 8

 

 

10 µg

29

± 3

 

 

33 µg

19

± 7

 

 

100 µg

27

± 1 D

 

 

333 µg

26

± 4 D

 

 

1000 µg

52

± 9 D

 

 

2500 µg

44

± 3 D

 

 

5000 µg

14

± 3 D

 

4-NOPD

10 µg

332 ± 12

Key to Positive Controls

Key to Plate Postfix Codes

4-NOPD 4-nitro-o-phenylene-diamine

D

Densely coloured plate

Conclusions:
Test item did not induce gene mutations by base pair changes or frameshifts in the genome of the Salmonella typhimurium strains used.
Executive summary:

This study was performed to investigate the potential of test chemical to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98,TA 100, and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation (S9 mix). Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Experiment I: 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate

Reduced background growth was observed with and without S9 mix in strains TA 98 and TA 100 in experiment I. In experiment II, reduced background growth was observed with and without S9 mix in all strains used (cf. tables of results).

Toxic effects, evident as a reduction in the number of revertants, were observed at higher concentrations with and without metabolic activation in nearly all strains used. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with test chemical at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. It can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, test chemical is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.
Reason / purpose for cross-reference:
read-across source
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Justification for type of information:
Data is from secondary source
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
In virto gene toxicity study of test chemical in Salmonella typhimurium
GLP compliance:
not specified
Type of assay:
bacterial gene mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Liver S9-fraction from phenobarbital/β-naphthoflavone-induced rats was used as exogenous metabolic activation system.
Test concentrations with justification for top dose:
experiment I: 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate without and with S9-mix
experiment II: 33, 100, 333, 1000, 2500 and 5000 μg/plate without and with S9-mix
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: in accordance with the OECD guideline
Details on test system and experimental conditions:
Details on test system and conditions
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk
Experiment I was performed according to the direct plate incorporation test, experiment II with the pre-incubation method.

DURATION
Preincubation period: 60 minutes
Exposure duration: 48 hours incubation with and without S9 mix

NUMBER OF REPLICATIONS: triplicates in 2 individual experiments

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
Method: mitotic index; cloning efficiency; relative total growth; other: No data


OTHER EXAMINATIONS:
Determination of polyploidy: No data
Determination of endoreplication: No data

Other:
experiment I: direct plate incorporation with 48 h incubation without and with S9-mix
experiment II: pre-incubation method with 60 minutes pre-incubation and at least 48 h incubation without and with S9-mix

Test concentrations were based on the results of a pre-experiment with strains TA98 and TA100 for toxicity and mutation induction both without and with S9-mix. Toxicity was evaluated for 8 concentrations up to the prescribed maximum concentration of 5000 μg/plate on the basis of a reduction in the number of revertant colonies and/or clearing of the bacterial background lawn. Because
relevant toxic effects were not observed in any of the strains at the maximal concentration, 5000 μg/plate was used as the top concentration. Since in this pre-experiment evaluable plates were obtained for five concentrations or more in the strains used, the pre-experiment is reported in experiment I.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
Test concentrations were based on the results of a pre-experiment with strains TA98 and TA100 for toxicity and mutation induction both without and with S9-mix. Toxicity was evaluated for 8 concentrations up to the prescribed maximum concentration of 5000 μg/plate on the basis of a reduction in the number of revertant colonies and/or clearing of the bacterial background lawn. Because relevant toxic effects were not observed in any of the strains at the maximal concentration, 5000 μg/plate was used as the top concentration.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Test chemical was investigated for the induction of gene mutations in strains of Salmonella typhimurium (Ames test).

In the main tests toxic effects evident as clearing of the bacterial background lawn were observed in experiment I in TA98 and TA100 and in experiment II in all strains predominantly at higher concentrations. Toxic effects evident as a reduction in the number of revertants were observed at higher concentrations without and with metabolic activation in nearly all strains tested. A biologically relevant increase in revertant colonies was not observed in any of the strains tested at any dose level in the absence or presence of S9-mix in both experiments.

Under the experimental conditions used, test chemical was not mutagenic in this gene mutation tests in bacteria both in the absence and the presence of S9 metabolic activation
Executive summary:

Test chemcail was investigated for the induction of gene mutations in strains of Salmonella typhimurium(Ames test).

Salmonella typhimuriumTA98, TA100, TA102, TA1535 and TA1537 were used for the AMES assay. DMSO was used as a vehicle. Concentration: experiment I: 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate without and with S9-mix experiment II: 33, 100, 333, 1000, 2500 and 5000 μg/plate without and with S9-mix

Treatment: experiment I: direct plate incorporation with 48 h incubation without and with S9-mix experiment II: pre-incubation method with 60 minutes pre-incubation and at least 48 h incubation without and with S9-mix. Liver S9-fraction from phenobarbital/β-naphthoflavone-induced rats was used as exogenous metabolic activation system. Test concentrations were based on the results of a pre-experiment with strains TA98 and TA100 for toxicity and mutation induction both without and with S9-mix. Toxicity was evaluated for 8 concentrations up to the prescribed maximum concentration of 5000 μg/plate on the basis of a reduction in the number of revertant colonies and/or clearing of the bacterial background lawn. Because relevant toxic effects were not observed in any of the strains at the maximal concentration, 5000 μg/plate was used as the top concentration. Since in this pre-experiment evaluable plates were obtained for five concentrations or more in the strains used, the pre-experiment is reported in experiment I. Experiment I was performed according to the direct plate incorporation test, experiment II with the pre-incubation method. Negative and positive controls were in accordance with the OECD guideline.

In the main tests toxic effects evident as clearing of the bacterial background lawn were observed in experiment I in TA98 and TA100 and in experiment II in all strains predominantly at higher concentrations. Toxic effects evident as a reduction in the number of revertants were observed at higher concentrations without and with metabolic activation in nearly all strains tested. A biologically relevant increase in revertant colonies was not observed in any of the strains tested at any dose level in the absence or presence of S9-mix in both experiments. Under the experimental conditions used, test chemical was not mutagenic in this gene mutation tests in bacteria both in the absence and the presence of S9 metabolic activation.

Data source

Reference
Reference Type:
other: company data
Title:
WoE for in-vitro toxicity study for CAS no 84281-74
Author:
Sustainability Support Services (Europe) AB
Year:
2018
Bibliographic source:
WoE report, Sustainability Support Services (Europe) AB, 2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
WoE report is based on two in-vitro gene toxicity studies on rats
1. The experiments were performed to assess the potential of the test item to induce gene mutations by means of two independent Salmonella typhimurium reverse mutation assays.
Experiment I was performed as a plate incorporation assay. Since a negative result was obtained in this experiment, experiment II was performed as a pre-incubation assay.
2. In virto gene toxicity study of test chemical in Salmonella typhimurium
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
1,3-Benzenediamine, coupled with diazotized m-phenylenediamine, acetates
EC Number:
282-617-7
EC Name:
1,3-Benzenediamine, coupled with diazotized m-phenylenediamine, acetates
Cas Number:
84281-74-3
Molecular formula:
C26H34N8O8
IUPAC Name:
1,3-Benzenediamine, coupled with diazotized m-phenylenediamine, acetates
Details on test material:
- IUPAC Name: 1,3-Benzenediamine, coupled with diazotized m-phenylenediamine, acetates
- Common Name: Basic Brown 1
- Molecular formula : C26H34N8O8
- Molecular weight : 586.597 g/mol
- Smiles notation : CC(=O)O{-}.N{+}c1ccc(N=Nc2cccc(N=Nc3ccc(N{+}.O{-}C(C)=O)cc3N{+}.O{-}C(C)=O)c2)c(N{+}.O{-}C(C)=O)c1
- Substance type : Organic
- Physical state : liquid

Method

Target gene:
1. The histidine dependent strains are derived from S. typhimurium strain LT2 through a mutation in the histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation (deletion of the uvrB gene) causes an inactivation of the excision repair system. The latter alteration also includesa deletion in the nitrate reductase and biotin genes. In the strains TA 98, TA 100, and TA 102 the Rfactor plasmid pKM 101 carries umu DC analogous genes that are involved in error-prone repair and the ampicillin resistance marker. The strain TA 102 does not contain the uvrB--mutation and is excision repair proficient. Additionally, TA 102 contains the multicopy plasmid pAQ1 carrying the hisG428 mutation (ochre mutation in the hisG gene ) and a tetracycline resistance gene (5).In summary, the mutations of the TA strains used in this study can be described as follows:
Salmonella typhimurium
Strains Genotype Type of mutations indicated
TA 1537 his C 3076; rfa-; uvrB-: frame shift mutations
TA 98 his D 3052; rfa-; uvrB-;R-factor " "
TA 1535 his G 46; rfa-; uvrB-: base-pair substitutions
TA 102 his G 428; rfa-; uvrB+;R-factor " "
TA 100 his G 46; rfa-; uvrB-;R-factor " "
Regular checking of the properties of the strains regarding the membrane permeability,ampicillin- and tetracycline resistance as well as spontaneous mutation rates is performed in the laboratory of RCC Cytotest Cell Research according to B. Ames et al.and D.Maron and B. Ames.In this way it was ensured
that the experimental conditions set down by Ames were fulfilled.
The bacterial strains TA 1535, TA 1537 TA 98, TA 100 and TA 102 were obtained from Trinova Biochem GmbH (35394 Gießen, Germany).
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535
Remarks:
1.
Details on mammalian cell type (if applicable):
not specified
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 1537
Remarks:
1.
Details on mammalian cell type (if applicable):
not specified
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 98
Remarks:
1.
Details on mammalian cell type (if applicable):
not specified
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 100
Remarks:
1.
Details on mammalian cell type (if applicable):
not specified
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 102
Remarks:
1.
Details on mammalian cell type (if applicable):
not specified
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Remarks:
2.
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
not specified
Metabolic activation:
with and without
Metabolic activation system:
liver microsomal activation
Test concentrations with justification for top dose:
1. In the pre-experiment the concentration range of the test item was 3 – 5000 μg/plate. The pre-experiment is reported as part of experiment I. Since the criteria mentioned above were met 5000 μg/plate were chosen as maximal concentration.
The concentration range included two logarithmic decades. The following concentrations were tested:
Pre-Experiment: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment I: 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate

2.experiment I: 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate without and with S9-mix
experiment II: 33, 100, 333, 1000, 2500 and 5000 μg/plate without and with S9-mix
Vehicle / solvent:
1. - Vehicle(s)/solvent(s) used:
With metabolic activation
Strains: TA 1535, TA 1537, TA 98, TA 100, TA 102 - DMSO
Without metabolic activation
Strains: TA 1535, TA 100 - water deionised
Strains: TA 1537, TA 98 - DMSO
Strain: TA 102 - water deionised
- Justification for choice of solvent/vehicle:The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

2.DMSO
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
1.
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Remarks:
Concentration: 10 μg/plate
Positive control substance:
sodium azide
Remarks:
For Strains: TA 1535, TA 100 without metabolic activation
Untreated negative controls:
yes
Remarks:
1.
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Remarks:
Concentration: 10 μg/plate in TA 98, 50 μg/plate in TA 1537
Positive control substance:
other: 4-nitro-o-phenylene-diamine, 4-NOPD
Remarks:
For Strains: TA 1537, TA 98 without metabolic activation
Untreated negative controls:
yes
Remarks:
1.
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Remarks:
Concentration: 4.0 μL/plate
Positive control substance:
methylmethanesulfonate
Remarks:
For Strain: TA 102 without metabolic activation
Untreated negative controls:
yes
Remarks:
1.
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Remarks:
Concentration: 2.5 μg/plate (10.0 μg/plate in TA 102)
Positive control substance:
other: 2-aminoanthracene, 2-AA
Remarks:
For Strains: TA 1535, TA 1537, TA 98, TA 100, TA 102 with metabolic activation
Untreated negative controls:
yes
Remarks:
2.
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: in accordance with the OECD guideline
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as mpregnation on paper disk
Storage
The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO (MERCK, D-64293 Darmstadt) in liquid nitrogen.Precultures
From the thawed ampoules of the strains 0.5 mL bacterial suspension was transferred into 250 mL Erlen meyer flasks containing 20 mL nutrient medium. A solution of 20 μL ampicillin (25 μg/mL) was added to the strains TA 98, TA 100, and TA 102. Additionally 20 μL tetracycline (2 μg/mL) was added to strain TA 102. This nutrient medium contains per litre:
8 g Merck Nutrient Broth (MERCK, D-64293 Darmstadt)
5 g NaCl (MERCK, D-64293 Darmstadt)
The bacterial cultures were incubated in a shaking water bath for 4 hours at 37° C.
Selective Agar
The plates with the minimal agar were obtained from E. Merck, D-64293 Darmstadt.
Overlay Agar
The overlay agar contains per litre:
6.0 g MERCK Agar Agar*
6.0 g NaCl*
10.5 mg L-Histidine x HCl x H2O*
12.2 mg Biotin*
* (MERCK, D-64293 Darmstadt)
Sterilisations were performed at 121° C in an autoclave.
Experimental Performance
For each strain and dose level, including the controls three plates were used.
The following materials were mixed in a test tube and poured onto the selective agar plates:
- 100 μL Test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control),
- 500 μL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
- 100 μL Bacteria suspension (cf. test system, pre-culture of the strains),
- 2000 μL Overlay agar
In the pre-incubation assay 100 μL test solution, 500 μL S9 mix / S9 mix substitution buffer and 100μL bacterial suspension were mixed in a test tube and incubated at 37°C for 60 minutes. After pre-in
cubation 2.0 mL overlay agar (45° C) was added to each tube. The mixture was poured on minimal agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 37° C in the dark .

2.Details on test system and conditions
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk
Experiment I was performed according to the direct plate incorporation test, experiment II with the preincubation method.
DURATION
Preincubation period: 60 minutes
Exposure duration: 48 hours incubation with and without S9 mix
NUMBER OF REPLICATIONS: triplicates in 2 individual experiments
NUMBER OF CELLS EVALUATED: No data
DETERMINATION OF CYTOTOXICITY
Method: mitotic index; cloning efficiency; relative total growth; other: No data
OTHER EXAMINATIONS:
Determination of polyploidy: No data
Determination of endoreplication: No data
Other:
experiment I: direct plate incorporation with 48 h incubation without and with S9-mix experiment II: pre-incubation method with 60 minutes pre-incubation and at least 48 h incubation without and with S9-mix
Test concentrations were based on the results of a pre-experiment with strains TA98 and TA100 for toxicity and mutation induction both without and with S9-mix. Toxicity was evaluated for 8 concentrations up to the prescribed maximum concentration of 5000 μg/plate on the basis of a reduction in the number of revertant colonies and/or clearing of the bacterial background lawn. Because
relevant toxic effects were not observed in any of the strains at the maximal concentration, 5000 μg/plate was used as the top concentration. Since in this pre-experiment evaluable plates were obtained for five concentrations or more in the strains used, the pre-experiment is reported in experiment I.
Evaluation criteria:
1. The Salmonella typhimurium reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce a significant increase in mutant colony frequencies
Evaluation of Results
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and TA 102) or thrice (strains TA 1535 and TA1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reprod uced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However,
whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
1.According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100, and TA 102
Remarks:
1.
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
Remarks:
2.
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: The test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Applicant's summary and conclusion

Conclusions:
1,3-Benzenediamine, coupled with diazotized m-phenylenediamine, acetates does not exhibit gene mutation Salmonella typhimurium strains TA 1535, TA 1537, TA 98,TA 100, and TA 102 both without and with S9-mix.
Executive summary:

Study 1

This study was performed to investigate the potential of test chemical to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98,TA 100, and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation (S9 mix). Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Experiment I: 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate

Reduced background growth was observed with and without S9 mix in strains TA 98 and TA 100 in experiment I. In experiment II, reduced background growth was observed with and without S9 mix in all strains used (cf. tables of results).

Toxic effects, evident as a reduction in the number of revertants, were observed at higher concentrations with and without metabolic activation in nearly all strains used. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with test chemical at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. It can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, test chemical is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Study 2

Test chemcail was investigated for the induction of gene mutations in strains of Salmonella typhimurium(Ames test).

Salmonella typhimuriumTA98, TA100, TA102, TA1535 and TA1537 were used for the AMES assay. DMSO was used as a vehicle. Concentration: experiment I: 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate without and with S9-mix experiment II: 33, 100, 333, 1000, 2500 and 5000 μg/plate without and with S9-mix

Treatment: experiment I: direct plate incorporation with 48 h incubation without and with S9-mix experiment II: pre-incubation method with 60 minutes pre-incubation and at least 48 h incubation without and with S9-mix. Liver S9-fraction from phenobarbital/β-naphthoflavone-induced rats was used as exogenous metabolic activation system. Test concentrations were based on the results of a pre-experiment with strains TA98 and TA100 for toxicity and mutation induction both without and with S9-mix. Toxicity was evaluated for 8 concentrations up to the prescribed maximum concentration of 5000 μg/plate on the basis of a reduction in the number of revertant colonies and/or clearing of the bacterial background lawn. Because relevant toxic effects were not observed in any of the strains at the maximal concentration, 5000 μg/plate was used as the top concentration. Since in this pre-experiment evaluable plates were obtained for five concentrations or more in the strains used, the pre-experiment is reported in experiment I. Experiment I was performed according to the direct plate incorporation test, experiment II with the pre-incubation method. Negative and positive controls were in accordance with the OECD guideline.

In the main tests toxic effects evident as clearing of the bacterial background lawn were observed in experiment I in TA98 and TA100 and in experiment II in all strains predominantly at higher concentrations. Toxic effects evident as a reduction in the number of revertants were observed at higher concentrations without and with metabolic activation in nearly all strains tested. A biologically relevant increase in revertant colonies was not observed in any of the strains tested at any dose level in the absence or presence of S9-mix in both experiments. Under the experimental conditions used, test chemical was not mutagenic in this gene mutation tests in bacteria both in the absence and the presence of S9 metabolic activation.

Test chemical induced a reproducible, dose-related increase in his+ revertants over the corresponding solvent in the S. typhimurium tester strains TA100, TA1537, and TA98 in the presence of S9 metabolic activation system. No mutagenic potential was observed for the tester strain TA1537 in the presence and absence of S9 metabolic activation system. Based on these observations, test chemical is likely to be mutagenic.

Based on the data available from the read across, 1,3-Benzenediamine, coupled with diazotized m-phenylenediamine, acetates does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation. 

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