1-Hexyl 4,5-diamino pyrazole hemisulfate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Mutagenicity of 1-hexyl 4,5-diamine pyrazole sulfate was tested with the dihydrochloride instead of the hemisulfate salt.

Overall, the genotoxicity of 4,5-diamino-1-hexyl-1H-pyrazole, dihydrochloride is sufficiently investigated in valid genotoxicity tests for the 3 endpoints of genotoxicity: gene mutations, chromosome aberrations and aneuploidy. 4,5-Diamino-1-hexyl-1H-pyrazole, dihydrochloride did not induce gene mutations both in bacteria nor in mammalian cells. Treatment with 4,5 -diamino-1-hexyl-1H-pyrazole, dihydrochloride did also not result in an increase in cells with micronuclei in an in vitro micronucleus test in human blood cells.

Consequently, on the basis of these tests, 4,5-diamino-1-hexyl-1H-pyrazole, dihydrochloride can be considered to have no genotoxic potential and additional tests are

unnecessary.

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. Accordingly, 1-hexyl 4,5-diamine pyrazole sulfate is, therefore, considered not to pose a risk to humans with regard to genotoxicity.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

according to the OECD and German principles of GLP

Type of assay:

bacterial reverse mutation assay

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

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 / 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.

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.

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'.

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 ± 5^R	306 ± 39
		1000 μg	16 ± 6	9 ± 1	20 ± 3	67 ± 15^R	270 ± 23
		2500 μg	16 ± 1	7 ± 1^MR	9 ± 1^MR	54 ± 5^MR	89 ± 6^MR
		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 ± 16^R	298 ± 47
		333 μg	13 ± 6	11 ± 3	31 ± 9	20 ± 2^MR	239 ± 10^R
		1000 μg	16 ± 6	10 ± 5	22 ± 7	12 ± 3^MR	101 ± 3^MR
		2500 μg	0 ± 0^R	1 ± 1^MR	2 ± 3^MR	0 ± 0^MR	0 ± 0^MR
		5000 μg	0 ± 0^R	0 ± 0^MR	1 ± 2^MR	0 ± 0^MR	0 ± 0^RM
	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

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From May 03, 2011 to July 04, 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented study, followed guideline, GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Principles of method if other than guideline:

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

GLP compliance:

yes

Remarks:

according to Germany and OECD principles of GLP

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT locus in V79 cells

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM (minimal essential medium) containing Hank’s salts supplemented with 10 % foetal bovine serum (FBS), neomycin (5 μg/mL) and amphotericin B (1%).- Source: V79 cell line were supplied by Laboratory for Mutagenicity Testing; Technical University, 64287 Darmstadt, Germany.- Properly maintained: Yes, large stocks of V79 cell line were stored in liquid nitrogen in the cell bank of lab allowing the repeated use of the same cell culture batch in experiments..- Propagation of stock culture: Thawed stock cultures are propagated at 37 °C in 80 cm2 plastic flasks. About 5 × 10(5) cells were seeded into each flask with 15 mL of MEM (minimal essential medium) containing supplemented Hank’s salts. The cells were sub-cultured twice weekly. The cell cultures were incubated at 37°C in a 1.5 % carbon dioxide atmosphere (98.5 % air).- Periodically checked for Mycoplasma contamination: yes- Periodically checked for karyotype stability: yes- Periodically "cleansed" against high spontaneous background: yes; before freezing, the level of spontaneous mutants was depressed by treatment with HAT medium.

Additional strain / cell type characteristics:

other: stable karyotype with a modal chromosome number of 22; high proliferation rate (doubling time 12-16 h in stock cultures) and cloning efficiencies of untreated cells of usually more than 50%.

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Pre-Test for toxicity I: 20.0, 40.0, 80.0, 160.0, 320.0, 640.0, 1280.0 and 2560.0 μg/mL (with and without metabolic activation) Pre-Test for toxicity II: 0.31, 0.63, 1.30, 2.50, 5.00, 10.00, 20.00 and 40.00 μg/mL (without metabolic activation)Experiment I (4 h treatment):Without metabolic activation: 1.25, 2.5, 5.0, 10.0, 20.0, 30.0 and 40.0 µg/mL (Dose levels: 5.0, 10.0, 20.0, 30.0 and 40.0 µg/mL were chosen for the mutation rate analysis)With metabolic activation: 40.0, 80.0, 160.0, 320.0, 640.0, 960.0 and 1280.0 µg/mL (Dose levels: 40.0, 80.0, 160.0, 320.0, 640.0 µg/mL were chosen for the mutation rate analysis)Experiment II:Without metabolic activation (24 h treatment): 0.63, 1.25, 2.5, 5.0, 10.0, 15.0 and 20.0 µg/mL (Dose levels: 2.5, 5.0, 10.0, 15.0 and 20.0 µg/mL were chosen for the mutation rate analysis)With metabolic activation (4 h treatment): 80.0, 160.0, 320.0, 640.0, 720.0, 800.0 and 880.0 µg/mL (Dose levels: 320.0, 640.0, 720.0, 800.0 and 880.0 µg/mL were chosen for the mutation rate analysis)

Vehicle / solvent:

- Vehicle used: Deionised water

Negative solvent / vehicle controls:

yes

Remarks:

nutrient medium

Positive controls:

yes

Remarks:

Without metabolic activation

Positive control substance:

ethylmethanesulphonate

Remarks:

Ethylmethanesulfonate (EMS) at a final concentration of 0.15 mg/mL was used.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

With metabolic activation

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

7,12-dimethylbenzanthracene (DMBA) at final concentration of 1.1 µg/mL in DMSO

Details on test system and experimental conditions:

METHOD OF APPLICATION: In complete culture medium, MEM (minimal essential medium) containing Hank’s salts, neomycin (5 μg/mL) and amphotericin B (1%). EXPERIMENTAL PROCEDURE AND DURATION: The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without metabolic activation and a treatment period of 4 h. The second experiment was performed with a treatment time of 4 h with and 24 h without metabolic activation.- Seeding: Two days after sub-cultivation stock cultures were trypsinized at 37°C for 5 minutes. Then the enzymatic digestion was stopped by adding complete culture medium with 10 % FBS and a single cell suspension was prepared. - Treatment: After 24 hours the medium was replaced with serum-free medium containing the test substance, either without S9 mix or with 50 μL/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. After 4 hours this medium was replaced with complete medium following two washing steps with "saline G". In the second experiment the cells were exposed to the test substance for 24 hours in complete medium, supplemented with 10 % FBS, in the absence of metabolic activation.- Expression and growth: Four days after treatment 1.5 × 10(6) cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 mL medium. Following the expression time of 7 days five 80 cm² cell culture flasks were seeded with about 3 – 5 × 10(5) cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in nonselective medium to determine the viability.The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 for about 8 days. The colonies were stained with 10% methylene blue in 0.01 % KOH solution.SELECTION AGENT: 11 μg/mL 6-Thioguanine (6-TG)NUMBER OF REPLICATIONS: 2DETERMINATION OF CYTOTOXICITY- Method: Relative cloning efficiency

Evaluation criteria:

- A test substance is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible positive response for one of the test points.- A test substance producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered to be non-mutagenic in this system.- A mutagenic response is described as follows:i) The test substance is classified as mutagenic if it induces reproducibly with one of the concentrations a mutation frequency that is three times higher than the spontaneous mutation frequency in the experiment. If the mutation frequency exceeds the historical range of the solvent controls, an induction factor of >2 is considered sufficient to indicate a mutagenic response.ii) The test substance is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that the above described two/threefold increase of the mutant frequency is not observed.However, in a case by case evaluation this decision depends on the level of the corresponding negative control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study is also taken into consideration.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test substance was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS:- The osmolarity in the solvent control and the maximum concentration (2560 µg/mL) in the pre-experiment was 283 and 339 respectively.- The pH-value in the solvent control and the maximum concentration (2560 µg/mL) in the pre-experiment after adjustment to neutral with NaOH was 7.36 and 7.37 respectively.- No precipitation of the test substance was observed up to the maximum concentration in the main experiment.Further details are provided in the study report.PRE-EXPERIMENTAL TOXICITY STUDIES:- Relevant cytotoxic effects were observed at 20.0 μg/mL and above in the absence of metabolic activation and at 1280 μg/mL and above with metabolic activation (4 h treatment). Following continuous treatment without metabolic activation (24 h) severe cytotoxic effects occurred at 20.0 μg/mL and above.- Turbidity occurred at 160 and 320 μg/mL in the absence of metabolic activation. At concentrations of 640 μg/mL and above precipitation was observed. Turbidity was also noted at 1280 and 2560 μg/mL in the presence of metabolic activation (4 h treatment). Following continuous treatment (24 h) without metabolic activation, neither precipitation nor turbidity occurred up to the maximum concentration.- No relevant shift of the osmolarity of the medium was observed in the pre-experiment at the two highest concentrations used (1280 and 2560 μg/mL).MAIN EXPERIMENT RESULTS: Cytotoxicity:- Relevant cytotoxic effects indicated by a relative cloning efficiency below 50% in at least one of the parallel cultures occurred in experiment I at 20.0 μg/mL and above without metabolic activation, and at 640 μg/mL with metabolic activation. - In the second experiment cytotoxic effects were observed at 15.0 μg/mL and above without metabolic activation and at 640 μg/mL and 800 μg/mL and above with metabolic activation. The recommended cytotoxic range of approximately 10-20% relative cloning efficiency was covered with and without metabolic activation. Even though the relative cloning efficiency was down to 0% at 30 and 40 μg/mL in the first experiment without metabolic activation and at 20 μg/mL in the second experiment without metabolic activation the data were judged as valid as the relative cell density at the first sub-cultivation following treatment was acceptable. The relative cell density is used as second parameter of cytotoxicity measuring the cell density of the cultures four days after removal of the test substance.Mutation frequency :- No relevant increase of the mutation frequency was detected in any of the experimental parts with and without metabolic activation. All absolute values of the mutation frequency remained within the historical solvent control range. The induction factor remained below 2.0 up to the maximum concentration with and without metabolic activation.- A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.RESULTS OF NEGATIVE AND POSITIVE CONTROLS:- In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 13.9 up to 33.2 mutants per 10(6) cells; the range of the groups treated with the test item was from 6.6 up to 36.2 mutants per 10(6) cells.- EMS (150 μg/mL) and DMBA (1.1 μg/mL) showed a distinct increase in induced mutant colonies.

Remarks on result:

other: strain/cell type: Chinese hamster lung fibroblasts (V79)

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):other: negative with and without metabolic activation4,5-Diamino-1-hexyl-1 H-pyrazole, dihydrochloride was considered non-mutagenic in the HPRT assay using the Chinese hamster V79 cell line, in the absence and presence 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 in vitro mammalian gene mutation test of 4,5-Diamino-1-hexyl-1 H-pyrazole, dihydrochloridewas determined following the OECD guideline 476 (In vitro Mammalian Cell Gene Mutation Test).

The assay was performed using Chinese hamster V79 cells in two independent experiments, using two parallel cultures each. The first experiment (Experiment I) was performed with and without metabolic activation and a treatment period of 4 h. The second experiment (Experiment II) was performed with a treatment time of 4 h with and 24 h without metabolic activation.

Based on the pre-test toxicity (conducted in the range 20.0 – 2560 µg/mL) results, the following concentrations of the test substance were selected for the main test:

Experiment I (4 h treatment):

Without metabolic activation: 1.25, 2.5, 5.0, 10.0, 20.0, 30.0 and 40.0 µg/mL (Dose levels: 5.0, 10.0, 20.0, 30.0 and 40.0 µg/mL were chosen for the mutation rate analysis)

With metabolic activation: 40.0, 80.0, 160.0, 320.0, 640.0, 960.0 and 1280.0 µg/mL (Dose levels: 40.0, 80.0, 160.0, 320.0, 640.0 µg/mL were chosen for the mutation rate analysis)

Experiment II:

Without metabolic activation (24 h treatment): 0.63, 1.25,2.5, 5.0, 10.0, 15.0 and 20.0 µg/mL (Dose levels:2.5, 5.0, 10.0, 15.0 and 20.0µg/mLwerechosen for the mutation rate analysis)

With metabolic activation (4 h treatment): 80.0, 160.0, 320.0, 640.0, 720.0, 800.0 and 880.0 µg/mL (Dose levels: 320.0, 640.0, 720.0, 800.0 and 880.0 µg/mLwere chosen for the mutation rate analysis)

The concentration range of the main experiments was limited by cytotoxic effects of the test substance.

Deionized water served as the vehicle control. Ethylmethanesulfonate (without metabolic activation) and 7,12-dimethylbenz(a)anthracene (with metabolic activation) served as positive controls. Each concentration, including the negative, positive and solvent controls was tested in duplicate.

Relevant cytotoxic effects indicated by a relative cloning efficiency value below 50% in at least one of the parallel cultures occurred in experiment I at 20.0 μg/mL and above without metabolic activation, and at 640 μg/mL with metabolic activation. In the second experiment cytotoxic effects were observed at 15.0 μg/mL and above without metabolic activation and at 640 μg/mL and 800 μg/mL and above with metabolic activation. The recommended cytotoxic range of approximately 10-20% relative cloning efficiency was covered with and without metabolic activation.

Even though the relative cloning efficiency was down to 0% at 30 and 40 μg/mL in the first experiment without metabolic activation and at 20 μg/mL in the second experiment without metabolic activation the data were judged as valid as the relative cell density at the first sub-cultivation following treatment was acceptable. The relative cell density, that is measuring the cell density of the cultures four days after removal of the test substance, is used as second parameter of cytotoxicity.

No relevant increase in the mutation frequency was detected in any of the experiments with and without metabolic activation. All absolute values of the mutation frequency remained within the historical solvent control range. The induction factor remained below 2.0 up to the maximum concentration with and without metabolic activation.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.

The positive controls induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

Based on above, 4,5-Diamino-1-hexyl-1 H-pyrazole, dihydrochloridewas considered non-mutagenic in the HPRT assay using the Chinese hamster V79 cell line, in the absence and presence of metabolic activation.

This mammalian gene mutation test is classified as acceptable, and satisfies the guideline requirements of the OECD 476 method.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Aug. 03, 2010 to Sep. 03, 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented study, followed guideline, GLP

Qualifier:

according to guideline

Guideline:

other: OECD Test Guideline 487 (In Vitro Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Remarks:

according to US FDA and OECD principles of GLP

Type of assay:

in vitro mammalian cell micronucleus test

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

Species / strain / cell type:

other: Peripheral blood lymphocytes were obtained from a healthy non-smoking 24-year-old adult male.

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI-1640 complete medium supplemented with 2% phytohaemagglutinin (PHA)- The donor had no recent history of radiotherapy, viral infection or the administration of drugs. This system has been demonstrated to be sensitive to the clastogenic activity of a variety of chemicals

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Preliminary toxicity assay: 0, 0.255, 0.765, 2.55, 7.65, 25.5, 76.5, 255, 765 and 2550 µg/mLMicronucleus Assay:Concurrent cytotoxicity Without S9: 4 h treatment + 20 h recovery: 0, 3.13, 6.25, 12.5, 25, 50, 100, 150, 175 and 200 µg/mL24 h treatment + 0 h recovery: 0, 24 0 3.13, 6.25, 12.5, 25, 50, 60, 70, 80, 90 and 100 µg/mLWith S9:4 h treatment + 20 h recovery: 0, 3.13, 6.25, 12.5, 25, 50, 100, 150, 175 and 200 µg/mLBased on the results of the concurrent cytotoxicity, following test doses were selected for micronucleus analysis: Without S9: 4 h treatment + 20 h recovery: 0, 25, 50, 100 and 150 µg/mL24 h treatment + 0 h recovery: 0, 12.5, 25, 50 and 80 µg/mLWith S9:4 h treatment + 20 h recovery: 0, 25, 50, 100 and 175 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Water- Justification for choice of solvent/vehicle: Water was used as the solvent based on the information provided by the Sponsor and compatibility with the target cells. The test substance was soluble in water at a concentration of 25.5 mg/mL, the maximum concentration prepared in the assay.

Negative solvent / vehicle controls:

yes

Remarks:

water

Positive controls:

yes

Remarks:

Without metabolic activation

Positive control substance:

other: Mitomycin C and Vinblastine

Remarks:

Mitomycin C at final concentration of 0.4 and 0.5 µg/mL; Vinblastine at final concentration of 10 and 20 ng/mL

Positive controls:

yes

Remarks:

With metabolic activation

Positive control substance:

cyclophosphamide

Remarks:

Cyclophosphamide at final concentration of 10 µg/mL in DMSO

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium PREPARATION OF TEST SOLUTION: All test substance dilutions were prepared in water by serial dilution immediately before use and delivered to the test system under yellow light.EXPERIMENTAL PROCEDURE: - Cell preparation: Approximately 0.5 mL heparinized blood was inoculated into centrifuge tubes containing 5 mL RPMI-1640 complete medium supplemented with 2% phytohemagglutinin (PHA). The tubes were incubated at 37±1°C in a humidified atmosphere of 5±1% CO2 in air for 44-48 hours.- Treatment: At the time of test substance treatment the culture tubes were centrifuged, the supernatant was aspirated, and the cells were resuspended in either 4.5 mL of fresh RPMI-1640 complete medium for the non-activated study or 4.5 mL S9 reaction mixture (3.5 mL serum free medium + 1 mL of S9 cofactor pool), to which was added 500 μL test substance dosing solution in solvent or solvent alone. The cells were treated for 4 and 24 hours in the non-activated test system and for 4 hours in the S9-activated test system. - Removal of test material and cytokinesis blocking: At the end of 4 hour treatments, the cells were centrifuged at approximately 1200 rpm for about 5 minutes and the treatment medium was aspirated, the cells washed with 3-5 mL of CMF-PBS and 5 mL RPMI-1640 complete medium with cytochalasin B (CYB) at 6 μg/mL was added to each tube. The cultures were returned to the incubator for 20 hours. For the 24 hour treatment in the absence of S9, CYB at 6 μg/mL was added at the beginning of the treatment.- Harvesting: At the end of the 24 hour from the initiation of the treatment, the cultures were centrifuged, supernatant was aspirated and the cell pellet was disrupted by flicking the tube. The cells were resuspended in 5 mL 0.075M KCl and treated for 3 minutes at room temperature. At the end of the KCl treatment and immediately prior to centrifuging, the cells were gently mixed and approximately 0.5 mL of cold acetic alcohol fixative (methanol:glacial acetic acid, 25:1, v/v) was added to each tube. The tubes were centrifuged, the supernatant aspirated, the cell pellet disrupted and 3-5 mL of fixative added to each tube. The cells were centrifuged again, the supernatant aspirated, the cells resuspended in 3-5 mL of fresh fixative and stored overnight or longer at approximately 2-8°C or the slides were prepared immediately after harvest. - Number of replications: DuplicateSLIDE PREPARATION: Prior to slide preparation, the fixed cells in 15 mL centrifuge tubes were centrifuged and the supernatant was aspirated leaving approximately 0.2 mL above the cell pellet. The cells were resuspended and the cells suspension was applied on clean microscope slides. The slides were left to air dry at room temperature.STAINING: The slides were stained with acridine orange stain at least 1 day prior to use. One set of slides were stained for 1-2 minutes in the acridine orange stain solution and then rinsed for several minutes in calcium and magnesium-free Dulbecco’s phosphate buffered-saline (CMF-DPBS). The second set of slides was packaged for storage until the final report issued. Stained slides were stored prior to and between scoring at 2-8°C.DETERMINATION OF CYTOTOXICITY: Cytokinesis blocked proliferation index (CBPI) was used to measure the relative frequencies of mononucleate, binucleated, and multi-nucleate cells in the culture. 500 cells per culture were assessed to determine the test substance effect on cell cycle kinetics for all test substance treatment groups and the controls.SCORING OF MICRONUCLEUS INDUCTION: The slides from three test substance treatment groups per treatment condition were scored for the presence of micronuclei. Selection of the highest dose for scoring was based on toxicity. The top dose selected for scoring was the lowest dose level that results with approximately 55±5% cytotoxicity based on CBPI. Two additional lower dose levels were also scored. Whenever possible a minimum of 1000 definitively binucleated cells (defined as the cell with intact cytoplasm containing two nuclei of approximately same size) from each culture will be examined and scored for the presence of micronuclei.

Evaluation criteria:

The test substance was considered to induce a positive response if the percentage of cells with micronuclei in one or more of the treatment concentrations was statistically significantly increased (p ≤ 0.05) compared to the solvent control group. Test substances not demonstrating a statistically significant increase in micronuclei were concluded to be negative.

Statistics:

Statistical analysis of the percentage of micronucleated binucleated cells was performed using Fisher's exact test. The Fisher's test was used to compare pairwise the percent of micronucleated cells of each treatment group with that of the cytochalasin B containing solvent control.

Species / strain:

other: Human peripheral blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS: Preliminary toxicity assay: - The test substance was soluble in water and in the treatment medium at all dose levels tested at the beginning of the treatment period. - At the conclusion of the treatment period, in the non-activated and S9-activated 4-hour treatment groups, visible precipitate was observed in treatment medium at dose levels ≥ 765 μg/mL, while dose levels ≤ 255 μg/mL were soluble in treatment medium. In the non-activated 24-hour treatment group, visible precipitate was observed in treatment medium at dose levels ≥ 255 μg/mL, while dose levels ≤ 76.5 μg/mL were soluble in treatment medium. Precipitation was determined under light microscope and at each precipitating dose level; black substances of different sizes were floating in the treatment medium. - Also, at the conclusion of the treatment period, hemolysis was observed at dose levels ≥ 765 μg/mL in the non-activated and S9-activated 4-hour treatment groups, and at dose levels ≥ 255 μg/mL in the non-activated 24-hour treatment group.- At beginning and conclusion of the treatment period, the osmolality and pH value were similar for treatment and vehicle control group.Micronucleus assay:- The test substance was soluble in water and in the treatment medium at all concentrations tested at the beginning of the treatment period. - At the conclusion of the treatment period, in the nonactivated 24-hour treatment groups, visible precipitate was observed in treatment medium at dose levels ≥ 80 μg/mL, while dose levels ≤ 70 μg/mL were soluble in treatment medium. In the non-activated and S9-activated 4-hour treatment groups, the test substance was soluble in the treatment medium at all dose levels tested.- At beginning and conclusion of the treatment period, the osmolality and pH value were similar for treatment and vehicle control group.RESULT OF PRELIMINARY TOXICITY ASSAY: Selection of dose levels for the micronucleus assay was based on 55±5% cytotoxicity based on cytokinesis blocked proliferation index (CBPI). Substantial toxicity (≥ 55±5% cytotoxicity relative to the solvent control) was observed at dose levels ≥ 255 μg/mL in the non-activated and S9-activated 4-hour exposure groups, and at dose levels ≥ 76.5 μg/mL in the non-activated 24-hour exposure group. Based on these findings, the doses chosen for the micronucleus assay ranged from 3.13 to 200 μg/mL for the non-activated and S9-activated 4-hour exposure groups, and from 3.13 to 100 μg/mL for the non-activated 24-hour exposure group.RESULT OF MICRONUCLEUS ASSAY:The results of cytotoxicity evaluated concurrently are as follows:Non-activated 4-hour exposure: At the highest test concentration evaluated microscopically for micronuclei (150 μg/mL), CBPI was 1.269 and cytotoxicity was 53% relative to the solvent control. S9-activated 4-hour exposure: At the highest test concentration evaluated microscopically for micronuclei (175 μg/mL), CBPI was 1.259 and cytotoxicity was 54% relative to the solvent control. Non-activated 24-hour exposure: At the highest test concentration evaluated microscopically for micronuclei (80 μg/mL), CBPI was 1.271 and cytotoxicity was 59% relative to the solvent control. Based on concurrent cytotoxicity, the dose levels selected for analysis of micronuclei were:Non-activated 4-hour exposure: 0, 25, 50, 100 and 150 µg/mLNon-activated 24-hour exposure: 0, 12.5, 25, 50 and 80 µg/mLS9-activated 4-hour exposure: 0, 25, 50, 100 and 175 µg/mLMicronucleus induction:- The percentage of cells with micronucleated binucleated cells in the test substance-treated groups was not statistically significantly increased relative to solvent control at any dose level (p>0.05, Fisher’s Exact test) in the 4-hour treatment group either in the presence of or the absence of S9 or in the 24-hour treatment group in the absence of S9.RESULTS OF CONTROL GROUPS:All the positive control compounds induced statistically significant increases in the proportion of cells with micronuclei.The results for the positive and negative controls indicate that all criteria for a valid assay were met.

Remarks on result:

other: strain/cell type: Human peripheral blood lymphocytes

Remarks:

Migrated from field 'Test system'.

Table 1: Micronucleus analysis of human peripheral blood lymphocytes treated with test substance (study # 67610)

Treatment conditions	Treatment	Concentration (μg/mL)	CBPI	Cytotoxicity	Percentage of MNBN Cells per Total BN Cells Counted
In the absence of S9, 4-hour treatment, 24-hour harvest	Water	-	1.57	-	0.10%
	Test substance	25	1.488	14%	0.20%
		50	1.469	18%	0.20%
		100	1.398	30%	0.30%
		150	1.269	53%	0.40%
	MMC	0.5	1.295	48%	5.2%**
	Vinblastine	0.02	1.326	43%	2.0%**
In the presence of S9, 4-hour treatment, 24-hour harvest	Water	-	1.565	-	0.20%
	Test substance	25	1.495	12%	0.10%
		50	1.44	22%	0.20%
		100	1.383	32%	0.10%
		175	1.259	54%	0.30%
	CP	10	1.239	58%	2.1%**
In the absence of S9, 24-hour treatment, 24-hour harvest	Water	12.5	1.553	17%	0.20%
	Test substance	25	1.549	18%	0.30%
		50	1.481	28%	0.30%
		80	1.271	59%	0.50%
	MMC	0.4	1.271	59%	4.9%**
	Vinblastine	0.01	1.304	55%	2.2%**

CBPI = Cytokinesis-Block Proliferation Index

MNBN = micronucleated binucleated

BN = binucleated

*p ≤ 0.05; **p ≤ 0.01, Fisher's exact test, relative to the solvent control.

Conclusions:

Interpretation of results (migrated information):other: negative with and without metabolic activationUnder the conditions of this in vitro micronucleus assay using human peripheral blood lymphocytes, 4,5-Diamino-1-hexyl-1 H-pyrazole, dihydrochloride was negative for the induction of micronuclei in both non-activated and S9-activated test systems.
This 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 in-vitro micronucleus test of 4,5-Diamino-1-hexyl-1 H-pyrazole, dihydrochloride was conducted following the OECD guideline 487 (In vitromicronucleus test) using human lymphocyte cultures in the absence and presence of a rat liver metabolizing system (S-9).

Peripheral blood lymphocytes were obtained from a healthy non-smoking 24-year-old adult male. The human lymphocyte cultures were prepared by inoculating heparinized blood into centrifuge tubes containing RPMI-1640 complete medium supplemented with 2% phytohaemagglutinin (PHA). The tubes were incubated at 37±1°C in a humidified atmosphere of 5±1% CO2 in air for 44-48 hours.

The cells were treated for 4 and 24 hours in the non-activated test system and for 4 hours in the S9-activated test system. Cytochalasin B (CYB, 6 μg/mL) was added to the cultures to block cytokinesis after removal of test substance in the 4 hour treatment with and without S9. For the 24 hour treatment in the absence of S9, CYB was added at the beginning of the treatment. All cells were harvested 24 hours after treatment initiation.

The following test concentrations were selected based on the results obtained in a preliminary cytotoxicity assay:

4 h treatment + 20 h recovery (with and without S9): 0, 3.13, 6.25, 12.5, 25, 50, 100, 150, 175 and 200 µg/mL

24 h treatment + 0 h recovery (without S9): 0, 24 0 3.13, 6.25, 12.5, 25, 50, 60, 70, 80, 90 and 100 µg/mL

Water served as the vehicle control. Mitomycin C and Vinblastine for non-activated system, and Cyclophosphamide for S9-activated system served as positive controls. 

The dose level selection for analysis of micronuclei was based on the cytokinesis blocked proliferation index (CBPI) (the lowest dose with at least 55±5% reduction in CBPI, relative to the solvent control and three lower doses). The results of concurrent cytotoxicity at the highest test concentration relative to the solvent control evaluated microscopically for micronuclei are as follows:

Non-activated 4-hour exposure: CBPI was 1.269 and cytotoxicity was 53% at 150 μg/mL.

S9-activated 4-hour exposure: CBPI was 1.259 and cytotoxicity was 54% at 175 μg/mL.

Non-activated 24-hour exposure: CBPI was 1.271 and cytotoxicity was 59% at 80 μg/mL.

Based on concurrent cytotoxicity, the dose levels selected for analysis of micronuclei were:

Non-activated 4-hour exposure: 0, 25, 50, 100 and 150 µg/mL

Non-activated 24-hour exposure: 0, 12.5, 25, 50 and 80 µg/mL

S9-activated 4-hour exposure: 0, 25, 50, 100 and 175 µg/mL

The percentage of cells with micronucleated binucleated cells in the test substance-treated groups was not statistically significantly increased relative to the solvent control at any dose level (p>0.05, Fisher’s Exact test) in the 4-hour treatment group either in the presence of or the absence of S9 or in the 24-hour treatment group in the absence of S9.

All the positive control compounds induced statistically significant increases in the proportion of cells with micronuclei. The results for the positive and negative controls indicate that all criteria for a valid assay were met.

Under the conditions of this in vitro micronucleus assay using human peripheral blood lymphocyte, 4,5-Diamino-1-hexyl-1 H-pyrazole, dihydrochloride was negative for the induction of micronuclei in both non-activated and S9-activated test systems.

This In Vitro Micronucleus Test is classified as acceptable, and satisfies the guideline requirements of the OECD 487 method.

Reference 4

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 10, 2010 to April 06, 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study well documented, followed guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline for the testing of chemicals. Draft proposal for a new Guideline 487: In vitro micronucleus test

Deviations:

no

GLP compliance:

no

Remarks:

As this study was conducted as an exploratory study it did not fall within the scope of the FDA/EPA Good Laboratory Practice (GLP) regulations

Type of assay:

in vitro mammalian cell micronucleus test

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

Species / strain / cell type:

lymphocytes: obtained from a healthy non-smoking 26 year old adult male on 08 March 2010 for the preliminary toxicity assay and on 22 March 2010 for the definitive toxicity assay.

Details on mammalian cell type (if applicable):

- Type and identity of media: 0.5 mL heparinized blood was inoculated into centrifuge tubes containing 5 mL RPMI-1640 complete medium supplemented with 2% Phytohemagglutinin (PHA).- Properly maintained: Yes- Any known disease in donors: No donor had recent history of radiotherapy, viral infection or the administration of drugs

Metabolic activation:

with and without

Metabolic activation system:

Mammalian liver post-mitochondrial fraction (S9) prepared from male Sprague Dawley rats induced with Aroclor 1254

Test concentrations with justification for top dose:

Pre-Experiment:without S9: 0.255, 0.765, 2.55, 7.65, 25.5, 76.5, 255, 765 and 2550 μg/mLwith S9: 0.255, 0.765, 2.55, 7.65, 25.5, 76.5, 255, 765 and 2550 μg/mLDefinitive Experiment:without S9: 5, 12.5, 25, 50, 75, 100, 125, 150, 175, 200, 250 and 300 μg/mL with S9: 5, 12.5, 25, 50, 75, 100, 125, 150, 175, 200, 250 and 300 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Water- Justification for choice of solvent/vehicle: Based on the solubility of the substance and compatibility with the target cells

Negative solvent / vehicle controls:

yes

Remarks:

water

Positive controls:

yes

Positive control substance:

other: In absence of S9: 0.4 and 0.5 µg/mL Mitomycin C, 10 and 20 ng/mL Vinblastine; In presence of S9: 10 and 15 µg/mL Cyclophosphamide.

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium (5 mL RPMI-1640 complete medium supplemented with 2% Phytohemagglutinin (PHA)).TEST TREATMENT VOLUME: 500 µL DURATION- Treatment with mitogen: Prior to test substance treatment, cells were exposed to mitogen (phytohemagglutinin (PHA)) for 44-48 h.- Exposure duration: 24 h (in absence of S9) and 4 h (in presence of S9)TEST TEMPERATURE: Cultures were incubated at 37 ±1°C in a humidified atmosphere of 5 ± 1% CO2 in air. NUMBER OF REPLICATIONS: DuplicateHARVESTING METHOD: At the end of 4 h treatments, the cells were centrifuged at approx. 1200 rpm for about 5 min and the treatment medium was aspirated, the cells washed with 3-5 mL of CMF-PBS and 5 mL RPMI-1640 complete medium with Cytochalasin B (Cyto-B) at 6 μg/mL was added to each tube. The cultures were returned to the incubator for 20 h. For the 24 h treatment in the absence of S9, Cyto-B at 6 μg/mL was added at the beginning of the treatment. At the end of the 24 h from the initiation of the treatment, the cultures were centrifuged, supernatant was aspirated and the cell pellet was disrupted by flicking the tube. The cells were resuspended in 5 mL 0.075 M KCl and treated for 3 min at room temperature.FIXATION: At the end of the KCl treatment and immediately prior to centrifuging, the cells were gently mixed and approx. 0.5 mL of cold acetic alcohol fixative (methanol:glacial acetic acid, 25:1, v/v) was added to each tube. The tubes were centrifuged, the supernatant aspirated, the cell pellet disrupted and 3-5 mL of fixative added to each tube. The cells were centrifuged again, the supernatant aspirated, the cells resuspended in 3-5 mL of fresh fixative and stored overnight or longer at approximately 2-8°C or the slides were prepared immediately after harvest. SLIDE PREPERATION: Prior to slide preparation, the fixed cells in 15 mL centrifuge tubes were centrifuged and the supernatant was aspirated leaving approx. 0.2 mL above the cell pellet. The cells were resuspended and the cells suspension was applied on clean microscope slides. The slides were left to air dry at room temperature. STAINING: The slides were stained with acridine orange stain at least 1 d prior to use. Slides were stained for 1-2 min in the acridine orange stain solution and then rinsed for several minutes in calcium and magnesium-free Dulbecco’s phosphate buffered-saline (CMF-DPBS). Stained slides were stored prior to and between scoring at 2-8°C.NUMBER OF CELLS EVALUATED: The frequency of lymphocytes containing one, two, or more than two nuclei in 200 cells per culture in the preliminary toxicity assay and 500 cells per culture in the definitive assay was assessed to determine the test article effect on cell cycle kinetics for all test substance treatment groups and the controls.

Evaluation criteria:

The test substance was considered to induce clastogenic and/or aneugenic events if:1. A statistically significant increase in the frequency of MNBN cells at one or more concentrations is observed.2. An incidence of cells with micronuclei at such a concentration that exceeds the negative control historical range established by the test facility is observed.3. A concentration-related increase in the proportion of cells with micronuclei is observed.- The test substance was considered as positive if all of the above criteria are met. - The test substance was considered as negative if none of the above criteria are met. Results which only partially satisfy the above criteria were dealt with on a case-by-case basis. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result. Biological relevance was taken into account, for example consistency of response within and between concentrations, or effects occurring only at high or very toxic concentrations.

Statistics:

Statistical analysis of the percentage of micronucleated binucleated cells was performed using Fisher's exact test. The Fisher's test was used to compare pairwise the percent of micronucleated cells of each treatment group with that of the Cytochalasin B containing solvent control.

Species / strain:

lymphocytes: obtained from a healthy non-smoking 26 year old adult male

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

PRELIMINARY STUDY RESULTS:- Effects of pH: The pH of the highest concentration (2550 μg/mL) of test substance in treatment medium was approx. 7.- Effects of osmolality: The osmolality in treatment medium of the highest concentration tested, 2550 μg/mL, was 252 mmol/kg. The osmolality of the solvent (Water) in treatment medium was 266 mmol/kg. The osmolality of the test material concentrations in treatment medium are acceptable because they did not exceed the osmolality of the solvent by more than 20%.- Precipitation: Visible precipitate was not observed in treatment medium at any dose levels and was soluble in treatment medium at the beginning of the treatment period. Precipitation and hemolysis were observed at doses ≥ 765 μg/mL in the S9 activated 4 h treatment and at doses ≥ 255 μg/mL in the non-activated 24 h treatment.- Cytotoxicity: Substantial toxicity (i.e., 55 ± 5% cytotoxicity based on CPBI was observed at dose levels ≥255 μg/mL in both the non-activated 24 h exposure group and in the S9-activated 4 h exposure group. Based upon the results of the toxicity study and in consultation with the Sponsor, the concentrations selected for testing in the micronucleus assay (definitive assay) were as follows 5, 12.5, 25, 50, 75, 100, 125, 150, 175, 200, 250 and 300 μg/mL.DEFINITIVE STUDY - Precipitation: Visible precipitate was not observed in treatment medium at any dose levels and test substance was soluble in treatment medium at the beginning and end of treatment in the S9 activated 4 h treatment. Visible precipitate was not observed in treatment medium at any dose levels and test substance was soluble in treatment medium at the beginning in the non-activated 24 h treatment. However, precipitation was observed at doses ≥ 100 μg/mL and hemolysis at doses ≥ 250 μg/mL at the end of the non-activated 24 h treatment. - Cytotoxicity: Substantial toxicity (i.e., 55 ± 5% cytotoxicity based on CPBI was observed at dose levels ≥175 μg/mL in the S9-activated 4 h exposure group and at doses ≥75 μg/mL in the non-activated 24 h exposure group. Selections of dose levels for microscopic analysis were 12.5, 50 and 175 μg/mL for the 4 h S9-activated exposure group and 5, 25 and 75 μg/mL for the non-activated 24 h exposure group. Details on the result are provided in 'Table 1 and Table 2' under "Any other information on results incl. tables".Percentage of cells with micronuclei: The percentage of cells with micronuclei in the S9-activated 4 h exposure group and non-activated 24 h exposure group was not significantly increased relative to the solvent control at any dose level (p>0.05, Fisher's Exact test).COMPARISON WITH HISTORICAL CONTROL DATA: Solvent control and positive control data in this study were comparable with the historical control data (From 2007 to 2009).

Remarks on result:

other: strain/cell type:

Remarks:

Migrated from field 'Test system'.

Table 1: 4,5-Diamino-1 -hexyl-1H-pyrazole, dihydrochloride, in the absence of exogenous metabolic activation 4 h treatment, 24 h harvest (Study # 66860)

Treatment Condition (µg/mL)	Total # of Cells Counted	Count per total cells           Cells with # of nuclei			CBPI^1	Cytotoxicity^2	Percentage of MNBN^3Cells/Total BN^4Cells Counted
		1	2	>2
Water	500	252	235	13	1.509	-	0.2
	500	262	228	10
4,5-Diamino-1-hexyl-1H-pyrazole, dihydrochloride
5	500	280	189	31	1.502	1%	0.1
	500	278	193	29
12.5	500	326	154	20	1.388	24%	Not evaluated
	500	331	144	25
25	500	340	143	17	1.365	28%	0.2
	500	331	150	19
50	500	368	119	13	1.275	46%	Not evaluated
	500	378	114	8
75	500	392	106	2	1.216	58%	0.3
	500	399	96	5
100	500	437	60	3	1.129	75%	Not evaluated
	500	437	63	0
125	500	445	55	0	1.103	80%	Not evaluated
	500	452	48	0
150	500	460	40	0	1.082	84%	Not evaluated
	500	458	42	0
175	500	468	32	0	1.062	88%	Not evaluated
	500	471	28	1
200	500	482	18	0	1.039	92%	Not evaluated
	500	479	21	0
250	500	490	10	0	1.022	96%	Not evaluated
	500	488	12	0
300	500	500	0	0	1	100%	Not evaluated
	500	500	0	0
MMC, 0.4	500	393	104	3	1.208	59%	5.3
	500	402	98	0
MMC, 0.5	500	426	74	0	1.149	71%	Not evaluated
	500	425	75	0
VB, 10 (ng/mL)	500	358	136	6	1.28	45%	2.4
	500	373	122	5
VB, 20 (ng/mL)	500	468	30	2	1.065	87%	Not evaluated
	500	470	29	1

Table 2: 4,5 -Diamino-1-hexyl-1H-pyrazole, dihydrochloride, in the presence of exogenous metabolic activation 4 h treatment, 24 h harvest (Study # 66860)

Treatment Condition (µg/mL)	Total # of Cells Counted	Count per total cells			CBPI^1	Cytotoxicity ^2	Percentage of MNBN^3Cells/Total BN^4Cells Counted
		Cells with # of nuclei
		1	2	>2
Water	500	246	248	6	1.516	-	0.2
	500	247	250	3
4,5-Diamino-1-hexyl-1H-pyrazole, dihydrochloride
5	500	267	228	5	1.486	6%	Not evaluated
	500	261	230	9
12.5	500	259	230	11	1.498	3%	0.2
	500	260	234	6
25	500	305	187	8	1.399	23%	Not evaluated
	500	307	190	3
50	500	327	170	3	1.348	33%	0.3
	500	332	164	4
75	500	342	156	2	1.331	36%	Not evaluated
	500	334	161	5
100	500	336	162	2	1.333	35%	Not evaluated
	500	335	163	2
125	500	347	151	2	1.309	40%	Not evaluated
	500	346	154	0
150	500	357	143	0	1.285	45%	Not evaluated
	500	358	142	0
175	500	379	121	0	1.252	51%	0.2
	500	370	129	1
200	500	385	115	0	1.224	57%	Not evaluated
	500	392	107	1
250	500	419	81	0	1.157	70%	Not evaluated
	500	424	76	0
300	500	436	64	0	1.123	76%	Not evaluated
	500	441	59	0
CP, 10	500	405	95	0	1.198	62%	2.6
	500	397	103	0
CP, 15	500	420	80	0	1.155	70%	Not evaluated
	500	425	75	0

¹CBPI = Cytokinesis-Block Proliferation Index

²Relative to the solvent control

³MNBN = micronucleated binucleated

⁴BN = binucleated

Not evaluated =Dose levels were tested but not evaluated for MNBN Cells.

CP = Cyclophosphamide

Conclusions:

Interpretation of results (migrated information):negative4,5-Diamino-1-hexyl-1H-pyrazole, dihydrochloride was considered non-mutagenic in human peripheral blood lymphocyte micronucleus assay (with and without metabolic activation). The toxic potential of dihdrochloride and sulfateh 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 in-vitro micronucleus test of 4,5-Diamino-1-hexyl-1H-pyrazole, dihydrochloride, following method equivalent or similar to OECD guideline 487 (In vitro micronucleus test) was conducted using duplicate human lymphocyte cultures prepared from blood of male donor.

The cells were treated for 24 h in the non-activated test system and for 4 h in the S9-activated test system, and all cells were harvested 24 h after treatment initiation. Following dose levels were tested:

Without S9: 5, 12.5, 25, 50, 75, 100, 125, 150, 175, 200, 250 and 300 μg/mL

With S9: 5, 12.5, 25, 50, 75, 100, 125, 150, 175, 200, 250 and 300 μg/mL

The above dose levels were selected on the basis of apreliminary toxicity test.

Vehicle control (water) and the following positive controls were also included in the study:

In absence of S9: 0.4 and 0.5 µg/mL Mitomycin C, 10 and 20 ng/mL Vinblastine

In presence of S9: 10 and 15 µg/mL Cyclophosphamide.

Visible precipitate was not observed in treatment medium at any dose level and test substance was soluble in treatment medium at the beginning and end of treatment in the S9 activated 4 h treatment. Visible precipitate was not observed in treatment medium at any dose levels and test substance was soluble in treatment medium at the beginning in the non-activated 24 h treatment. However, precipitation was observed at doses ≥ 100 μg/mL and hemolysis at doses ≥ 250 μg/mL at the end of the non-activated 24 h treatment.

Substantial toxicity [i.e., 55±5% cytotoxicity based on Cytokinesis-Block Proliferation Index (CPBI)] was observed at dose levels ≥175 μg/mL in the S9 -activated 4 h exposure group and at doses ≥75 μg/mL in the non-activated 24 h exposure group. Selections of dose levels for microscopic analysis were 12.5, 50 and 175 μg/mL for the 4 h S9-activated exposure group and 5, 25 and 75 μg/mL for the non-activated 24 h exposure group.

The percentage of cells with micronuclei in the S9-activated 4 h exposure group and non-activated 24-h exposure group was not significantly increased relative to the solvent control at any dose level (p>0.05, Fisher's Exact test).

In conclusion, 4,5-Diamino-1-hexyl-1H-pyrazole, dihydrochloride was considered non-mutagenic inhuman peripheral blood lymphocyte micronucleus assay (with and without metabolic activation).

This mammalian gene mutation test is classified as acceptable, and satisfies the guideline requirements of the OECD 487 method.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)