2-benzyl-2-methyl-3-butenitrile

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:

2010-09-28 to 2011-01-10

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Test according to EU-Method B.13/14 adopted 31. May, 2008 "Mutagenicity - Reverse mutation test using bacteria", Test according to OECD Guideline for Testing of Chemicals Part 471, adopted 21. Jul. 1997 "Bacterial Reverse Mutation Test", Test according to "Revised OECD Principles of Good Laboratory Practice" (Paris, 1997).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

CITROWANIL B, colourless to pale yellow liquid

IUPAC Name:

CITROWANIL B, colourless to pale yellow liquid

Constituent 2

Reference substance name:

2-benzyl-2-methyl-3-butennitril

IUPAC Name:

2-benzyl-2-methyl-3-butennitril

Constituent 3

Reference substance name:

2-benzyl-2-methyl-3-butenitrile

EC Number:

407-870-4

EC Name:

2-benzyl-2-methyl-3-butenitrile

Cas Number:

97384-48-0

Molecular formula:

C12H13N

IUPAC Name:

2-benzyl-2-methylbut-3-enenitrile

Details on test material:

Citrowanil B; C12H13N

Method

Target gene:

TA97a: hisD6610, uvrB, pKM 101, rfa
TA98: hisD3052, uvrB, pKM 101, rfa
TA100: hisG46, uvrB, pKM 101, rfa
TA102: hisG428,pKM 101, rfa
TA1535: hisG46, uvrB, rfa

Species / strain

Species / strain 1

Species / strain / cell type:

other: S. typhimurium TA97a

Additional strain / cell type characteristics:

other: hisD6610, uvrB, pKM 101, rfa

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

other: hisG428,pKM 101, rfa

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: hisG46, uvrB, pKM 101, rfa

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: hisD3052, uvrB, pKM 101, rfa

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

other: hisG46, uvrB, rfa

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix with 4% S9, produced from the livers of male Spraque-Dawley rats wich were treated with 500 mg Aroclor 1254/kg body weight intraperitoneally

Test concentrations with justification for top dose:

First Experiment: 4998 / 1499 / 500 / 150 / 50 µg/plate
Second Experiment: 501 / 150 / 50 / 15 / 5 µg/plate
Third Experiment: 504 / 252 / 126 / 63 / 32 / 16 / 8 µg/plate

Vehicle / solvent:

DMSO

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Migrated to IUCLID6: 20 µg/plate in DMSO for TA98 with metabolic activation

Controls 2

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Migrated to IUCLID6: 1 µg/plate in water for TA100, TA1535 without metabolic activation

Controls 3

Positive controls:

yes

Positive control substance:

other: 1 µg/plate 2-Amino-anthracene in DMSO for TA97a, TA100, TA102, TA1535 with metabolic activation

Controls 4

Positive controls:

yes

Positive control substance:

other: 20 µg/plate 4-Nitro-1,2-phenylene diaminein DMSO for TA97a, TA98, TA102 without metabolic activation

Details on test system and experimental conditions:

Per strain and dose, four plates with and four plates without S9 were used.
The first and the second experiment were carried out according to the plate incorporation method, the third experiment was carried out according to the Pre-incubation method.
Each test item solution was membrane filtrated before use to accomplish sterility.

Plate incorporation method:
0.1 mL of the appropriate solution of the test item was given into a sterile tube. After mixing with 0.1 mL overnight culture of the respective strain and 0.5 mL phosphate buffer (treatement without S9) or 0.5 mL S9 mix (treatement with S9), 2 mL Top-Agar, containing 10 mL 0.5 mMol histidine-biotin-solution per 100 mL, were added.
Pre-incubation method:
0.1 mL of the appropriate solution of the test item was given into a sterile tube. After mixing with 0.1 mL overnight culture of the respective strain and 0.5 mL phosphate buffer (treatement without S9) or 0.5 mL S9 mix (treatement with S9) were added. The mixture was incubated in an incubating chamber at 37°C for 20 minutes. During this time the vessles were aerated through careful shaking. Then 2 mL Top-Agar, containing 10 mL 0.5 mMol histidine-biotin-solution per 100 mL, was added.
In each case the mixture was vortexed gently, then poured on a minimal glucose plate and distributed evenly, rotating the plate. The plates were closed and left to harden for a few minutes, then inverted and placed in the dark incubator at 37°C.

Evaluation criteria:

The colonies were counted visually after 48 hours incubation, the numbers were recorded.

A test item is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor f(I) ≥ 2) in at least one strain can be observed. A concentration-releated increase over the range tested can also be taken as a sign of mutagenic activity.

Statistics:

A spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control.
The increase factor f(I) of revertant induction (mean revetants divided by mean spontaneous revertants [vehicle control]) and the absolute number of revertants (mean revetants less mean spontaneous revertants [vehicle control]) were calculated, too.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: yes at 4998 / 1499 µg/plate, no at 500 / 150 / 50 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: yes at 4998 / 1499 µg/plate, no at 500 / 150 / 50 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: yes at 4998 / 1499 µg/plate, no at 500 / 150 / 50 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: yes at 4998 / 1499 µg/plate, no at 500 / 150 / 50 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

other: S. typhimurium TA97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: other: Plate incorporation and Pre-incubation method

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Survey of the Findings:

The mean revertant values of the four replicates are presented in the following table.

 

Exp.	Strain		TA97a		TA98		TA100		TA102		TA1535
	Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
1	H2O	Mean	124	115	11	17	128	113	218	233	14	12
	DMSO	Mean	112	105	11	14	112	117	191	294	12	13
	Pos. Contr.	Mean f(I)	768 6.9	789 7.5	752 68.4	142 10.1	633 5.0	496 4.2	817 4.3	657 2.2	242 17.3	144 11.1
	4998 µg/pl.	Mean f(I)	110 0.98	67 0.64	1 0.09	0 0.00	0 0.00	0 0.00	20 0.10	0 0.00	0 0.00	0 0.00
	1499 µg/pl.	Mean f(I)	57 0.51	115 1.10	0 0.00	0 0.00	0 0.00	27 0.23	23 0.12	15 0.05	0 0.00	0 0.00
	500 µg/pl.	Mean f(I)	101 0.90	111 1.06	9 0.82	10 0.71	123 1.10	129 1.10	229 1.20	217 0.74	10 0.83	15 1.15
	150 µg/pl.	Mean f(I)	113 1.01	132 1.26	10 0.91	11 0.79	130 1.16	130 1.11	206 1.08	241 0.82	11 0.92	12 0.92
	50 µg/pl.	Mean f(I)	116 1.04	113 1.08	9 0.82	12 0.86	125 1.12	111 0.95	282 1.48	234 0.80	13 1.08	13 1.00
2	H2O	Mean	112	101	11	15	99	115	208	200	14	14
	DMSO	Mean	105	97	11	16	90	111	253	176	10	12
	Pos. Contr.	Mean f(I)	310 3.0	414 4.3	305 27.7	260 16.3	674 6.8	1084 9.8	639 2.5	796 4.5	226 16.1	122 10.2
	501 µg/pl.	Mean f(I)	114 1.09	107 1.10	10 0.91	12 0.75	108 1.20	99 0.89	151 0.60	139 0.79	10 1.00	13 1.08
	150 µg/pl.	Mean f(I)	117 1.11	125 1.29	11 1.00	15 0.94	100 1.11	148 1.33	190 0.75	176 1.00	13 1.30	9 0.75
	50 µg/pl.	Mean f(I)	109 1.04	99 1.02	11 1.00	12 0.75	126 1.40	83 0.75	158 0.62	168 0.95	13 1.30	14 1.17
	15 µg/pl.	Mean f(I)	110 1.05	91 0.94	11 1.00	13 0.81	88 0.98	113 1.02	179 0.71	182 1.03	11 1.10	18 1.50
	5 µg/pl.	Mean f(I)	94 0.90	105 1.08	12 1.09	12 0.75	111 1.23	84 0.76	213 0.84	205 1.16	13 1.30	16 1.33
3	H2O	Mean	110	108	11	16	78	127	199	174	16	14
	DMSO	Mean	105	105	14	11	98	106	196	186	17	17
	Pos. Contr.	Mean f(I)	320 3.1	303 2.9	231 16.5	117 10.6	254 3.3	221 2.1	886 4.5	514 2.8	237 14.8	208 12.2
	504 µg/pl.	Mean f(I)	115 1.10	135 1.29	6 0.43	9 0.82	29 0.30	91 0.86	222 1.13	224 1.2	9 0.53	10 0.59
	252 µg/pl.	Mean f(I)	106 1.01	104 0.99	5 0.36	12 1.09	29 0.30	84 0.79	230 1.17	188 1.01	13 0.76	14 0.82
	126 µg/pl.	Mean f(I)	121 1.15	111 1.06	8 0.57	13 1.18	79 0.81	104 0.98	268 1.37	192 1.03	10 0.59	19 1.12
	63 µg/pl.	Mean f(I)	111 1.06	112 1.07	10 0.71	14 1.27	99 1.01	126 1.19	159 0.81	174 0.94	11 0.65	16 0.94
	32 µg/pl.	Mean f(I)	108 1.03	107 1.02	10 0.71	13 1.18	108 1.10	88 0.83	211 1.08	258 1.39	11 0.65	14 0.82
	18 µg/pl	Mean f(I)	112 1.07	108 1.03	9 0.64	18 1.64	88 0.90	82 0.77	219 1.12	228 1.23	12 0.71	14 0.82
	8 µg/pl	Mean f(I)	112 1.07	113 1.08	8 0.57	16 1.45	107 1.09	110 1.04	218 1.11	156 0.84	11 0.65	12 0.71

 f(I) = increase factor

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation
negative with metabolic activation

CITROWANIL B is not mutagenic in the Bacterial Reverse Mutation Test using Salmonella typhimurium, strains TA97a, TA98, TA100, TA102 and TA1535.

Executive summary:

Three valid experiments were performed to evaluate the mutagenic potential of CITROWANIL B in the Bacterial Reverse Mutation Test.

 

First Experiment:

Five concentrations of the test item, dissolved in dimethyl sulfoxid (DMSO) (ranging from 4998 to 50 µg/plate) were used. Five genetically manipulated strains ofSalmonella typhimurium(TA97a, TA98, TA100, TA102 and TA1535) were exposed to the test item both in the presence and in the absence of a metabolic activation system (S9) for 48 hours using the plate incorporation method.

Signs of toxicity towards the bacteria could be observed in the two highest concentrations (4998 and 1499 µg/plate).

None of the three last concentrations (500, 150 and 50 µg/plate) caused a significant increase in the numbers of revertant colonies in the tested strains. The test item didn’t show any mutagenic effect in the first experiment.

The sterility control and the determination of the titre didn’t show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were within the normal range. All positive controls showed mutagenic effects with and without metabolic activation.

 

Second Experiment:

Because of the results of the first experiment, a second experiment was performed, using five concentrations of the test item (ranging from 501 to 5 µg/plate), using the plate incorporation method, too.

The test item didn’t show mutagenic effects in the second experiment, either.

No signs of toxicity towards the bacteria could be observed.

The sterility control and the determination of the titre didn’t show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were within the normal range. All positive controls showed mutagenic effects with and without metabolic activation.

 

Third Experiment:

To verify the first and the second experiment, a third experiment was performed, using seven concentrations of the test item (ranging from 504 to 8 µg/plate) and a modification in study performance(pre-incubation method).

The test item didn’t show mutagenic effects in the third experiment, either.

No signs of toxicity towards the bacteria could be observed.

The sterility control and the determination of the titre didn’t show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were within the normal range. All positive controls showed mutagenic effects with and without metabolic activation.

 

Under the conditions of the test, the test item didn’t show mutagenic effects in Salmonella typhimurium, strains TA97a, TA98, TA100, TA102 and TA1535.

Therefore, no concentration-effect relationship could be determined.

 

The test item CITROWANIL B is considered as “not mutagenic under the conditions of the test”.