Octene, hydroformylation products, low-boiling

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:

22 January 2009 and 10 February 2009.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)).

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Octene, hydroformylation products, low-boiling

EC Number:

273-110-1

EC Name:

Octene, hydroformylation products, low-boiling

Cas Number:

68938-03-4

IUPAC Name:

Octene, hydroformylation products, low-boiling

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): Oxooil LS9
- Physical state: Clear colourless liquid
-- Stability under test conditions: Stable
- Storage condition of test material: Room temperature in the dark

Method

Target gene:

Histidine for Salmonella.
Tryptophan for E.Coli

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/beta­naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
main test:
Experiment one: 50, 150, 500, 1500 and 5000 µg/plate
Experiment two: 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: The test material was immiscible in dimethyl sulphoxide at 50 mg/ml but was fully miscible in acetone at the same concentration in solubility checks performed in house. Distilled water was not evaluated as a potential vehicle in this test system as information provided by the sponsor suggested it was immiscible with the test material.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Remarks:

2-Aminoanthracene

Remarks:

With S9 mix

Controls 2

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With S9 mix

Controls 3

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 mix

Controls 4

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix

Controls 5

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 10h
- Exposure duration: 48 - 72 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 48 -72 hrs


SELECTION AGENT (mutation assays): Not applicable.


NUMBER OF REPLICATIONS: Triplicate plating.


NUMBER OF CELLS EVALUATED: Not applicable.


DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.


OTHER EXAMINATIONS: None

Evaluation criteria:

Acceptance Criteria:

The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
All tester strain cultures should be in the approximate range of 1 to 9.9 x 109 bacteria per ml.
Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
There should be a minimum of four non-toxic test material dose levels.
There should not be an excessive loss of plates due to contamination.

Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.

Statistics:

Standard deviation

Results and discussion

Test results

Test results 1

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

Tested up to maximum recommended dose of 5000 micro.g/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

Tested up to maximum recommended dose of 5000 micro.g/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Distilled water was not evaluated as a potential vehicle in this test system as information provided by the sponsor suggested it was immiscible with the test material.
- Precipitation: No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9- mix.



RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test:The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). The test material formulation and S9-mix used in this experiment were both shown to be sterile.


COMPARISON WITH HISTORICAL CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the
activity of the S9-mix and the sensitivity of the bacterial strains.


ADDITIONAL INFORMATION ON CYTOTOXICITY: None

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. Spontaneous Mutation Rates (Concurrent Negative Controls)

EXPERIMENT 1

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA^-		TA98		TA1537
95		20		32		23		13
98	(99)	29	(24)	30	(29)	29	(29)	10	(12)
103		24		26		36		12

EXPERIMENT 2

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA^-		TA98		TA1537
86		25		18		23		10
118	(104)	32	(25)	30	(26)	24	(24)	13	(12)
109		19		29		25		13

Test Results: Experiment 1 – Without Metabolic Activation

Test Period		From: 01 February 2009						To: 04 February 2009
With out S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		WP2uvrA^‑			TA98		TA1537
-	0	101 103 88	(97) 8.1#	22 22 25	(23) 1.7	26 24 29	(26) 2.5		20 16 15	(17) 2.6	12 9 9	(10) 1.7
-	50	86 89 87	(87) 1.5	19 23 22	(21) 2.1	22 22 31	(25) 5.2		18 14 16	(16) 2.0	9 14 8	(10) 3.2
-	150	89 91 102	(94) 7.0	16 25 26	(22) 5.5	25 23 30	(26) 3.6		18 18 15	(17) 1.7	10 8 7	(8) 1.5
-	500	106 95 90	(97) 8.2	25 20 27	(24) 3.6	25 23 26	(25) 1.5		16 15 18	(16) 1.5	10 10 9	(10) 0.6
-	1500	90 101 91	(94) 6.1	25 18 19	(21) 3.8	30 21 32	(28) 5.9		18 19 18	(18) 0.6	7 10 10	(9) 1.7
-	5000	79 81 81	(80) 1.2	21 18 21	(20) 1.7	30 32 26	(29) 3.1		16 16 20	(17) 2.3	13 8 9	(10) 2.6
Positive controls S9-Mix -	Name Concentration (μg/plate) No. colonies per plate	ENNG		ENNG		ENNG			4NQO		9AA
		3		5		2			0.2		80
		670 759 826	(752) 78.3	658 467 395	(507) 135.9	384 352 348	(361) 19.7		198 246 192	(212) 29.6	521 531 531	(528) 5.8

ENNG N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

#        Standard deviation

 

Test Results: Experiment 1 – With Metabolic Activation

Test Period		From: 01 February 2009					To: 04 February 2009
With S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		WP2uvrA^‑			TA98		TA1537
+	0	106 96 100	(101) 5.0#	13 14 10	(12) 2.1	35 35 31		(34) 2.3	33 22 30	(28) 5.7	9 9 9	(9) 0.0
+	50	99 89 98	(95) 5.5	9 12 15	(12) 3.0	34 30 34		(33) 2.3	33 32 27	(31) 3.2	11 10 7	(9) 2.1
+	150	84 90 99	(91) 7.5	7 9 11	(9) 2.0	34 33 34		(34) 0.6	30 21 32	(28) 5.9	9 8 8	(8) 0.6
+	500	89 88 101	(93) 7.2	14 15 10	(13) 2.6	34 31 32		(32) 1.5	21 36 21	(26) 8.7	10 8 7	(8) 1.5
+	1500	85 95 97	(92) 6.4	11 10 11	(11) 0.6	32 33 34		(33) 1.0	25 33 22	(27) 5.7	8 8 10	(9) 1.2
+	5000	89 98 86	(91) 6.2	10 11 12	(11) 1.0	36 33 34		(34) 1.5	23 33 31	(29) 5.3	8 9 8	(8) 0.6
Positive controls S9-Mix +	Name Concentration (μg/plate) No. colonies per plate	2AA		2AA		2AA			BP		2AA
		1		2		10			5		2
		2779 2359 2834	(2657) 259.8	181 206 217	(201) 1.8.4	1720 1652 1646		(1673) 41.1	225 241 267	(244) 21.2	523 511 563	(532) 27.2

2AA    2-Aminoanthracene

BP      Benzo(a)pyrene

#        Standard deviation

 

Test Results: Experiment 2 – Without Metabolic Activation

Test Period		From: 07 February 2009						To: 10 February 2009
With out S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		WP2uvrA^‑			TA98		TA1537
-	0	120 103 104	(109) 9.5#	27 34 31	(31) 3.5	33 33 31	(32) 1.2		18 22 22	(21) 2.3	12 12 14	(13) 1.2
-	50	119 104 103	(109) 9.0	27 31 26	(28) 2.6	31 36 31	(33) 2.9		20 24 20	(21) 2.3	10 11 12	(11) 1.0
-	150	117 118 84	(106) 19.3	21 37 29	(29) 8.0	24 26 25	(25) 1.0		16 12 19	(16) 3.5	10 12 10	(11) 1.2
-	500	101 101 99	(100) 1.2	23 30 31	(28) 4.4	29 27 29	(28) 1.2		23 19 15	(19) 4.0	10 11 11	(11) 0.6
-	1500	128 109 110	(116) 10.7	30 24 27	(27) 3.0	35 29 33	(32) 3.1		19 23 18	(20) 2.6	14 10 9	(11) 2.6
-	5000	115 121 90	(109) 16.4	30 26 25	(27) 2.6	34 32 34	(33) 1.2		19 21 22	(21) 1.5	10 12 14	(12) 2.0
Positive controls S9-Mix -	Name Concentration (μg/plate) No. colonies per plate	ENNG		ENNG		ENNG			4NQO		9AA
		3		5		2			0.2		80
		1190 1279 1220	(1230) 45.3	1771 1697 1518	(1662) 130.1	2145 2228 2073	(2149) 77.6		213 233 207	(218) 13.6	1226 1215 1227	(1223) 6.7

 

ENNG N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

#        Standard deviation

 

Test Results: Experiment 2 – With Metabolic Activation

Test Period		From: 07 February 2009					To: 10 February 2009
With S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		WP2uvrA^‑			TA98		TA1537
+	0	99 97 99	(98) 1.2#	10 14 9	(11) 2.6	41 40 34		(38) 3.8	30 30 30	(30) 0.0	14 16 15	(15) 1.0
+	50	92 84 91	(89) 4.4	12 8 15	(12) 3.5	35 41 34		(37) 3.8	24 24 31	(26) 4.0	15 16 14	(15) 1.0
+	150	88 93 95	(92) 3.6	9 11 13	(11) 2.0	40 46 30		(39) 8.1	30 24 31	(28) 3.8	15 12 15	(14) 1.7
+	500	93 92 97	(94) 2.6	11 12 9	(11) 1.5	42 37 43		(41) 3.2	24 31 24	(26) 4.0	10 10 12	(11) 1.2
+	1500	102 93 95	(97) 4.7	9 16 11	(12) 3.6	35 40 36		(37) 2.6	22 26 35	(28) 6.7	11 12 11	(11) 0.6
+	5000	91 102 96	(96) 5.5	14 11 13	(13) 1.5	38 44 35		(39) 4.6	27 25 25	(26) 1.2	12 10 10	(11) 1.2
Positive controls S9-Mix +	Name Concentration (μg/plate) No. colonies per plate	2AA		2AA		2AA			BP		2AA
		1		2		10			5		2
		2481 3433 3619	(3178) 610.5	184 273 245	(234) 45.5	468 622 552		(547) 77.1	145 227 216	(196) 44.5	328 588 552	(489) 140.9

 

2AA    2-Aminoanthracene

BP      Benzo(a)pyrene

#        Standard deviation

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test material was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Introduction. Thethod conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It alsoets the requirents of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) Number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Methods.Salmonella typhimuriumstrains TA1535, TA1537, TA98, TA100 andEscherichia colistrain WP2uvrA^-were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations. 

Results.The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

Conclusion.The test material was considered to be non-mutagenic under the conditions of this test.