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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:
From Aug. 31, 2009 to Sept. 08, 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study well documented, followed guideline, GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
according to the OECD and German principles of GLP
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material: 4,5-Diamino-1-hexyl-1H-pyrazole, dihydrochloride; Pyrazole HECL/Hexylpyrazole (Code # A016370)- TSIN: 804026- Substance type: Pure active substance- Physical state: Light pink powder- Stability under test conditions: The substance is considered to be stable for more than 5 years if stored dry and protected from light at room temperature.- Stability in solution: The test substance is assumed to be stable over a total time period of 7 d in water and in water/ethanol (1:1) on the conditions applied. However, the test substance is not stable in water which was corrected to pH 7.- Storage condition of test material: At room temperature- Solubility: Solubility in different solvents is as follows: > 10 weight% in water (pH 9) > 10 weight% in ethanol/water 1:1
Specific details on test material used for the study:
The in vitro mutagenicity tests were performed with 1-hexyl-1H-pyrazole-4,5-diamine dihydrocloride, i.e. the dihydrochloride instead of the hemisulfate salt. The toxic potential of both salts of 1-hexyl-1H-pyrazole-4,5-diamine is attributable to the free base component. Therefore, the results of the in-vitro mutagenicity studies carried out with 1-hexyl-1H-pyrazole-4,5-diamine dihydrochloride were taken into consideration for the risk assessment of 1-hexyl 4,5-diamine pyrazole sulfate

Method

Target gene:
The specific target genes of Salmonella Typhimurium tester strains are as follows:TA 1537: his C 3076 (Frame shift mutation)TA 98: his D 3052 (Frame shift mutation)TA 1535 and TA 100: his G 46 (Base-pair substitution)TA 102: his G 428 (Base-pair substitution)
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: Faulty lipopolysaccharide envelope, inactivation of excision repair system, nitrate reductase and biotin deficient, (error prone repair and ampicillin resistance marker in TA 98 and TA 100 only)
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
other: Faulty lipopolysaccharide envelope, nitrate reductase and biotin deficient, excision repair proficient and error prone repair with ampicillin resistance marker, multicopy plasmid pAQ1 and tetracycline resistance
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-Naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Pre-Experiment/Experiment I and II: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate.In this study, the pre-experiment was considered as main Experiment I because evaluable plates (> 0) with colonies were observed in all strains used at five concentrations or more.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Deionised water- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
sodium azide
Remarks:
Migrated to IUCLID6: Prepared by dissolving in deionised water and used at a concentration of 10 µg/plate for strains TA 1535, TA 100
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
other: 4-nitro-o-phenylenediamine solution prepared by dissolving in DMSO and used at a concentration of 10 and 50 µg/plate for strains TA 98 and TA 1537 respectively
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
methylmethanesulfonate
Remarks:
Migrated to IUCLID6: Prepared in deionised water at concentration of 3 µL/plate, used for strain TA102
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
with metabolic activation
Positive control substance:
other: 2-aminoanthracene solution prepared by dissolving in DMSO at concentrations of 2.5 µg/plate for strains TA 1535, TA 1537, TA 100, TA 98 and at 10 µg/plate for strain TA 102
Details on test system and experimental conditions:
METHOD OF APPLICATION: Direct plate incorporation (Experiment I) and pre-incubation method (Experiment II)MAINTENENCE OF TESTER STRAIN: Tester bacterial strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 were obtained from Trinova Biochem GmbH (35394 Gieβen, Germany). The strains were precultured and stored as follows:- Storage: The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO in liquid nitrogen. - Periodic checking: Regular checking of the properties of the strains regarding the membrane permeability, ampicillin and tetracycline-resistance as well as spontaneous mutation rates was performed in the laboratory of Harlan CCR according to B. Ames et al. and D. Maron and B. Ames to ensure the experimental conditions set down by Ames.- 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 mL ampicillin (25 µg/mL) was added to the strains TA 98, TA 100, and TA 102. This nutrient medium contains per litre: 8 g Merck Nutrient Broth (MERCK, D-64293)5 g NaCI (MERCK, D-64293). The bacterial cultures were incubated in a shaking water bath for 4 h at 37˚C.EXPERIMENTAL DURATION AND PROCEDURE: For each strain and dose level, including the controls three plates were used.- Plate incorporation method (Experiment I): 100 µL test solution/negative control/positive control, 500 µL S9 mix (for test with metabolic activation)/S9 mix substitution buffer (for test without metabolic activation), 100 µL bacterial suspension and 2000 µL of overlay agar was mixed in a test tube and poured onto the selective agar plates. - Preincubation assay (Experiment II): In the pre-incubation assay, 100 µL test solution/negative control/positive control, 500 µL S9 mix (for test with metabolic activation)/S9 mix substitution buffer (for test without metabolic activation) and 100 µL bacterial suspension were mixed in a test tube and incubated at 37˚C for 1 h.After pre-incubation 2mL overlay agar (45˚C) was added to each tube. The mixture was poured on minimal agar plates. The plates were incubated upside down for at least 48 h at 37˚C in the dark after solidification.SELECTION MEDIUM:Selective agar: Selective agar was obtained from Merck, D-64293 DarmstadtOverlay Agar: The overlay agar (Merck, D-64293 Darmstadt) contains/L: 6 g MERCK Agar Agar 6 g NaCl 10.5 mg L-Histidine × HCl ×H2O 12.2 mg Biotin Sterilizations were performed at 121˚C in an autoclave.NUMBER OF REPLICATIONS: TriplicateDETERMINATION OF CYTOTOXICITY - Pre-experiment: Toxicity of test material was tested in triplicates/strain/dose group with all strains used. Eight concentrations were evaluated for toxicity. The experimental conditions of pre-experiment were the same as described for the Experiment I above (plate incorporation test). - Method for evaluation: Reduction in the number of spontaneous revertants or clearing of bacterial background lawn.
Evaluation criteria:
- A test substance 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.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 2500 and 333-2500 µg/plate in TA 98 and TA 100 respectively in Experiment 1 and at 2500-5000 µg/plate in TA 1535, TA 1537, TA 98 and at 333-5000 µg/plate in TA 100 in Experiment 2
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 2500 µg/plate in Experiment 1 and at 1000-5000 µg/plate in Experiment 2
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test substance was observed either in the test tubes or on the incubated agar plates.
RESULTS OF PRE-EXPERIMENT/EXPERIMENT I AND II:
i) Results of background growth:The plates incubated with the test substance showed normal background in all strains used in the presence of metabolic activation. Reduced background growth was observed in all strains at higher concentrations in the absence of metabolic activation. For details refer to ‘Table 1’ under ‘Any other information on results incl. tables’ section.
ii) Results of toxicity study:Toxicity of the test substance can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn. In this study, evaluable plates (> 0) with colonies were observed in all strains used at five concentrations or more. Based on these results, pre-experiment was reported as main Experiment I and eight concentrations were tested in Experiment II and 5000 µg/plate was chosen as maximal concentration.Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5) occurred in all strains at higher concentrations in the absence of metabolic activation. No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with metabolic activation. For details refer to ‘Table 2’ under ‘Any other information on results incl. tables’ section.
iii) Genotoxicity Results: No substantial increase in revertant colony numbers of the five tester strains was observed following treatment with test substance 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.
COMPARISON WITH HISTORICAL CONTROL DATA: Negative control, solvent control and positive control data in this study were comparable with the historical control data (From January 2008 until October 2008).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Results of background growth in reverse mutation assay of 4,5-diamino-1-hexyl-1H-pyrazole, dichloride (Study # 66101)

Strain

Experiment I

Experiment II

without S9 mix

with S9 mix

without S9 mix

with S9 mix

TA 1535

5000

/

2500 - 5000

/

TA 1537

2500 - 5000

/

2500 - 5000

/

TA 98

2500 - 5000

/

2500 - 5000

/

TA 100

333 - 5000

/

100 - 5000

/

TA 102

2500 - 5000

/

333 - 5000

/

/ = no reduced background growth

Table 2: Toxic effects observed in reverse mutation assay of 4,5-diamino-1-hexyl-1H-pyrazole, dichloride (Study # 66101)

Strain

Experiment I

Experiment II

without S9 mix

with S9 mix

without S9 mix

with S9 mix

TA 1535

/*

/

2500 - 5000

/

TA 1537

/*

/

2500 - 5000

/

TA 98

2500*

/

2500 - 5000

/

TA 100

333 – 2500*

/

333 - 5000

/

TA 102

2500*

/

1000 - 5000

/

/ = no toxic effects, evident as a reduction in the number of revertants (below the induction

factor of 0.5)

* analysis at 5000 µg/plate not possible due to reduced background growth

Table 3: Number of revertant colonies in reverse mutation assay of 4,5-diamino-1-hexyl-1H-pyrazole, dichloride in Pre-Experiment/Experiment 1 (Study # 66101)

Metabolic Activation

Test Group

Dose Level (per plate)

Revertant Colony Counts (Mean± SD)

TA 1535

TA 1537

TA 98

TA 100

TA 102

Without Activation

Deionised Water

 

15 ± 5

11 ± 5

28 ± 8

159 ± 16

416 ± 20

Untreated

 

12 ± 2

9 ± 1

29 ± 7

146 ± 8

389 ± 27

4,5-diamino-1- hexyl-1H-pyrazole dihydrochloride

3 μg

14 ± 1

8 ± 2

28 ± 5

156 ± 6

382 ± 26

10 μg

13 ± 3

15 ± 1

25 ± 2

154 ± 10

399 ± 22

33 μg

14 ± 3

10 ± 2

32 ± 3

149 ± 10

436 ± 22

100 μg

13 ± 3

9 ± 1

28 ± 6

151 ± 16

377 ± 14

333 μg

13 ± 3

14 ± 2

27 ± 7

54 ± 5R

306 ± 39

1000 μg

16 ± 6

9 ±

1

20 ± 3

67 ± 15R

270 ± 23

2500 μg

16 ± 1

7 ± 1MR

9 ± 1MR

54 ± 5MR

89 ± 6MR

5000 μg

NR

NR

NR

NR

NR

NaN3

10 μg

1611 ± 35

 

 

1904 ± 50

 

4-NOPD

10 μg

 

 

306 ± 18

 

 

4-NOPD

50 μg

 

71 ± 10

 

 

 

MMS

3 μL

 

 

 

 

2881 ± 214

With Activation

Deionised

ater

 

15± 6

9 ± 4

36 ± 2

170 ± 14

607 ± 20

Untreated

 

15± 2

10 ± 5

41 ± 9

138 ± 10

561 ± 27

4,5-diamino-1- hexyl-1H-pyrazole dihydrochloride

3 μg

18 ± 3

11 ± 5

39 ± 5

148 ± 14

570 ± 32

10 μg

20 ± 7

9 ± 2

37 ± 1

168 ± 17

560 ± 44

33 μg

21 ± 6

11

 5

41

± 1

175 ± 15

684 ± 34

100 μg

13 ± 2

11 ± 3

33 ± 2

159 ± 4

688 ± 16

333 μg

12 ± 2

10 ± 3

37 ± 9

151 ± 21

588 ± 12

1000 μg

17 ± 5

8 ± 2

36 ± 8

153 ± 19

566 ± 19

2500 μg

19 ± 3

11 ± 3

35 ± 2

122 ± 16

518 ± 23

5000 μg

14 ± 2

13 ± 1

31 ± 3

145 ±

8

306 ± 1

2-AA

2.5 μg

345 ± 21

228 ± 5

2170 ± 424

2366 ± 277

 

2-AA

10 μg

 

 

 

 

1937 ± 143

 

Table 4: Number of revertant colonies in reverse mutation assay of 4,5-diamino-1-hexyl-1H-pyrazole, dichloride in Experiment 2 (Study # 66101)

Metabolic Activation

Test Group

Dose Level (per plate)

Revertant Colony Counts (Mean± SD)

TA 1535

TA 1537

TA 98

TA 100

TA 102

Without Activation

Deionised Water

 

13± 5

9 ± 3

34 ± 7

144 ± 5

408 ± 24

Untreated

 

16 ± 2

12 ± 2

31 ± 9

143 ± 14

384 ± 32

4,5-diamino-1- hexyl-1H-pyrazole, dihydrochloride

3 μg

15 ± 1

13 ± 1

31 ± 8

158 ± 22

355 ± 30

10 μg

15 ± 5

13 ± 1

33 ± 8

150 ± 14

412 ± 7

33 μg

14 ± 2

14 ± 2

26 ± 3

140 ± 12

402 ± 14

100 μg

16 ± 1

13 ± 3

27 ± 2

70 ± 16R

298 ± 47

333 μg

13 ± 6

11 ± 3

31 ± 9

20 ± 2MR

239 ± 10R

1000 μg

16 ± 6

10 ± 5

22 ± 7

12 ± 3MR

101 ± 3MR

2500 μg

0 ± 0R

1 ± 1MR

2 ± 3MR

0 ± 0MR

0 ± 0MR

5000 μg

0 ± 0R

0 ± 0MR

1 ± 2MR

0 ± 0MR

0 ± 0RM

NaN3

10 μg

1918 ± 36

 

 

2173 ± 65

 

4-NOPD

10 μg

 

 

543 ± 75

 

 

4-NOPD

50 μg

 

90 ± 8

 

 

 

MMS

3 μL

 

 

 

 

3909 ± 152

With Activation

Deionised Water

 

16± 4

11 ± 3

38 ± 4

170 ± 15

639 ± 6

Untreated

 

18± 2

11 ± 4

47 ± 6

178 ± 11

575 ± 9

4,5-diamino-1- hexyl-1H-pyrazole, dihydrochloride

3 μg

14 ± 4

15 ± 2

43 ± 6

174 ± 13

574 ± 20

10 μg

22 ± 3

13 ± 4

43 ± 7

188 ± 10

637 ± 18

33 μg

20 ± 4

12 ± 3

42 ± 8

186 ± 7

687 ± 24

100 μg

17 ± 3

10 ± 3

39 ± 3

175 ± 2

722 ± 49

333 μg

17 ± 2

11 ± 4

47 ± 6

153 ± 19

607 ± 21

1000 μg

16 ± 3

12 ± 1

38 ± 4

153 ± 3

591 ± 47

2500 μg

18 ± 2

12 ± 2

30 ± 7

140 ± 8

568 ± 23

5000 μg

15 ± 4

11 ± 3

24 ± 0

134 ± 6

318 ± 25

2-AA

2.5 μg

385 ± 27

498 ± 5

3217 ± 466

2690 ± 141

 

2-AA

10 μg

 

 

 

 

2497 ± 136

NaN3 = Sodium azide

2 -AA = 2 -Aminoanthracene

MMS = Methyl methane sulfonate

4 -NOPD = 4 -Nitro-O-phenylene-diamine

R = Reduced background growth

M = Manual count

N = Analysis not possible

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):negative with and without metabolic activation4,5-diamino-1-hexyl-1H-pyrazole, dihydrochloride (Pyrazole HECL/Hexylpyrazole) was considered non-mutagenic in Samonella typhimurium reverse mutation assay, in the presence and absence of metabolic activation. The in vitro mutagenicity tests was performed with 1-hexyl-1H-pyrazole-4,5-diamine dihydrocloride, i.e. the dihydrochloride instead of the hemisulfate salt. The toxic potential of both salts of 1-hexyl-1H-pyrazole-4,5-diamine is attributable to the free base component. Therefore, the results of the in-vitro mutagenicity studies carried out with 1-hexyl-1H-pyrazole-4,5-diamine dihydrochloride were taken into consideration for the risk assessment of 1-hexyl 4,5-diamine pyrazole sulfate
Executive summary:

The bacterial reverse mutation test of 4,5-diamino-1-hexyl-1H-pyrazole, dihydrochloride (Pyrazole HECL/Hexylpyrazole) was determined following OECD guideline 471 (Bacterial Reverse Mutation Test) and EU Method B.13/14 (Mutagenecity - Reverse Mutation Test Using Bacteria).

A plate incorporation (Experiment I) and pre-incubation assay (Experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 was performed with and without liver microsomal activation in a selective agar media. Each concentration, including the negative, positive and solvent controls was tested in triplicate. The test material was tested at the following concentrations:

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

In this study, the pre-experiment was considered as main Experiment I because evaluable plates (> 0) with colonies were observed in all strains used at five concentrations or more.   

The plates incubated with the test substance showed normal background in all strains used in the presence of metabolic activation. Reduced background growth was observed in all strains at higher concentrations in the absence of metabolic activation.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5) occurred in all strains at higher concentrations in the absence of metabolic activation. No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with metabolic activation.

No substantial increase in revertant colony numbers of the five tester strains was observed following treatment with test substance at any dose level in the presence or 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.

Negative control, solvent control and positive control data in this study were comparable with the historical control data.

4,5-diamino-1-hexyl-1H-pyrazole, dihydrochloride did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Based on the above, 4,5-diamino-1-hexyl-1H-pyrazole, dihydrochloride (Pyrazole HECL/Hexylpyrazole) was considered non-mutagenic in this Samonella typhimurium reverse mutation assay in the presence or absence of metabolic activation.

This bacterial reverse mutation test is classified as acceptable, and satisfies the guideline requirements of the OECD 471 method.