Reaction mass of 1,1'-(2,2,4-trimethylhexane-1,6-diyl)bis-1H-pyrrole-2,5-dione and 1H-Pyrrole-2,5-dione, 1,1'-(2,4,4-trimethyl-1,6-hexanediyl)bis-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-05-30 to 2008-06-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21 Jul 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 08 Jun 2000

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 1,1'-(2,2,4-trimethylhexane-1,6-diyl)bis-1H-pyrrole-2,5-dione and 1,1'-(2,4,4-trimethylhexane-1,6-diyl)bis-1H-pyrrole-2,5-dione

EC Number:

915-671-3

Molecular formula:

C17H22N2O4

IUPAC Name:

Reaction mass of 1,1'-(2,2,4-trimethylhexane-1,6-diyl)bis-1H-pyrrole-2,5-dione and 1,1'-(2,4,4-trimethylhexane-1,6-diyl)bis-1H-pyrrole-2,5-dione

Method

Target gene:

his operon for S. typhimurium strains and trp operon for E. coli strain

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:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Experiment 1: 333, 1000, 3330 and 5000 µg/plate for all strains with and without metabolic activation.
Experiment 2:
10, 33, 100, 333 and 666 µg/plate for TA1535, TA98 and TA100 with and without metabolic activation,
3, 10, 33, 100 and 333 µg/plate for TA1537 with and without metabolic activation,
33, 100, 333, 1000 and 3330 µg/plate for WP2 uvrA with and without metabolic activation.
Experiment 3: 100, 333, 1000 and 3330 µg/plate for TA98 with and without metabolic activation

A dose range finding test was conducted in S. typhimurium strain TA100 and in E. coli strain WP2 uvrA using the concentrations 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the presence and absence of metabolic activation (S9 mix). In tester strain TA100, toxicity was observed at dose levels of 333 µg/plate and above in the presence and absence of S9 mix. In tester strain WP2 uvrA cytotoxicity was observed at 3330 µg/plate and above in the absence of S9 mix and at 5000 µg/plate in the presence of S9 mix. The test was reported as a part of the first experiment of the present study. The highest concentration was the level at which the test substance inhibited bacterial growth or the test substance exhibited limited solubility.

Vehicle / solvent:

- Vehicle/solvent used: DMSO
The test item doses were corrected for the purity, a correction factor of 1.13 was used.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene (2AA), +S9, 1 µg/plate in DMSO for TA98 and TA100, 2.5 µg/plate in DMSO for TA1535, TA1537 and TA100, 5 µg/plate in DMSO for TA1537 and 10 µg/plate in DMSO for WP2 uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h at 37.0 ± 1.0 °C

NUMBER OF REPLICATIONS: triplicates each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) the total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 and WP2 uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2 uvrA is greater than three (3) times the concurrent control.
b) In case a positive response will be repeated, the positive response should be reproducible in at least one independently repeated experiment.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥ 333 µg/plate with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥ 333 µg/plate with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥ 1000 µg/plate with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥ 333 µg/plate with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 3330 µg/plate without metabolic activation and at 5000 µg/plate with metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS:
- Precipitation and time of the determination: Precipitation was noted at 3330 and 5000 µg/plate at the end of the incubation period.

RANGE-FINDING/SCREENING STUDIES:
A dose range finding test was conducted in S. typhimurium strain TA100 and in E. coli strain WP2 uvrA using the concentrations 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the presence and absence of metabolic activation (S9 mix). In tester strain TA100, cytotoxicity was observed at dose levels of 333 µg/plate and above in the presence and absence of S9 mix. In tester strain WP2 uvrA cytotoxicity was observed at 3330 µg/plate and above in the absence of S9 mix and at 5000 µg/plate in the presence of S9 mix. The test was reported as a part of the first experiment of the present study.

HISTORICAL CONTROL DATA: please refer to Table No. 2 under “Any other information on results incl. tables”

Any other information on results incl. tables

Table 1: Experimental results

Experiment 1: Standard plate test (SPT)
Strain	TA 1535		TA 1537		TA 98		TA 100		WP2 uvrA
Metabolic activation	without S9	with S9	without S9	with S9	without S9	with S9	without S9	with S9	without S9	with S9
Vehicle control
DMSO mean	14	10	5	4	18	28	121	118	29	30
± SD	± 2	± 1	± 2	± 1	± 6	± 3	± 1	± 6	± 8	± 3
Test item [µg/plate]
3 mean							136	131	31	35
± SD							± 8	± 5	± 2	± 10
10 mean	10	9	5	4	24	27	148	116	28	31
± SD	± 4	± 3	± 3	± 2	± 5	± 3	± 2	± 8	± 8	± 9
33 mean	11	7	3	3	32	28	135	117	27	28
± SD	± 2	± 4	± 1	± 0	± 5	± 3	± 14	± 3	± 5	± 6
100 mean	8	9	4	5	22	27	126	125	27	35
± SD	± 3	± 3	± 2	± 1	± 6	± 6	± 5	± 6	± 6	± 6
333 mean	5 s	5 s	0 s	2 s	18	21	70 s	70 s	27	29
± SD	± 1	± 3	± 0	± 2	± 6	± 4	± 14	± 3	± 3	± 3
1000 mean	MC e	MC e	0 a	0 a	MC e	6 m	MC e	MC e	18	19
± SD			± 0	± 0		± 3			± 4	± 8
3330 mean SP	0 a	0 a	0 a	0 a	0 a	0 a	0 a	0 a	MC e	9
± SD	± 0	± 0	± 0	± 0	± 0	± 0	± 0	± 0		± 5
5000 mean SP							0 a	0 a	MC e	29 s
± SD							± 0	± 0		± 10
Positive control
^§mean	960	408	234	312	999	923	1013	1196	1037	581
± SD	± 16	± 42	± 40	± 22	± 60	± 51	± 40	± 123	± 56	± 85
Experiment 2: Standard plate test (SPT)
Strain	TA 1535		TA 1537		TA 98		TA 100		WP2 uvrA
Metabolic activation	without S9	with S9	without S9	with S9	without S9	with S9	without S9	with S9	without S9	with S9
Vehicle control
DMSO mean	7	9	9	7	26	29	125	93	30	33
± SD	± 2	± 2	± 4	± 2	± 4	± 3	± 13	± 14	± 5	± 5
Test item
3 mean			8
± SD			± 3	±
10 mean	9	8	8	7	20	27	124	71
± SD	± 4	± 4	± 4	± 2	± 4	± 4	± 7	± 8
33 mean	10	7	5	8	21	29	133	74	30	22
± SD	± 3	± 3	± 2	± 3	± 4	± 4	± 23	± 3	± 5	± 1
100 mean	6	5	6	6	25	28	121	72	34	32
± SD	± 2	± 1	± 3	± 1	± 7	± 6	± 6	± 12	± 9	± 4
333 mean	4 s	5 s	0 s	5	17	29	39 m	55 m	34	23
± SD	± 1	± 2	± 1	± 1	± 1	± 7	± 9	± 9	± 1	± 8
666 mean	MC e	MC e		3 s	14	21	MC e	MC e
± SD				± 1	± 2	± 1
1000 mean									30	21
± SD									± 10	± 8
3330 mean SP									MC e	12
± SD										± 5
Positive control
^§mean	859	185	314	236	993	389	677	573	1152	264
± SD	± 10	± 12	± 42	± 108	± 40	± 114	± 58	± 161	± 28	± 67
Experiment 3: Standard plate test (SPT)
Strain	TA 1535		TA 1537		TA 98		TA 100		WP2 uvrA
Metabolic activation	without S9	with S9	without S9	with S9	without S9	with S9	without S9	with S9	without S9	with S9
Vehicle control
DMSO mean					25	28
± SD					± 3	± 3
Test item
100 mean					25	28
± SD					± 3	± 3
333 mean					16	27
± SD					± 2	± 3
1000 mean					MC a	6 m
± SD						± 2
3330 mean SP					0 e	0 a
± SD					± 0	± 0
Positive control
^§mean					1156	662
± SD					± 10	± 65
^§= information on respective positive control is reported in the Method section above (Controls, positive control substances)
^b= thinning of the background lawn of non-revertant cells was observed
MC: Microcolonies
e: Bacterial background lawn extremely reduced
a: Bacterial background lawn absent
m: Bacterial background lawn moderately reduced
s: Bacterial background lawn slightly reduced
SP: slight precipitate

Table 2: Historical control data

Strain	TA 1535		TA 1537		TA 98		TA 100		WP2 uvrA
Metabolic activation	without S9	with S9	without S9	with S9	without S9	with S9	without S9	with S9	without S9	with S9
Vehicle control (DMSO)
HCD [range] mean ± SD	3 - 28 12 ± 14	3 - 29 12 ± 14	3 - 19 7 ± 9	3 - 21 7 ± 10	12 - 45 21 ± 19	12 - 51 26 ± 21	63 - 194 121 ± 85	60 - 195 107 ± 94	4 - 39 17 ± 19	4 - 44 17 ± 20
Positive controls
	SA	2AA	9AC	2AA	NF	2AA	MMS	2AA	4-NQO	2AA
HCD [range] mean ± SD	375 - 1923 1119 ± 688	58 - 538 185 ± 227	79 - 927 335 ± 417	57 - 833 353 ± 418	286 - 1790 1072 ± 648	171 - 1703 698 ± 935	452 - 1593 1074 ± 526	223 - 2061 1098 ± 1021	67 - 1387 755 ± 805	56 - 760 262 ± 272
SA: sodium azide; 2AA: 2 -aminoanthracene; 9AC: 9 -aminoacridine; NF: 2 -nitrofluorene; MMS: methylmethanesulfonate; 4 -NQO: 4 -nitroquinoline-N-oxide

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative with and without metabolic activation
The test substance did not show mutagenic activity in Salmonella typhimurium TA1535, TA1537, TA98 or TA100 or in Escherichia coli WP2 uvrA with and without metabolic activation.