1-butyl-2,3-dicyclohexyl-1-methylguanidine

Genetic toxicity: in vitro

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 18 March 2009 to 16 July 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Method

Target gene:

The histidine gene

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

other: histidine deficient (His G 46), rfa and uvrb mutations

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

other: histidine deficient (His G 46), rfa and uvrb mutations, addition of the plasmid pKM101

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 102

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

other: histidine deficient (His G 428 (pAQ1), rfa mutation, addition of the plasmid pKM101

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 1537

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

other: histidine deficient (His C 3076), rfa and uvrb mutations

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

other: histidine deficient (His D 3052), rfa and uvrb mutations, addition of the plasmid pKM101

Metabolic activation:

with and without

Metabolic activation system:

S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254

Test concentrations with justification for top dose:

- Preliminary toxicity test: 10, 100, 500, 1000, 2500 or 5000 µg/plate
- Mutagenicity experiments: Since the test item was toxic in the preliminary test, the choice of the highest dose-level retained for the main experiments was based on the level of toxicity. See "Any other information" for details.

Vehicle / solvent:

Vehicle(s)/solvent(s) used: DMSO (Batch number K36449950707 and K36994550731, supplier: Merck KGaA, Darmstadt, Germany)

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without S9 mix: sodium azide (1 µg/plate) for TA 1535 and TA 100; 9-Aminoacridine (50 µg/plate) for TA 1537; 2-Nitrofluorene (0.5 µg/plate) for TA 98: Mitomycin C (0.5 µg/plate) for TA 102.

Remarks:

In the absence of S9 Mix

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: With S9 mix: 2-Anthramine (2 µg/plate) for TA 1535, TA 1537 and TA 98; Benzo(a)pyrene (5 µg/plate) for TA 100 and 2-Anthramine (10 µg/plate) for TA 102.

Remarks:

In the presence of S9 Mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) except for the second test with S9 mix, which was performed according to the preincubation method

DURATION
- Preincubation period:
Plate incorporation method: not applicable
Preincubation method: 60 minutes at 37 °C
- Exposure duration: 48 to 72 hours at 37°C

NUMBER OF REPLICATIONS: triplicates

DETERMINATION OF CYTOTOXICITY
- Method: observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

SELECTION AGENT (mutation assays):
- histidine production

Evaluation criteria:

A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive
result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

Statistics:

No data

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: no precipitate was observed in the Petri plates when scoring the revertants at any dose-level.
- Other confounding effects: no data

PRELIMINARY TOXICITY TEST: A moderate to strong toxicity (decrease in the number of revertants and thinning of the bacterial lawn) was noted at dose-levels ≥ 1000 μg/mL in the TA 100 strain and at dose-levels ≥ 2500 μg/mL in the TA 98 and TA 102 strains, either with or without S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Without S9 mix: A slight increase in the number of revertants was noted in the TA 1537 strain in the second experiment (2.2-fold the vehicle control value). Since this increase did not reach the threshold of 3-fold the vehicle control value and since it was due to only one out of the three plates used, and was neither dose-related nor observed in the first experiment, it was not considered as biologically relevant. A slight increase in the number of revertants was noted in the TA 98 strain in the second experiment (2-fold the vehicle control value). This increase reached the threshold of 2- fold the vehicle control value but it was neither dose-related, nor observed in the first and third experiment. Therefore, it was considered as non-biologically relevant.
- With S9 mix: In the first experiment, using the direct plate incorporation method, a moderate to strong toxicity was noted at dose-levels ≥ 1250 μg/plate in the TA 1535 and TA 100 strains and at dose-levels ≥ 2500 μg/plate in the TA 1537, TA 98 and TA 102 strains. In the second experiment, performed using the preincubation method, a moderate to strong toxicity was noted at dose-levels ≥ 312.5 μg/plate in the TA 1535, TA 1537, TA 98 and TA 100 strains and at dose-levels ≥ 625 μg/plate towards the TA 102 strain.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Without S9 mix: A moderate to strong toxicity was noted at dose-levels ≥ 625 μg/plate in the TA 1537, TA 98, TA 100 and TA 102 strains and at dose-levels ≥ 1250 μg/plate in the TA 1535 strain.
- With S9 mix: In the first experiment, using the direct plate incorporation method, a moderate to strong toxicity was noted at dose-levels ≥ 1250 μg/plate in the TA 1535 and TA 100 strains and at dose-levels ≥ 2500 μg/plate in the TA 1537, TA 98 and TA 102 strains. In the second experiment, performed using the preincubation method, a moderate to strong toxicity was noted at dose-levels ≥ 312.5 μg/plate in the TA 1535, TA 1537, TA 98 and TA 100 strains and at dose-levels ≥ 625 μg/plate towards the TA 102 strain.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 7.6.1/1: Preliminary toxicity test – Direct plate incorporation method

Strain	Compound	Dose level per plate	S-9 mix	Mean revertant colony count	SD	Ratio treated / solvent	Individual revertant colony count
TA 98	DMSO TEST ITEM             DMSO TEST ITEM	10 µg 100 µg 500 µg 1000 µg 2500 µg 5000 µg     10 µg 100 µg 500 µg 1000 µg 2500 µg 5000 µg	- - - - - - -   + + + + + + +	20 47 25 32 22 5 0   41 30 44 48 37 14 0	- - - - - - -   - - - - - - -	2.4 1.3 1.6 1.1 0.3 0.0     0.7 1.1 1.2 0.9 0.3 0.0	20 47 25 32 22 5ST 0ST   41 30 44 48 37 14MT 0ST
TA 100	DMSO TEST ITEM             DMSO TEST ITEM	10 µg 100 µg 500 µg 1000 µg 2500 µg 5000 µg     10 µg 100 µg 500 µg 1000 µg 2500 µg 5000 µg	- - - - - - -   + + + + + + +	145 139 187 145 109 36 0   147 159 141 164 132 5 0	- - - - - - -   - - - - - - -	1.0 1.3 1.0 0.8 0.2 0.0     1.1 1.0 1.1 0.9 0.0 0.0	145 139 187 145 109MT 36ST 0ST   147 159 141 164 132 5 St 0 St
TA 102	DMSO TEST ITEM             DMSO TEST ITEM	10 µg 100 µg 500 µg 1000 µg 2500 µg 5000 µg     10 µg 100 µg 500 µg 1000 µg 2500 µg 5000 µg	- - - - - - -   + + + + + + +	396 405 432 322 325 85 0   517 526 578 519 395 157 0	- - - - - - -   - - - - - - -	1.0 1.1 0.8 0.8 0.2 0.0     1.0 1.1 1.0 0.8 0.3 0.0	396 405 432 322 325 85ST 0ST   517 526 578 519 395 157MT 0ST

SD: Standard Deviation

MT: Moderate toxicity

ST: Strong toxicity

- Absence of S9

+ Presence of S9

 

Table 7.6.1/2: First experiment – Direct plate incorporation method

Strain	Compound	Dose level per plate	S-9 mix	Mean revertant colony count	SD	Ratio treated / solvent	Individual revertant colony count
TA 1535	DMSO TEST ITEM         NAN3   DMSO TEST ITEM         2 AM	78.1 µg 156.3 µg 312.5 µg 625 µg 1250 µg 1 µg     156.3 µg 312.5 µg 625 µg 1250 µg 2500 µg 2 µg	- - - - - - -   + + + + + + +	26 21 33 25 32 20 681   21 22 20 17 15 6 175	8 8 8 9 3 3 18   3 2 2 7 3 5 23	0.8 1.3 1.0 1.2 0.8 25.8     1.0 1.0 0.8 0.7 0.3 8.3	22, 35, 22 30, 16,16 41, 26, 32 31, 30, 15 34, 34, 28 18, 18, 24 687, 695, 660   18, 23, 22 23, 22, 20 19, 20, 22 23, 19, 10 14MT, 18MT, 13MT 11ST, 2ST, 5ST 183, 150, 193
TA 1537	DMSO TEST ITEM           9AA   DMSO TEST ITEM         2 AM	39.1 µg 78.1 µg 156.3 µg 312.5 µg 625 µg 1250 µg 50 µg     156.3 µg 312.5 µg 625 µg 1250 µg 2500 µg 2 µg	- - - - - - - -   + + + + + + +	10 7 13 14 12 15 8 1036   7 13 9 14 12 7 177	8 1 3 3 5 1 5 273   3 1 5 3 2 3 8	0.7 1.4 1.4 1.2 1.5 0.9 107.1     1.7 1.3 1.9 1.7 1.0 24.1	6, 9, 14 7, 8, 6 11, 16, 13 13, 11, 17 17, 11, 8 14, 16, 14 6MT, 5MT, 14MT 911, 847, 1359   10, 5, 7 14, 12, 12 13, 4, 11 12, 12, 18 13, 14, 10 11ST, 5ST, 5ST 181, 181, 168
TA 98	DMSO TEST ITEM         2NF   DMSO TEST ITEM         2 AM	78.1 µg 156.3 µg 312.5 µg 625 µg 1250 µg 0.5 µg     156.3 µg 312.5 µg 625 µg 1250 µg 2500 µg 2 µg	- - - - - - -     + + + + + +	31 36 23 25 28 25 201   41 34 42 34 23 7 1606	10 3 9 7 3 5 12   1 7 7 8 8 2 174	1.2 0.7 0.8 0.9 0.8 6.5     0.8 1.0 0.8 0.6 0.2 38.9	38, 20, 35 40, 34, 34 32, 23, 14 32, 26, 18 29, 25, 30 20, 25, 30 190, 198, 214   41, 41, 42 32, 29, 42 35, 49, 42 32, 43, 28 20, 17, 32 8ST, 7ST, 5ST 1637, 1419, 1762
TA 100	DMSO TEST ITEM           NAN3   DMSO TEST ITEM           BAP	39.1 µg 78.1 µg 156.3 µg 312.5 µg 625 µg 1250 µg 1 µg     78.1 µg 156.3 µg 312.5 µg 625 µg 1250 µg 2500 µg 5 µg	- - - - - - - -   + + + + + + + +	157 132 153 145 153 104 21 1027   128 112 110 110 86 27 5 391	3 11 22 20 8 4 20 28   12 13 14 9 14 3 5 32	0.8 1.0 0.9 1.0 0.7 0.1 6.5     0.9 0.9 0.9 0.7 0.2 0.0 3.1	156, 155, 160 126, 144, 125 153, 175, 132 146, 125, 164 162, 152, 146 108MT, 102MT, 101MT 44ST, 11ST, 7ST 1035, 996, 1050   137, 114, 133 119, 97, 121 108, 125, 97 103, 120, 107 97, 71, 91 30MT, 26MT, 24MT 1ST, 10ST, 5ST 422, 358, 392
TA 102	DMSO TEST ITEM           MMC   DMSO TEST ITEM         2 AM	19.5 µg 39.1 µg 78.1 µg 156.3 µg 312.5 µg 625 µg 0.5 µg     156.3 µg 312.5 µg 625 µg 1250 µg 2500 µg 10 µg	- - - - - - - -   + + + + + + +	383 330 398 395 425 399 224 3227   439 316 596 554 321 139 3992	22 48 32 23 25 58 57 77   15 90 17 32 51 15 96	0.9 1.0 1.0 1.1 1.0 0.6 8.4     0.7 1.4 1.3 0.7 0.3 9.1	392, 400, 358 381, 323, 286 361, 413, 419 418, 396, 372 454, 411, 410 333, 441, 424 159MT, 249MT, 165MT 3286, 3140, 3256   544, 438, 425 365, 372, 212 611, 578, 598 543, 590, 528 262, 344, 356 126MT, 156MT, 135MT 4079, 4009, 3889

SD: Standard Deviation

MT: Moderate toxicity

ST: Strong toxicity

- Absence of S9

+ Presence of S9

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the experimental conditions, the test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

Executive summary:

In a reverse gene mutation assay in bacteria, strains TA1535, TA1537, TA98, TA100 and TA102 of S. typhimurium were exposed to the substance (> 98%).

A preliminary toxicity test was performed to define the dose-levels of the substance to be used for the mutagenicity study. The test item was then tested in three independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254. All experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method.

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered as valid.

Since the test item was toxic in the preliminary test, the choice of the highest dose-level retained for the main experiments was based on the level of toxicity, according to the criteria specified inthe international guidelines.

No precipitate was observed in the Petri plates when scoring the revertants at any of thedose-levels tested.

In the absence of S9 mix, a moderate to strong toxicity was noted at dose-levels ≥ 625μg/plate in the TA 1537, TA 98, TA 100 and TA 102 strains and at dose-levels ≥ 1250μg/plate in the TA 1535 strain. A slight increase in the number of revertants was noted in the TA 1537 strain in the second experiment (2.2-fold the vehicle control value). Since this increase did not reach the threshold of 3-fold the vehicle control value and since it was due to only one out of the three plates used, and was neither dose-related nor observed in the first experiment, it was not considered as biologically relevant. A slight increase in the number of revertants was noted in the TA 98 strain in the second experiment (2-fold the vehicle control value). This increase reached the threshold of 2- fold the vehicle control value but it was neither dose-related, nor observed in the first andthird experiment. Therefore, it was considered as non-biologically relevant.

In the presence of S9 mix, in the first experiment, using the direct plate incorporation method, a moderate to strong toxicity was noted at dose-levels ≥ 1250μg/plate in the TA 1535 and TA 100 strains and at dose-levels ≥ 2500μg/plate in the TA 1537, TA 98 and TA 102 strains. In the second experiment, performed using the preincubation method, a moderate to strong toxicity was noted at dose-levels ≥ 312.5μg/plate in the TA 1535, TA 1537, TA 98 and TA 100 strains and at dose-levels ≥ 625μg/plate towards the TA 102 strain.

The test item did not induce any noteworthy increase in the number of revertants, in any of thefive tester strains.

Under the experimental conditions, the test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

This study is classified as acceptable. This study satisfies the requirement of OECD 471 guideline for in vitro mutagenicity (bacterial reverse gene mutation).