1,4-dibromobutane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test:

The test chemical did not induce mutation in the Salmonella typhimurium strains both in the presence and absence of S9 metabolic activation system and hence is not likely to be mutagenic under the conditions of this study.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from experimental report.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

To evaluate the mutagenic potential of test chemical in Salmonella typhimurium strain TA 1535, TA 100, TA 1537, TA 98 by Ames test.

GLP compliance:

not specified

Type of assay:

bacterial forward mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 100, TA 1537, TA 98

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

other: All strains show a considerably reduced hydrophilic polysaccharide layer (rfa), which leads to an increase in permeability to lipophilic substances.

Cytokinesis block (if used):

not specified

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix was prepared from Sprague-Dawley rats livers induced with Aroclor 1254.

Test concentrations with justification for top dose:

1st Experiment
Strains: TA 1535. TA 100. TA 1537, TA 98
Dose.: 0, 20, 100, 500. 2500 and 5000 µg/plate

2nd Experiment
Strains: TA 100, TA 98
Domes: 0, 20. 100, 500 and 1000 µg/plate

3rd Experiment
Strain: TA 100
Doses: 0, 50, 100, 500, 1000 and 2500 µg/plate

4th Experiment
Strains: TA 1535, JA 1537
Doses: 0, 20, 100, 500, 1000 and 1500 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Complete solubility of the test substance in DMSO.

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: + S-9 mix 10 µg 2-aminoanthracene for the strains TA 100, TA 98, TA 1537 and TA 1535 - S-9 mix 5µg N-methyl-N-nitro-Nnitroso;TA 100 and TA 1535 10 µg 4 -nitro-o-phenylenediamine ;strain TA 98 100 µg 9-aminoacridine chloride monohydrate ; TA 1537.

Details on test system and experimental conditions:

METHOD OF APPLICATION: Standard Plate method
- Cell density at seeding (if applicable): >108 bacteria/ml

DURATION
- Preincubation period: 16 hours
- Exposure duration:2days

DETERMINATION OF CYTOTOXICITY
- Method: A bacteriotoxic effect OTHER EXAMINATIONS:

Evaluation criteria:

A substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate
- dose-response relationship
- reproducibility of the results.

Statistics:

Yes ,,Mean ±standard deviation was observed.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Positive controls validity:

other: As though the test substace was used as a positive control

Additional information on results:

No mutagenic effect were observed in the salmonella typhimurium strain TA 100 used in expriment number three , when exposed to test substance.

Remarks on result:

other: No mutagenic effect were observed.

TABLE 1

STRAIN: TA 1535

AMES TEST WITH: 86/221

METHOD: STANDARD PLATE TEST

 

DOSE MCG/PL	REVERTANTS/PLATES						TITER DIL	QUOTIENT
	-S9	M	SD	+S9*	M	SD	EXP-6	-S9	+S9*
NEGATIVE CONTROL DMSO	15 18 21	18	3	21 17 19	19	2	45 41 3	1.0	1.0
20	18 16 14	16	2	21 21 26	23	3		0.9	1.2
100	18 20 14	17	3	22 22 24	23	1		1.0	1.2
500	20 24 21	22	2	28 28 24	27	2		1.2	1.4
2500	B B B	-	-	13B 12B 8B	11	3	0 0 0	-	0.6
5000	B B B	-	-	B B B	-	-	0 0 0	-	-
POSITIVE CONTROL 2-AA 10				320 265 242	276	40			14.5
POSITIVE CONTROL MNNG 5	1960 2350 2300	2203	212					122.4

* : S-9 FRACTION/COFACTORS = 3: 7         EXP :EXP. TO 10

B: REDUCED his BACKGROUND           

           

 

 

 

 

 

 

 

TABLE 2

STRAIN: TA 100

AMES TEST WITH: 86/221

METHOD: STANDARD PLATE TEST

DOSE MCG/PL	REVERTANTS/PLATES						TITER DIL	QUOTIENT
	-S9	M	SD	+S9*	M	SD	EXP-6	-S9	+S9*
NEGATIVE CONTROL DMSO	94 109 117	107	12	110 104 107	107	3	15 28 11	1.0	1.0
20	113 119 111	114	4	102 118 125	115	12		1.1	1.1
100	116 117 111	112	7	119 125 113	119	6		1.1	1.1
500	113 124 146	128	17	137 139 138	138	1		1.2	1.3
2500	B B B	-	-	74B 73B 80B	76	4	0 0 0	-	0.7
5000	B B B	-	-	B B B	-	-	0 0 0	-	-
POSITIVE CONTROL 2-AA 10				1780 1860 1910	1850	66			17.3
POSITIVE CONTROL MNNG 5	1740 1930 1850	1840	95					17.3

 

* : S-9 FRACTION/COFACTORS = 3: 7         EXP :EXP. TO 10

B: REDUCED his BACKGROUND                       

 

 

 

 

 

 

TABLE 3

STRAIN: TA 1537

AMES TEST WITH: 86/221

METHOD: STANDARD PLATE TEST

DOSE MCG/PL	REVERTANTS/PLATES						TITER DIL	QUOTIENT
	-S9	M	SD	+S9*	M	SD	EXP-6	-S9	+S9*
NEGATIVE CONTROL DMSO	13 13 10	12	2	8 10 10	9	1	16 13 11	1.0	1.0
20	9 13 8	10	3	15 11 8	11	4		0.8	1.2
100	11 10 11	11	1	8 10 8	9	1		0.9	0.9
500	16 9 11	12	4	9 9 16	11	4		1.0	1.2
2500	B B B	-	-	5B 8B 9B	7	2	0 0 0	-	0.8
5000	B B B	-	-	B B B	-	-	0 0 0	-	-
POSITIVE CONTROL 2-AA 10				199 194 195	196	3			21.0
POSITIVE CONTROL MNNG 5	744 661 833	746	86					62.2

 

* : S-9 FRACTION/COFACTORS = 3: 7         EXP :EXP. TO 10

B: REDUCED his BACKGROUND                       

 

 

 

 

 

 

TABLE 4

STRAIN: TA 98

AMES TEST WITH: 86/221

METHOD: STANDARD PLATE TEST

DOSE MCG/PL	REVERTANTS/PLATES						TITER DIL	QUOTIENT
	-S9	M	SD	+S9*	M	SD	EXP-6	-S9	+S9*
NEGATIVE CONTROL DMSO	22 21 21	21	1	39 32 31	34	4	62 53 57	1.0	1.0
20	21 29 22	24	4	31 34 32	32	2		1.1	1.0
100	21 21 -	21	0	40 32 29	34	6		1.0	1.0
500	20 24 23	22	2	35 30 28	31	4		1.0	0.9
2500	B B B	-	-	17B 23B 17B	19	3	0 0 0	-	0.6
5000	B B B	-	-	B B B	-	-	0 0 0	-	-
POSITIVE CONTROL 2-AA 10				1992 1800 2000	1907	101			56.1
POSITIVE CONTROL MNNG 5	781 839 899	840	59					39.4

 

* : S-9 FRACTION/COFACTORS = 3: 7         EXP :EXP. TO 10

B: REDUCED his BACKGROUND                       

 

Table – 5

AMES TEST WITH 86/221

METHOD: STANDARD PLATE TEST STRAIN: TA 100

DOSE MCG/ PL	REVERTANTS / PLATE						QUOT IENT
	-S9	M	SD	+S9*	M	SD	-S9	+S9*
NEGATIVE CONTROL DMSO	116	114	10	105	103	8	1.0	1.0
	103			94
	123			109
20	99	102	13	115	110	8	0.9	1.1
	91			101
	117			113
100	93	103	10	97	104	8	0.9	1.0
	112			104
	103			112
500	109	111	10	112	106	6	1.0	1.0
	102			103
	121			102
1000	93	98	9	141	129	10	0.9	1.3
	92			123
	108			124
POSITIVE CONTROL 2-AA				2100	2033	76		19.8
				2050
				1950
10
POSITIVE CONTROL MNNG	1670	1703	42				14.9
	1750
	1690
5

* : S9 FRACTION/COFACTORS = 3. 7

 

Table – 6

AMES TEST WITH 86/33

METHOD: STANDARD PLATE TEST STRAIN: TA 100

DOSE MCG/ PL	REVERTANTS / PLATE						QUOT IENT
	-S9	M	SD	+S9*	M	SD	-S9	+S9*
NEGATIVE CONTROL DMSO	116	114	10	105	103	8	1.0	1.0
	103			94
	123			109
20	122	116	6	117	113	6	1.0	1.1
	113			-
	112			108
100	96	110	21	125	132	8	1.0	1.3
	100			140
	134			132
500	127	128	17	137	152	17	1.1	1.5
	112			148
	145			171
1000	149	145	7	207	206	2	1.3	2.0
	150			206
	137			204
POSITIVE CONTROL 2-AA				2100	2033	76		19.8
				2050
				1950
10
POSITIVE CONTROL MNNG	1670	1703	42				14.9
	1750
	1690
5

* : S9 FRACTION/COFACTORS = 3. 7 - : CONTAMINATION

Table – 7

AMES TEST WITH 86/221

METHOD: STANDARD PLATE TEST STRAIN: TA 98

DOSE MCG/ PL	REVERTANTS / PLATE						QUOT IENT
	-S9	M	SD	+S9*	M	SD	-S9	+S9*
NEGATIVE CONTROL DMSO	25	23	2	35	34	4	1.0	1.0
	22			38
	21			30
20	30	25	6	34	32	2	1.1	0.9
	25			32
	19			31
100	24	20	4	40	38	3	0.7	1.1
	19			36
	17			-
500	17	18	2	29	29	2	0.8	0.8
	20			30
	17			27
1000	12	10	3	33	30	3	0.5	0.9
	7			30
	12			27
POSITIVE CONTROL 2-AA				1433	1420	26		41.4
				1440
				1390
10
POSITIVE CONTROL MNNG	1060	1150	95				50.7
	1140
	1250
10

* : S9 FRACTION/COFACTORS = 3. 7 - : CONTAMINATION

Table – 7

AMES TEST WITH 86/221

METHOD: STANDARD PLATE TEST STRAIN: TA 100

DOSE MCG/ PL	REVERTANTS / PLATE						TITER DIL	QUOT IENT
	-S9	M	SD	+S9*	M	SD	EXP-6	-S9	+S9*
NEGATIVE CONTROL DMSO	117	119	8	110	108	3	14	1.0	1.0
	128			104			15
	112			109			18
50	91	105	16	121	113	7		0.9	1.0
	122			109
	101			108
100	123	131	7	103	124	26		1.1	1.1
	132			115
	137			153
500	113	126	14	108	129	18		1.1	1.2
	124			140
	140			138
1000	127	122	8	139	154	13	22	1.0	1.4
	113			162			31
	127			162			17
5000	B	-	-	77	74	9	21	-	0.7
	B			81			16
	B			63			11
POSITIVE CONTROL 2-AA				1660	1630	79			15.1
				1690
				1540
10
POSITIVE CONTROL MNNG	2150	2015	191					16.9
	1880
	-
5

 

*: S59 FRACTION/COFACTORS = 3.7 EXP : EXP. T0 10

-: CONTAMINATION B: REDUCED his- BACKOROUND

Table – 9

AMES TEST WITH 87/33

METHOD: STANDARD PLATE TEST STRAIN: TA 100

DOSE MCG/ PL		REVERTANTS / PLATE						QUOT IENT
	-S9	M	SD	+S9*	M	SD	TITER DIL Exp-6	-S9	+S9*
NEGATIVE CONTROL DMSO	117	119	8	110	108	3	14	1.0	1.0
	128			104			15
	112			109			18
50	127	119	7	120	119	8		1.0
	115			126
	114			110
100	116	113	3	136	132	5		0.9	1.2
	110			127
	113			133
500	135	141	9	132	156	26		1.2	1.4
	152			152
	137			183
1000	182	173	18	157	170	19	33	1.5	1.6
	153			192			31
	137			161			34
2500	211	210	16	235	250	13	26	1.8	2.3
	225			253			32
	193			261			31
POSITIVE CONTROL 2-AA				1660	1630	79			15.1
				1690
				1540
10
POSITIVE CONTROL MNNG	2150	2015	191					16.9
	1880
	-
5

 

 

 

 

 

 

 

 

Table – 10

AMES TEST WITH 86/221

METHOD: STANDARD PLATE TEST STRAIN: TA1535

 

DOSE MCG/ PL		REVERTANTS / PLATE						QUOT IENT
	-S9	M	SD	+S9*	M	SD	TITER DIL Exp-6	-S9	+S9*
NEGATIVE CONTROL DMSO	20	19	3	23	20	3	49	1.0	1.0
	16			17			44
	21			19			51
20	21	23	2	17	17	2		1.2	0.8
	24			15
	23			18
100	20	20	2	19	17	2		1.1	0.8
	22			16
	18			15
500	18	19	3	19	20	3		1.0	1.0
	17			18
	23			24
1000	22	20	3	18	18	1	55	1.1	0.9
	17			17			64
	21			19			48	1.2	1.0
1500	24	23	1	20	20	4	56
	22			24			57
	22			17			48
POSITIVE CONTROL 2-AA				577	481	140			24.4
				545
				320
10
POSITIVE CONTROL MNNG	2050	2233	176					117.5
	2250
	2400
5

 

 

 

 

 

 

 

 

 

Table – 11

AMES TEST WITH 86/221

METHOD: STANDARD PLATE TEST STRAIN: TA1535

 

DOSE MCG/ PL		REVERTANTS / PLATE						QUOT IENT
	-S9	M	SD	+S9*	M	SD	TITER DIL Exp-6	-S9	+S9*
NEGATIVE CONTROL DMSO	14	14	2	14	15	1	33	1.0	1.0
	12			15			27
	16			15			27
20	15	15	1	14	14	1		1.1	1.0
	14			13
	16			15
100	14	13	1	13	13	3		1.0	0.9
	13			16
	13			10
500	15	13	2	10	13	3		0.9	0.9
	13			15
	11			13
1000	10	11	2	12	13	2	32	0.8	0.9
	11			12			33
	13			16			44
1500	12	14	2	14	15	2	24	1.0	1.0
	14			18			32
	15			14			30
POSITIVE CONTROL 2-AA				203	221	16			15.1
				234
				226
10
POSITIVE CONTROL MNNG	661	702	109					50.1
	825
	619
5

 

 

Conclusions:

Test chemical was evaluated for its mutagenic potential in Salmonella typhimurium strain TA 1535, TA 100, TA 1537, TA 98 by Ames test. Test chemical, which is known to be positive in the Ames test, was investigated in parallel as a positive control substance (chemical class control) using the strain TA 100. The test result was considered to be negative in the presence and absence of metabolic activation.

Executive summary:

Test chemical was evaluated for its mutagenic potential in Salmonella typhimurium strain TA 1535, TA 100, TA 1537, TA 98 by Ames test. The test chemical, which is known to be positive in the Ames test, was investigated in parallel as a positive control substance (chemical class control) using the strain TA 100. The test result was considered to be negative in the presence and absence of metabolic activation.Genetic toxicity study was assessed for its possible mutagenic potential. For this purpose AMES test was performed according to guideline OECD 471. The test material was exposed at different concentration in 4 individual experiments. These concentrations are mentioned below :

1st Experiment

Strains: TA 1535. TA 100. TA 1537, TA 98

Doses: 0, 20, 100, 500. 2500 and 5000 µg/plate

 

2nd Experiment

Strains: TA 100, TA 98

Doses: 0, 20. 100, 500 and 1000 µg/plate

 

3rd Experiment

Strain: TA 100

Doses: 0, 50, 100, 500, 1000 and 2500 µg/plate

 

4th Experiment

Strains: TA 1535, JA 1537

Doses: 0, 20, 100, 500, 1000 and 1500 µg/plate

The test material which is known to be positive in the Ames test, was investigated in parallel as a positive control substance (chemical class control) using the strain TA 100. The test material was exposed in the presence and absence of metabolic activation. No mutagenic effects were observed in any of the strain. Thereforetest chemical was considered to be non mutagenic in Salmonella typhimurium strain TA 1535, TA 100, TA 1537, TA 98 by Ames test. Hence the substance cannot be classified as gene mutant in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Data available for the test chemicals were reviewed to determine the toxic nature of the test chemical. The studies are as mentioned below:

1)Test chemical was evaluated for its mutagenic potential in Salmonella typhimurium strain TA 1535, TA 100, TA 1537, TA 98 by Ames test. The test chemical, which is known to be positive in the Ames test, was investigated in parallel as a positive control substance (chemical class control) using the strain TA 100. The test result was considered to be negative in the presence and absence of metabolic activation.Genetic toxicity study was assessed for its possible mutagenic potential. For this purpose AMES test was performed according to guideline OECD 471. The test material was exposed at different concentration in 4 individual experiments. These concentrations are mentioned below :

1st Experiment

Strains: TA 1535. TA 100. TA 1537, TA 98

Doses: 0, 20, 100, 500. 2500 and 5000 µg/plate

 2nd Experiment

Strains: TA 100, TA 98

Doses: 0, 20. 100, 500 and 1000 µg/plate

 3rd Experiment

Strain: TA 100

Doses: 0, 50, 100, 500, 1000 and 2500 µg/plate

 4th Experiment

Strains: TA 1535, JA 1537

Doses: 0, 20, 100, 500, 1000 and 1500 µg/plate

The test material which is known to be positive in the Ames test, was investigated in parallel as a positive control substance (chemical class control) using the strain TA 100. The test material was exposed in the presence and absence of metabolic activation. No mutagenic effects were observed in any of the strain. Thereforetest chemicalwas considered to be non mutagenic in Salmonella typhimurium strain TA 1535, TA 100, TA 1537, TA 98 by Ames test. Hence the substance cannot be classified as gene mutant in vitro.

2)The spot test was performed by on Salmonella Typhimurum with strain TA1530 (hisG46, Δgal, ΔuvrB), TA1535 (hisG46, rfa, ΔuvrB) and T100 (hisG46, rfa, ΔuvrB, pKM101) for the test compound . Spot tests were performed by spreading of a stationary cell suspension (ca. 10⁹/ml) of the indicator strains TA1530, TA1535 or TA100 on mutation plates (containing traces of L-histidine), followed by the addition of the desired amount of the test chemical in the middle of the plate. The plates were then incubated for 48 h at 37°C, and the number of his + revertant colonies were determined. An increase in the number of revertants was noted with the test compound and hence is likely to classify for gene mutation in vitro.

3) Salmonella typhimurium mutation test was performed to determine the mutagenic nature of the test compound. The study was performed using Salmonella typhimurium strain TA1535 both in the presence and absence of S9 metabolic activation system.The test compound induced base pair substitution in Salmonella typhimurium strain TA1535 and hence is likely to be mutagenic in nature.

4)Bacterial gene mutation assay was performed to determine the mutagenic nature of the test compound. Preincubation assay was performed in the Salmonella typhimurium strain TA98, TA100, TA1535 and TA1537 and E. col Wp2 uvrA in the presence and absence of S9 metabolic activation system at doses of 0.0763-5000 µg/plate.The test compound induced mutation in the Salmonella typhimurium strains TA1535, E. col Wp2 uvrA (±S9; at all doses) and TA100 (+ S9; 9.77- 1250µg/plate) and failed to induce mutation in the Salmonella typhimurium strains TA98, TA100, and TA1537.

5)The aim of the study was to determine the mutagenic activity of test chemical in Salmonella typhimurium NM5004 strain by introducing a plasmid containing both rat GSH glutathione S-transferase (GST) 5-5 cDNA and the umuC lacZ operon into the host strain Salmonella typhimurium TA1535 and used to examine whether or not GST modified the genotoxic activities of test chemical. Liver microsomes was obtained from Male Sprague-Dawley rats. The bacterial suspension was incubated with 0.1mM test chemical (dissolved in DMSO) at 37 °C for 2 h and the expressed β-galactosidase activity was determined by the method of Miller and expressed in those units or absorbance at 420 nm using o-nitrophenyl-β-D-galactopyranoside as a substrate. The genotoxicities of chemical carcinogens and mutagens were determined by measuring induction of an umu gene expression and Ames test in the two Salmonella tester strains. Cytotoxicity was observed to be 65 -80% in GST activated gene and 50 -60% in GST inactivated gene in Salmonella typhimurium NM5004 tester strain. 0 -40 revertants were observed in Salmonella typhimurium TA1535/pSK1002 strain which is considered to be negligible. The test chemical gave different responses in the two tester strains, but it could not be defined whether these chemicals are activated or inactivated by GST 5-5. Thus, the test chemical can be considered negative as satisfactory results to understand the classification were not obtained.

6) In Gene mutation study, a plasmid vector containing a cDNA for the rat f3 class GSH S-transferase (GST)5-5 was constructed and expressed in the normal [GST 5-5(+)] or in the reverse orientation [GST 5-5(-)] in SalmonellatyphimuriumTA1535 strain to observe the mutagenic nature of the test chemical. The experiment was carried out according to preincubation assay. Dibromomethane was used as a reference point for mutation system because it expressed the GST 5-5 (+) gene. Very small number of revertants were observed after the experiment. Therefore, The test chemical was a very weak mutagen in both the GST 5-5(+) and GST 5-5(-) S. typhimurium TA 1535 strains.

Based on the data available, the test chemical did not induce gene mutation in sallmonela strain in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Chromosomal Abberation:

In vitro chromosomal aberration assay was performed to determine the mutagenic nature of the test compound both in the presence and absence of S9 metabolic activation system. The test chemical was used at dose levels of 0.05-0.2 mg/mL using CHL cells in 6 hr treatment, 18 hr recovery period (With metabolic activation) and 0.025-0.2 mg/mL using CHL cells in 24 hr and 48 hr continuous treatment period.The test compound induced structural changes in 6 hr treatment, 18 hr recovery period and structural changes in 24 hr continuous treatment period and structural and ploidy changes in the CHL cells in 48 hrs continuous treatment period and hence is positive for gene mutation in vitro.

Based on the data available, the test chemical induced chromosome aberrations in Chinese hamster cell line in the presence and absence of S9 metabolic activation system and hence it is likely to classify as a gene mutant in vitro.

Justification for classification or non-classification

Based on the experimental data available from the latest study, the target substance cannot be classified as mutagenic in nature. As though other supporting studies reviewed for the target has the positive result for gene mutation .These supporting studies have used low concentration in experiment and also detailed information are not available as per guideline. Considering all these factors for classification the target substance cannot be classified as mutant in nature. Thus the test chemical is not likely to classify as a gene mutant in vitro.