[No public or meaningful name is available]

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 January 30,2012 to March 12, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was performed according to the OECD 471 with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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:

Condensation products of cyclopentanone and pentaldehyde, fractionation pitch

Molecular formula:

not applicable, UVCB

IUPAC Name:

Condensation products of cyclopentanone and pentaldehyde, fractionation pitch

Details on test material:

- Physical state: Red-brownish liquid
- Analytical purity: UVCB
- Expiration date of the lot/batch: 11 May 2013
- Stability under storage conditions: Stable
- Storage condition of test material: At room temperature in the dark

Method

Target gene:

Histidine mutation :
TA1537: hisC3076, TA98: hisD3052/R-factor*, TA1535: hisG46, TA100: hisG46/R-factor*.

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Experiment 1: 3, 10, 33, 100, 333, 1000, 3330, and 5000 µL/plate
Experiment 2: 33, 100, 333, 1000, 3330, and 5000 µL/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO; physiol. saline; ethanol.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

(The vehicle of the test substance, which was ethanol).

Negative solvent / vehicle controls:

yes

Remarks:

(Saline)

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without metabolic activation

Migrated to IUCLID6: TA1535

Controls 2

Untreated negative controls:

yes

Remarks:

(The vehicle of the test substance, which was ethanol).

Negative solvent / vehicle controls:

yes

Remarks:

(DMSO)

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

With metabolic activation

Migrated to IUCLID6: TA1535

Controls 3

Untreated negative controls:

yes

Remarks:

(The vehicle of the test substance, which was ethanol).

Negative solvent / vehicle controls:

yes

Remarks:

(DMSO)

Positive controls:

yes

Positive control substance:

other: ICR-191 (TA1537)

Remarks:

With and without metabolic activation

Controls 4

Untreated negative controls:

yes

Remarks:

(The vehicle of the test substance, which was ethanol).

Negative solvent / vehicle controls:

yes

Remarks:

(DMSO)

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

With and without metabolic activation

Migrated to IUCLID6: TA98

Controls 5

Untreated negative controls:

yes

Remarks:

(The vehicle of the test substance, which was ethanol).

Negative solvent / vehicle controls:

yes

Remarks:

(DMSO)

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

With and without metabolic activation

Migrated to IUCLID6: TA100

Controls 6

Untreated negative controls:

yes

Remarks:

(The vehicle of the test substance, which was ethanol).

Negative solvent / vehicle controls:

yes

Remarks:

(DMSO)

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

With and without metabolic activation

Migrated to IUCLID6: WP2uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation). Top agar in top agar tubes was melted by heating to 45°C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (10E9 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test substance in ethanol and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli were counted.

NUMBER OF REPLICATIONS: Triplicates.

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

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 times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three times the concurrent vehicle 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 tester strain TA100 is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three times the concurrent vehicle control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

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:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
The dose range of the test material used in the preliminary toxicity study was 3, 10, 33, 100, 333, 1000, 3330 and 5000 μg/plate in the absence and presence of S9-mix. The test material exhibited toxicity at 3330 and 5000 μg/plate to the strain of Salmonella used (TA100). In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested.


COMPARISON WITH HISTORICAL CONTROL DATA:
The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Any other information on results incl. tables

Table 1. Experiment 1: Mutagenic response of Condensation products of cyclopentanone and pentaldehyde, fractionation pitch in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Dose                             Mean number of revertant colonies/3 replicate plates (± S.D.) with (µg/plate)                      different strains of Salmonella typhimurium and one Escherichia coli Strain
	TA1535	TA1537	TA98	TA100	WP2uvrA
Without S9-mix
Positive control	807 ± 25	920 ± 32	858 ± 40	812 ± 17	977 ± 9
Solvent control	10 ± 0	7 ± 2	23 ± 3	107 ± 3	21 ± 5
3				92 ± 8	23 ± 2
10				88 ± 8	18 ± 5
33	12 ± 2	4 ± 1	20 ± 3	91 ± 4	20 ± 3
100	6 ± 2	6 ± 2	19 ± 2	95 ± 16	22 ± 6
333	8 ± 2	3 ± 3	17 ± 3	81 ± 5	21 ± 6
1000	8 ± 2	4 ± 2	16 ± 3	62 ± 8	23 ± 5
3330	4 ± 1 SP	1 ± 2 s	8 ± 5 SP	6 ± 2 SP	19 ± 2 SP
5000	5 ± 2 s SP	1 ± 2 e SP	9 ± 2 SP	7 ± 1 SP	14 ± 4 SP
With S9-mix^1
Positive control	252 ± 9	394 ± 23	886 ± 20	955 ± 51	381 ± 35
Solvent control	7 ± 2	5 ± 2	22 ± 2	146 ± 13	21 ± 2
3				99 ± 3	23 ± 5
10				85 ± 3	18 ± 5
33	8 ± 1	2 ± 1	20 ± 1	88 ± 7	22 ± 2
100	8 ± 2	3 ± 1	24 ± 5	93 ± 8	25 ± 1
333	7 ± 3	6 ± 2	20 ± 4	98 ± 7	24 ± 2
1000	10 ± 3	4 ± 3	17 ± 3	84 ± 13	27 ± 1
3330	10 ± 1 SP	2 ± 2	12 ± 2 SP	57 ± 36 SP	17 ± 2 SP
5000	8 ± 3 SP	2 ± 1 SP	17 ± 3 SP	23 ± 8 SP *	24 ± 5 SP

Solvent control: 0.1 mL ethanol

¹   The S9-mix contained 5% (v/v) S9 fraction

s    Bacterial background lawn slightly reduced

e    Bacterial background lawn extremely reduced

SP  Slight Precipitate

sp* Slight Precipitate: The bacterial background lawn could not be determined

Table 2. Experiment 2: Mutagenic response of Condensation products of cyclopentanone and pentaldehyde, fractionation pitch in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Dose                             Mean number of revertant colonies/3 replicate plates (± S.D.) with (µg/plate)                      different strains of Salmonella typhimurium and one Escherichia coli strain
	TA1535	TA1537	TA98	TA100	WP2uvrA
Without S9-mix
Positive control	698 ± 45	543 ± 61	989 ± 45	842 ± 63	977 ± 104
Solvent control	5 ± 1	4 ± 1	15 ± 4	90 ± 14	13 ± 4
33	7 ± 3	3 ± 2	16 ± 1	81 ± 6	17 ± 5
100	5 ± 2	2 ± 1	16 ± 4	80 ± 13	14 ± 2
333	7 ± 1	1 ± 0	19 ± 1	81 ± 6	16 ± 4
1000	3 ± 2	2 ± 1	16 ± 2	72 ± 6	13 ± 2
3330 SP	1 ± 1	0 ± 0 m	14 ± 3	31 ± 6	16 ± 3
5000 SP	2 ± 1	3 ± 1 m	8 ± 2	13 ± 3	14 ± 4
With S9-mix^1
Positive control	171 ± 13	329 ± 31	715 ± 23	1138 ± 183	429 ± 13
Solvent control	4 ± 2	3 ± 1	19 ± 3	115 ± 7	15 ± 2
33	5 ± 3	4 ± 2	21 ± 6	96 ± 15	20 ± 1
100	3 ± 1	2 ± 1	25 ± 2	108 ± 5	16 ± 4
333	5 ± 2	4 ± 2	24 ± 5	138 ± 17	17 ± 4
1000	2 ± 1	3 ± 1	23 ± 4	145 ± 21	15 ± 2
3330 SP	2 ± 1	2 ± 1 s	17 ± 5	103 ± 13	18 ± 3
5000 SP	4 ± 2	2 ± 1 m	18 ± 3	97 ± 13	19 ± 4

Solvent control: 0.1 mL ethanol

¹The S9-mix contained 10% (v/v) S9 fraction

s   Bacterial background lawn slightly reduced

m  Bacterial background lawn moderately reduced

SP  Slight Precipitate

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative

Based on the results of this study it is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

Evaluation of the mutagenic activity of the test substance in the Salmonella typhimurium and the Escherichia coli reverse mutation assay (with independent repeat) was determinated according to OECD 471, EC B.13/14 Guidelines with GLP.

Four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and a tryptophan-requiring strain of Escherichia coli (WP2uvrA) were used for the assay. The test was performed in two independent experiments in the presence and absence of S9-mix (5% (v/v) S9 fraction in expertiment 1 and 10% (v/v) S9 fraction in experiment 2). The dose range was determined in a prelimirary toxicity assay and was 3 to 5000 µg/plate in the first experiment. The experiment was repeated using a dose range of 33 to 5000 µg/plate.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that Condensation products of cyclopentanone and pentaldehyde, fractionation pitch is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.