1,1'-(1,1-dimethyl-3-methylene-1,3-propanediyl)bisbenzene

Genetic toxicity: in vitro

Administrative data

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:

other: Study performed according to GLP and guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1,1'-(1,1-dimethyl-3-methylene-1,3-propanediyl)bisbenzene

EC Number:

228-846-8

EC Name:

1,1'-(1,1-dimethyl-3-methylene-1,3-propanediyl)bisbenzene

Cas Number:

6362-80-7

Molecular formula:

C18H20

IUPAC Name:

(4-methyl-4-phenylpent-1-en-2-yl)benzene

Details on test material:

- Name of test material (as cited in study report): , 2,4-diphenyl-4-methyl-1-pentene [English name: 1,1’-(1,1-dimethyl-3-methylene-1,3-propanediyl)bisbenzene
- Analytical purity: 96.97%
- Impurities (identity and concentrations): 2,4-Diphenyl-4-methyl-2-pentene 2.54%)
- Storage condition of test material: Stored in a cabinet in the test substance storage room of the testing facility at room temperature (nominal value: 23°C, measured values 20.0 - 25.3°C) and protected from light.

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Dose finding test:
Test concentrations in the dose-finding test were set in accordance with Methods for Testing New Chemical Substances (November 21, 2003) with 5000 μg/plate as the highest concentration for each strain and lower concentrations at 1250, 312.5, 78.1, 19.5, 4.88, 1.22 and 0.305 μg/plate in a common ratio of 4 for a total of 8 concentrations both with and without addition of S9 mix.

Main test (I) and Main Test (II):
From the results of the dose-finding test, 6 concentrations in a common ratio of 2 were set for the main tests (I) and (II) with at least 4 concentrations expected to show no growth inhibition of bacteria both with and without addition of S9 mix. Without S9 mix, concentrations were set at 9.77, 19.5, 39.1, 78.1, 156.3 and 312.5 μg/plate for TA100 and TA1535, at 156.3, 312.5, 625, 1250, 2500 and 5000 μg/plate for WP2uvrA, and at 2.44, 4.88, 9.77, 19.5, 39.1 and 78.1 μg/plate for TA98 and TA1537. With S9 mix, they were set at 39.1, 78.1, 156.3, 312.5, 625 and 1250 μg/plate for TA100, at 156.3, 312.5, 625, 1250, 2500 and 5000 μg/plate for TA1535 and WP2uvrA, at 9.77, 19.5, 39.1, 78.1, 156.3 and 312.5 μg/plate for TA98, and at 2.44, 4.88, 9.77, 19.5, 39.1 and 78.1 μg/plate for TA1537. Negative controls and positive controls were provided for each strain.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: 9-aminoacridine hydrochloride and 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide

Controls 2

Untreated negative controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Details on test system and experimental conditions:

The test was conducted by the preincubation method without metabolic activation (without S9 mix) and with metabolic activation (with S9 mix). (1) 0.1 mL of sample solution, (2) 0.5 mL of high-pressure steam sterilized 0.1 mol/L sodium phosphate buffer solution (pH 7.4) (without metabolic activation) or 0.5 mL of S9 mix (with metabolic activation) and (3) 0.1 mL of bacterial suspension were added in that order to dry-heat sterilized test tubes (15.5100 mm, clean test tube Rarubo, Terumo Corp.), which were incubated at 37°C for 20 minutes using a shaker in reciprocating mode. Then 2 mL of top agar warmed to 45°C was added and mixed, spread on the minimum glucose agar plate medium, and the plate was overturned and incubated for about 48 hours in an incubator set at 37°C.

After the end of incubation, the presence or absence of precipitation on the plate was observed visually, then the number of revertant colonies was counted with a colony analyzer (CA-11D, System Science Co., Ltd.), and the bacteria were observed for growth inhibition under a 100x microscope. The plates were also visually observed for precipitation at the start of incubation.

The number of plates for each strain at each concentration with and without metabolic activation was one in the dose-finding test and three in both main tests (I) and (II). Test tubes and plates were identified by colors marked in oil-based ink for each strain.

Evaluation criteria:

Test results were considered positive if the number of revertant colonies in the plates treated with test substance was twice or more that of the negative control, and dose-dependent increase was observed

Statistics:

Mean and standard deviation of the numbers of revertant colonies were calculated for each concentration. Significance of difference was not tested because the following judgment criteria were used

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Dose-Finding Test (Table 1)

Precipitation on the Plate
Without S9 mix, a clear oily precipitate was observed on the plates at concentrations of 1250 μg/plate and above both at the start and end of incubation. With S9 mix, a white oily film of precipitate was observed at 1250 μg/plate and above at the start of incubation, and a fine white precipitate was observed at the highest concentration of 5000 μg/plate at the end of incubation.

Bacterial Growth Inhibition
Bacterial growth inhibition was observed without S9 mix at 312.5 μg/plate and above for TA100 and TA1535, at the highest concentration of 5000 μg/plate for WP2uvrA, and at 78.1 μg/plate and above for TA98 and TA1537. With S9 mix, it was observed at 1250 μg/plate and above for TA100, at the highest concentration of 5000 μg/plate for TA1535 and WP2uvrA, at 312.5 μg/plate and above for TA98, and at 78.1 μg/plate and above for TA1537.

Number of Revertant Colonies
The number of revertant colonies was less than twice that of the negative control for any strains with or without S9 mix.

Sterility Test
No bacterial contamination of the highest concentration of test substance solution (50 μg/mL) or the S9 mix was observed in the sterility test.

Negative Control and Positive Controls
The positive control substances showed clear increases in the numbers of revertant colonies, and the numbers of revertant colonies in the negative control and positive controls were within the range of the background data of the test facility.

Main Test (I) (Tables 2-1 to 2-2 and Main Test (II) (Tables 3-1 to 3-2 )

Precipitation on the Plate
Without S9 mix, a clear oily precipitate was observed on the plates at concentrations of 625 μg/plate and above at the start of incubation and at 1250 μg/plate and above at the end of incubation. With S9 mix, a white oily film of precipitate was observed at 1250 μg/plate and above at the start of incubation, and a fine white precipitate was observed at 2500 μg/plate and above at the end of incubation.

Bacterial Growth Inhibition
Bacterial growth inhibition was observed without S9 mix at 156.3 μg/plate and above for TA100 and TA1535, at the highest concentration of 5000 μg/plate for WP2uvrA, and at 39.1 μg/plate and above for TA98 and TA1537. With S9 mix, it was observed at 625 μg/plate and above for TA100, at 2500 μg/plate and above for TA1535, at the highest concentration of 5000 μg/plate for WP2uvrA, at the highest concentration of 312.5 μg/plate for TA98, and at the highest concentration of 78.1 μg/plate for TA1537.

Number of Revertant Colonies
The number of revertant colonies was less than twice that of the negative control for all strains with or without S9 mix.

Sterility Test
No bacterial contamination of the highest concentration of test substance solution (50 μg/mL) or the S9 mix was observed in the sterility test.

Negative Control and Positive Controls
The positive control substances showed clear increases in the numbers of revertant colonies, and the numbers of revertant colonies in the negative control and positive controls were within the range of the background data of the test facility.

Any other information on results incl. tables

Table 1: Reverse mutation test of 1,1’-(1,1-dimethyl-3-methylene-1,3-propanediyl) bisbenzene in bacteria (dose range finding test)

With (+) or without (-) S9 mix	Compound concentration (µg/plate)	Number of revertants (number of colonies/plate)
		Base-pair substitution type			Frameshift type
		TA100	TA1535	WP2uvrA	TA98	TA1537
S9 mix (-)	Negative control	111	10	51	19	13
	0.305	136	17	48	17	3
	1.22	146	8	46	9	6
	4.88	125	16	35	17	9
	19.5	118	13	47	20	13
	78.1	137	17	44	13*	14*
	312.5	114*	10*	33	22*	8*
	1250#	126*	13*	49	16*	7*
	5000#	125*	16*	48*	22*	6*
S9 mix (+)	Negative control	146	9	41	29	20
	0.305	137	16	36	26	15
	1.22	138	16	44	31	18
	4.88	154	14	49	32	19
	19.5	127	11	46	29	21
	78.1	137	17	40	28	19*
	312.5	139	7	42	17*	20*
	1250#	123*	12	44	35*	10*
	5000#	137*	12*	36*	27*	4*
Positive control not requiring S9 mix	Name	AF-2	NaN3	AF-2	AF-2	9AA
	Concentration (µg/plate)	0.01	0.5	0.01	0.1	80
	Number of colonies/plate	531	628	157	485	571
Positive control requiring S9 mix	Name	2AA
	Concentration (µg/plate)	1	2	10	0.5	2
	Number of colonies/plate	957	312	843	352	187

Negative control: Dimethylsulfoxide

AF2:2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide; NaN₃sodium azide; 9AA: 9-aminoacridine hydrochloride; 2AA: 2-aminoanthracene.

*: Bacterial growth inhibition was observed

#: Clear oily precipitations were observed on the surface of agar plate

##: White fine precipitations were observed on the surface of agar plate

 

 

Table 2-1: Reverse mutation test of 1,1’-(1,1-dimethyl-3-methylene-1,3-propanediyl) bisbenzene in bacteria (mutagenicity test I: -S9 mix)

Compound concentration (µg/plate)	Number of revertants (number of colonies/plate)
	Base-pair substitution type			Frameshift type
	TA100	TA1535	WP2uvrA	TA98	TA1537
Negative control	117, 118, 143 (126 ± 14.7)	12, 17, 19 (16 ± 3.6)	43, 45, 46 (45 ± 1.5)	13, 19, 22 (18 ± 4.6)	13, 14, 20 (16 ± 3.8)
2.44				16, 21, 27 (21 ± 5.5)	17, 19, 24 (20 ± 4.6)
4.88				20, 25, 28 (24 ± 4.0)	19, 25, 28 (24 ± 4.6)
9.77	97, 104, 111 (104 ± 7.0)	15, 18, 24 (19 ± 4.6)		16, 20, 20 (19 ± 2.3)	16, 20, 23 (20 ± 5.9)
19.5	114, 116, 130 (120 ± 8.7)	20, 20, 25 (22 ± 2.9)		17, 19, 19 (18 ± 1.2)	18, 20, 29 (22 ± 5.9)
39.1	100, 100, 103 (101 ± 1.7)	13, 13, 17 (14 ± 2.3)		20*, 21*, 21* (21 ± 0.6)	21*, 22*, 23* (22 ± 5.9)
78.1	97, 105, 118 (107 ± 10.6)	12, 14, 18 (15 ± 3.1)		15*, 19*, 27* (20 ± 6.1)	15*, 17*, 24* (19 ± 4.7)
156.3	88*, 101*, 104* (98± 8.5)	11*, 16*, 19* (15 ± 4.0)	44, 50, 60 (51 ± 8.1)
312.5	90*, 109*, 109* (103 ± 11.0)	15*, 16*, 17* (16 ± 1.0)	39, 47, 51 (46 ± 6.1)
625			37, 51, 54 (47 ± 9.1)
1250#			54, 58, 67 (60 ± 6.7)
2500#			38, 41, 44 (41 ± 3.0)
5000#			46*, 55, 61* (54 ± 7.5)
Positive control
Name	AF2	NaN3	AF-2	AF-2		9AA
Concentration (µg/plate)	0.01	0.5	0.01	0.1		80
Number of colonies/plate	470, 544, 548 (521 ± 43.9)	578, 604, 614 (599 ± 18.6)	165, 168, 171 (168 ± 3.0)	401, 425, 441 (422 ± 20.1)		339, 393, 481 (404 ± 71.7)

Negative control: Dimethylsulfoxide

AF2:2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide; NaN₃sodium azide; 9AA: 9-aminoacridine hydrochloride.

(  ): Mean ± S.D

*: Bacterial growth inhibition was observed

#: Clear oily precipitations were observed on the surface of agar plate

 

 

 

 

Table 2-2: Reverse mutation test of 1,1’-(1,1-dimethyl-3-methylene-1,3-propanediyl) bisbenzene in bacteria (mutagenicity test I: ±S9 mix)

Compound concentration (µg/plate)	Number of revertants (number of colonies/plate)
	Base-pair substitution type			Frameshift type
	TA100	TA1535	WP2uvrA	TA98	TA1537
Negative control	123, 130, 145 (133 ± 11.2)	16, 17, 19 (17 ± 1.5)	34, 42, 60 (45 ± 13.3)	29, 36, 41 (35 ± 6.0)	20, 21, 21 (21 ± 0.6)
2.44					21, 33, 36 (30 ± 7.9)
4.88					28, 33, 38 (33 ± 5.0)
9.77				25, 37, 44 (35 ± 9.6)	24, 29, 32 (28 ± 4.0)
19.5				22, 26, 27 (25 ± 2.6)	24, 29, 32 (28 ± 4.2)
39.1	108, 126, 131 (122 ± 12.1)			36, 36, 42 (38 ± 3.5)	25, 26, 29 (27 ± 2.1)
78.1	122, 123, 128 (124 ± 3.2)			35, 36, 41 (37 ± 3.2)	30*, 32*, 33* (32 ± 1.5)
156.3	118, 124, 124 (122 ± 3.5)	13, 18, 20 (17 ± 3.6)	43, 51, 53 (49 ± 5.3)	29, 30, 41 (33 ± 6.7)
312.5	101, 111, 114 (109 ± 6.8)	9, 10, 14 (11 ± 2.6)	41, 46, 51 (46 ± 5.0)	19*, 26*, 35* (27 ± 8.0)
625	98*, 101*, 114* (104 ± 8.5)	18, 18, 21 (19 ± 1.7)	28, 37, 58 (41 ± 15.4)
1250#	82*, 86*, 97* (88 ± 7.8)	7, 13, 18 (13 ± 5.5)	42, 45, 54 (47 ± 6.2)
2500#		14*, 22*, 27* (21 ± 6.6)	51, 56, 57 (55 ± 3.2)
5000##		15*, 17*, 19* (17 ± 2.0)	42*, 53*, 69* (55 ± 13.6)
Positive control
Name	2AA
Concentration (µg/plate)	1	2	10	0.5		2
Number of colonies/plate	748, 777, 835 (787 ± 44.3)	303, 330, 367 (333 ± 32.1)	746, 759, 924 (810 ± 99.2)	364, 381, 385 (377 ± 11.2)		134, 142, 165 (147 ± 16.1)

Negative control: Dimethylsulfoxide

2AA: 2-Aminoanthracene

(  ): Mean ± S.D

*: Bacterial growth inhibition was observed

##: White fine precipitations were observed on the surface of agar plate

 

Table 3-1: Table 2-2: Reverse mutation test of 1,1’-(1,1-dimethyl-3-methylene-1,3-propanediyl) bisbenzene in bacteria (mutagenicity test II: -S9 mix)

Compound concentration (µg/plate)	Number of revertants (number of colonies/plate)
	Base-pair substitution type			Frameshift type
	TA100	TA1535	WP2uvrA	TA98	TA1537
Negative control	119, 119, 124 (121 ± 2.9)	11, 12, 13 (12 ± 1.0)	42, 49, 54 (48 ± 6.0)	20, 21, 21 (21 ± 0.6)	10, 16, 19 (15 ± 4.6)
2.44				12, 18, 19 (16 ± 3.8)	20, 22, 25 (22 ± 2.5)
4.88				17, 23, 30 (23 ± 6.5)	25, 26, 28 (26 ± 1.5)
9.77	106, 114, 120 (113 ± 7.0)	14, 16, 19 (16 ± 2.5)		19, 21, 24 (21 ± 2.5)	18, 20, 24 (21 ± 3.1)
19.5	126, 127, 130 (128 ± 2.1)	13, 13, 16 (14 ± 1.7)		16, 22, 25 (21 ± 4.6)	17, 21, 21 (20 ± 2.3)
39.1	118, 136, 137 (130 ± 10.7)	12, 14, 19 (15 ± 3.6)		20*, 22*, 25* (22 ± 2.5)	20*, 21*. 27* (22 ± 3.8)
78.1	118, 120, 122 (120 ± 2.0)	10, 14, 17 (14 ± 3.5)		18*, 21*, 25* (21 ± 3.5)	20*, 23*, 27* (23 ± 3.5)
156.3	119*, 128*, 134* (127 ± 7.5)	11*, 12*, 15* (13 ± 2.1)	50, 61, 63 (58 ± 7.0)
312.5	97*, 109*, 118* (108 ± 10.5)	9*, 11*, 12* (11 ± 1.5)	47, 55, 57 (53 ± 5.3)
625			51, 63, 63 (59 ± 6.9)
1250#			53, 64, 72 (63 ± 9.5)
2500#			62, 69, 76 (69 ± 7.0)
5000#			56*, 58*, 62* (59 ± 3.1)
Positive control
Name	AF-2	NaN3	AF-2	AF-2	9AA
Concentration (µg/plate)	0.01	0.5	0.01	0.1	80
Number of colonies/plate	539, 549, 595 (561 ± 29.9)	588, 604, 621 (604 ± 16.5)	153, 169, 174 (165 ± 11.0)	377, 387, 459 (408 ± 44.7)	377, 435, 482 (431 ± 52.6)

Negative control: Dimethylsulfoxide

AF2:2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide; NaN₃sodium azide; 9AA: 9-aminoacridine hydrochloride.

(  ): Mean ± S.D

*: Bacterial growth inhibition was observed

#: Clear oily precipitations were observed on the surface of agar plate

Table 3-2: Reverse mutation test of 1,1’-(1,1-dimethyl-3-methylene-1,3-propanediyl) bisbenzene in bacteria (mutagenicity test II: +S9 mix)

Compound concentration (µg/plate)	Number of revertants (number of colonies/plate)
	Base-pair substitution type			Frameshift type
	TA100	TA1535	WP2uvrA	TA98	TA1537
Negative control	107, 116, 140 (121 ± 17.1)	10, 12, 13 (12 ± 1.5)	48, 50, 52 (50 ± 2.0)	30, 37, 38 (35 ± 4.4)	20, 20, 27 (22 ± 4.0)
2.44					26, 31, 32 (30 ± 3.2)
4.88					23, 33, 35 (30 ± 6.4)
9.77				29, 31, 32 (31 ± 1.5)	25, 30, 37 (31 ± 6.0)
19.5				28, 32, 37 (32 ± 4.5)	32, 34, 41 (36 ± 4.7)
39.1	144, 155, 164 (154 ± 10.0)			33, 35, 42 (37 ± 4.7)	25, 28, 36 (30 ± 5.7)
78.1	128, 151, 159 (146 ± 16.1)			35, 39, 39 (38 ± 2.3)	31*, 34*, 37* (34 ± 3.0)
156.3	115, 136, 141 (131 ± 13.8)	8, 12, 19 (13 ± 5.6)	47, 53, 61 (54 ± 7.0)	29, 38, 40 (36 ± 5.9)
312.5	123, 131, 142 (132 ± 9.5)	8, 10, 11 (10 ± 1.5)	60, 64, 67 (64 ± 3.5)	25*, 38*, 38* (34 ± 7.5)
625	109*, 137*, 151* (132 ± 21.4)	9, 10, 12 (10 ± 1.5)	50, 56, 60 (55 ± 5.0)
1250#	116*, 124*, 137* (126 ± 10.6)	7, 9, 16 (11 ± 4.7)	45, 51, 61 (52 ± 8.1)
2500##		10*, 11*, 11* (11 ± 0.6)	34, 46, 47 (42 ± 7.2)
5000##		7*, 10*, 12* (10 ± 2.5)	48*, 56*, 59* (54 ± 5.7)
Positive control
Name	2AA
Concentration (µg/plate)	1	2	10	0.5	2
Number of colonies/plate	818, 903, 914 (878 ± 52.5)	297, 317, 338 (317 ± 20.5)	822, 832, 910 (855 ± 48.2)	375, 376, 399 (388 ± 13.6)	147, 153, 162 (154 ± 7.5)

Negative control: Dimethylsulfoxide

2AA: 2-Aminoanthracene

(  ): Mean ± S.D

*: Bacterial growth inhibition was observed

##: White fine precipitations were observed on the surface of agar plate

Applicant's summary and conclusion

Conclusions:

From these results, 2,4-diphenyl-4-methyl-1-pentene is judged to have no potential to induce genetic mutations under the conditions of this study