Reaction products of cyclododeca-1,5,9-triene and dinitrogen monoxide, distillation overheads

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:

Nov- Dec 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997-07-21

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt Rheinland-Pfalz

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of (4Z,8E)-dodeca-4,8,11-trienal and (4E,8Z)-dodeca-4,8,11-trienal

EC Number:

701-295-5

Molecular formula:

C12 H18 O

IUPAC Name:

Reaction mass of (4Z,8E)-dodeca-4,8,11-trienal and (4E,8Z)-dodeca-4,8,11-trienal

Specific details on test material used for the study:

Test material is the main constituent of the registered substance.

Method

Target gene:

trp / his

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:

Type and composition of metabolic activation system:
- Source of S9 : Prepared at BASF SE
- Method of preparation of S9 mix : Exposure of 5 male Wistar rats with 80 mg/kg bw phenobarbital (i.p.) and β-naphthoflavone (oral) on 3d; 24h after last administration rats were sacrificied and livers were prepared; Mixture of S9 fraction and S9 supplement prior to each experiment
- Preparation date of S9 fraction: 07 Sep 2018
- Content of protein in S9 fraction: 28.61 g/L
- Concentration or volume of S9 mix and S9 in the final culture medium : 0.5 mL S9 mix
- Sterility controls of S9 mix were performed

Test concentrations with justification for top dose:

Due to toxicity observed in a previous performed Non-GLP study (40M0072/18M062), the doses were adjusted in the 1st Experiment (standard plate test):
0.33; 1.0; 3.3; 10; 33 and 100 μg/plate (TA strains without S9 mix)
3.3; 10; 33; 100; 333 and 1000 μg/plate (E.coli without S9 mix)
1.0; 3.3; 10; 33; 100 and 333 μg/plate (TA strains with S9 mix)
33; 100; 333; 1000; 2500 and 5000 μg/plate (E.coli with S9 mix)

Due to toxicity observed in the standard plate test, the doses were adjusted in the preincubation test:
0; 0.33; 1.0; 3.3; 10; 33 and 100 μg/plate (TA strains with and without S9 mix)
0; 1; 3.3; 10; 33; 100 and 333 μg/plate (E. coli without S9 mix)
0; 10; 33; 100; 333; 1000 and 2500 μg/plate (E.coli with S9 mix)

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: DMSO had been demonstrated to be suitable in a bacterial reverse mutation test; historical data available

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: 2-aminoanthracene (2-AA); N-methyl-N'-nitro-N-nitrosoguanidine (MNNG); 4-nitro-o-phenylenediamine (NOPD)

Remarks:

positive control: 2-aminoanthracene was tested with S9 mix; other without S9 mix

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 3
- Number of independent experiments : 2 experiments plus 2 control settings

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Approximately 10^9 cells per mL
- Test substance added in soft agar (plate incorbation); preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 min
- Exposure duration/duration of treatment: 48 - 72 h

Evaluation criteria:

The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least
doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli
WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and
TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix
or after adding a metabolizing system.

A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the range of the historical negative
control data under all experimental conditions in at least two experiments carried out
independently of each other.

Toxicity detected by a
• decrease in the number of revertants (factor ≤ 0.6)
• clearing or diminution of the background lawn (= reduced his- or trp- background growth)

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:

cytotoxicity

Remarks:

at and above 3.3 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above 100 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above 1.0 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above 3.3 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

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:

cytotoxicity

Remarks:

at and above 333 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: No test substance precipitation was observed with and without S9 mix

STUDY RESULTS

Concurrent vehicle negative and positive control data :
-The number of revertant colonies in the negative controls was within the range of the
historical negative control data for each tested strain
- The positive control substances both with and without S9 mix induced a distinct increase in
the number of revertant colonies compatible with the range of the historical positive control
data or above
-The sterility controls revealed no indication of bacterial contamination

Ames test:
- Signs of toxicity : Bacteriotoxic effects were observed (dependend on bacterial strain and test condition) in the standard plate test and preincubation test

Any other information on results incl. tables

TOXICITY

Tab.1: Decreased revertant numbers were observed at following concentrations (μg/plate)

Experiment	S9	TA 1535	TA 100	TA 1537	TA 98	E.coli
1st-STP	Without	100	100	10 - 100	100	1000
	With	333	333	100 - 333	100 - 333	2500 - 5000
2nd-PIT	Without	10 - 100	100	1.0, 3.3, 33, 100	3.3, 33 - 100	333
	With	3.3, 10, 100	100	0.33 - 100	100	1000 - 2500

STP- standard plate test: PIT- preincubation test

Tab. 2: Reduced background growth was observed at following concentrations (μg/plate)

Experiment	S9	TA 1535	TA 100	TA 1537	TA 98	E.coli
1st-STP	Without	100	100	100	100	333 - 1000
	With	333	333	333	333	2500 - 5000
2nd-PIT	Without	100	100	100	100	333
	With	100	100	100	100	1000 - 2500

STP- standard plate test: PIT- preincubation test

STANDARD PLATE TEST

Tab. 3: Standard plate test with metabolic activation

Strain	Test group	Dose (µg/plate)	Mean revertants per plate	Standard deviation	Factor	Individual revertant colony counts
TA 1535	DMSO	-	12.7	6.0	-	19, 7, 12
	Test item	1.0	8.3	2.9	0.7	5, 10, 10
		3.3	14.0	8.7	1.1	19, 19, 4
		10	11.0	1.7	0.9	9, 12, 12
		33	10.0	1.0	0.8	10, 9, 11
		100	8.3	0.6	0.7	9, 8, 8
		333	2.0	1.7	0.2	1 B, 4 B, 1 B
	2-AA	2.5	148.0	1.0	11.7	148, 147, 149
TA 100	DMSO	-	104.7	5.5	-	99, 105, 110
	Test item	1.0	106.0	18.2	1.0	94, 97, 127
		3.3	114.7	13.9	1.1	130, 103, 111
		10	106.7	17.0	1.0	108, 123, 89
		33	101.7	6.7	1.0	96, 109, 100
		100	106.7	11.6	1.0	101, 120, 99
		333	0.0	0.0	0.0	0 B, 0 B, 0 B
	2-AA	2.5	1483.0	582.0	14.2	811, 1811, 1827
TA 1537	DMSO	-	5.7	0.6	-	6, 5, 6
	Test item	1.0	8.3	2.1	1.5	6, 10, 9
		3.3	7.0	4.6	1.2	6, 12, 3
		10	6.3	2.5	1.1	6, 9, 4
		33	4.3	1.5	0.8	4, 3, 6
		100	3.3	2.5	0.6	3, 6, 1
		333	0.0	0.0	0.0	0 B, 0 B, 0 B
	2-AA	2.5	158.0	37.2	27.9	180, 115, 179
TA 98	DMSO Test item	-	19.3	8.6	-	27, 10, 21
		1.0	22.0	1.0	1.1	22, 21, 23
		3.3	17.3	3.5	0.9	17, 21, 14
		10	15.0	6.6	0.8	9, 22, 14
		33	14.0	1.0	0.7	15, 13, 14
		100	7.3	4.5	0.4	12, 7, 3
		333	1.7	1.2	0.1	3 B, 1 B, 1 B
	2-AA	2.5	1178.3	174.0	60.9	1335, 1209, 991
E. coli	DMSO Test item	-	19.7	3.8	-	17, 24, 18
		33	20.7	3.1	1.1	18, 20, 24
		100	21.7	8.1	1.1	17, 31, 17
		333	22.7	5.5	1.2	28, 23, 17
		1000	14.3	10.5	0.7	25, 4, 14
		2500	7.7	3.1	0.4	7 B, 11 B, 5 B
		5000	0.0	0.0	0.0	0 B, 0 B, 0 B
	2-AA	60	212.0	35.2	10.8	172, 238, 226

Tab. 4: Standard plate test without metabolic activation

Strain	Test group	Dose (µg/plate)	Mean revertants per plate	Standard deviation	Factor	Individual revertant colony counts
TA 1535	DMSO	-	11.0	1.7	-	12, 12, 9
	Test item	0.33	9.3	2.1	0.8	10, 11, 7
		1.0	12.7	2.3	1.2	14, 10, 14
		3.3	9.3	3.5	0.8	9, 13, 6
		10	8.7	2.5	0.8	6, 11, 9
		33	14.3	0.6	1.3	14, 14, 15
		100	2.0	0.0	0.2	2 B, 2 B, 2 B
	MNNG	5.0	6079.0	61.0	552.6	6029, 6061, 6147
TA 100	DMSO	-	126.0	18.5	-	147, 119, 112
	Test item	0.33	123.3	15.9	1.0	141, 119, 110
		1.0	126.3	4.0	1.0	124, 124, 131
		3.3	118.3	26.3	0.9	88, 132, 135
		10	99.7	4.7	0.8	105, 96, 98
		33	101.7	8.5	0.8	92, 108, 105
		100	37.3	22.2	0.3	63 B, 25 B, 24 B
	MNNG	5.0	6004.7	222.7	47.7	5832, 6256, 5926
TA 1537	DMSO	-	10.3	3.1	-	7, 11, 13
	Test item	0.33	8.0	2.0	0.8	6, 8, 10
		1.0	7.3	3.1	0.7	10, 8, 4
		3.3	7.0	3.0	0.7	4, 10, 7
		10	4.7	2.9	0.5	3, 8, 3
		33	5.0	3.6	0.5	6, 1, 8
		100	2.7	1.2	0.3	4 B, 2 B, 2 B
	AAC	100	999.7	295.1	96.7	1090, 670, 1239
TA 98	DMSO Test item	-	17.0	1.7	-	19, 16, 16
		0.33	18.7	7.4	1.1	16, 27, 13
		1.0	12.7	4.2	0.7	8, 16, 14
		3.3	15.3	4.7	0.9	10, 17, 19
		10	13.7	1.5	0.8	12, 14, 15
		33	13.0	1.7	0.8	14, 14, 11
		100	4.3	2.9	0.3	1 B, 6 B, 6 B
	NOPD	10	855.0	56.3	50.3	916, 805, 844
E. coli	DMSO Test item	-	21.0	3.6	-	24, 17, 22
		3.3	18.0	5.6	0.9	23, 19, 12
		10	26.0	4.6	1.2	22, 31, 25
		33	25.0	5.6	1.2	19, 26, 30
		100	16.7	7.2	0.8	25, 13, 12
		333	14.7	7.4	0.7	23 B, 12 B, 9 B
		1000	0.0	0.0	0.0	0 B, 0 B, 0 B
	4-NQO	5	1528.3	45.9	72.8	1568, 1539, 1478

PREINCUBATION TEST

Tab. 5: Preincubation test without metabolic activation

Strain	Test group	Dose (µg/plate)	Mean revertants per plate	Standard deviation	Factor	Individual revertant colony counts
TA 1535	DMSO	-	15.3	4.9	-	21, 12, 13
	Test item	0.33	16.3	5.5	1.1	19, 10, 20
		1.0	11.0	3.6	0.7	8, 15, 10
		3.3	14.3	4.2	0.9	19, 11, 13
		10	9.3	2.1	0.6	11, 10, 7
		33	7.7	2.3	0.5	9, 9, 5
		100	1.0	0.0	0.1	1 B, 1 B, 1 B
	MNNG	5.0	6535.0	309.9	426.2	6886, 6299, 6420
TA 100	DMSO	-	105.3	12.9	-	96, 100, 120
	Test item	0.33	111.0	6.0	1.1	105, 117, 111
		1.0	112.7	14.0	1.1	117, 124, 97
		3.3	112.7	27.5	1.1	86, 141, 111
		10	100.3	14.6	1.0	90, 94, 117
		33	99.0	4.4	0.9	94, 102, 101
		100	0.0	0.0	0.0	0 B, 0 B, 0 B
	MNNG	5.0	5002.7	223.6	47.5	4745, 5118, 5145
TA 1537	DMSO	-	9.3	6.1	-	16, 4, 8
	Test item	0.33	13.7	4.0	1.5	16, 16, 9
		1.0	5.0	2.6	0.5	8, 4, 3
		3.3	5.3	2.9	0.6	7, 7, 2
		10	7.3	2.1	0.8	9, 8, 5
		33	5.3	0.6	0.6	6, 5, 5
		100	1.7	1.2	0.2	3 B, 1 B, 1 B
	AAC	100	953.3	234.4	102.1	1224, 819, 817
TA 98	DMSO	-	24.0	0.0	-	24, 24, 24
	Test item	0.33	16.7	4.0	0.7	19, 19, 12
		1.0	15.7	4.9	0.7	19, 10, 18
		3.3	14.7	4.9	0.6	18, 9, 17
		10	22.7	3.8	0.9	27, 21, 20
		33	12.3	1.2	0.5	11, 13, 13
		100	0.0	0.0	0.0	0 B, 0 B, 0 B
	NOPD	10	958.3	17.1	39.9	947, 950, 978
E. coli	DMSO	-	21.3	6.5	-	28, 15, 21
	Test item	1.0	26.0	3.6	1.2	25, 23, 30
		3.3	26.7	7.4	1.3	24, 35, 21
		10	27.3	4.0	1.3	23, 28, 31
		33	18.7	2.9	0.9	17, 17, 22
		100	26.0	12.2	1.2	32, 34, 12
		333	9.3	6.8	0.4	17 B, 4 B, 7 B
	4-NQO	5	655.3	0.6	30.7	655, 655, 656

 

Tab.6: Preincubation test with metabolic activation

Strain	Test group	Dose (µg/plate)	Mean revertants per plate	Standard deviation	Factor	Individual revertant colony counts
TA 1535	DMSO	-	15.3	4.9	-	21, 12, 13
	Test item	0.33	16.3	5.5	1.1	19, 10, 20
		1.0	11.0	3.6	0.7	8, 15, 10
		3.3	14.3	4.2	0.9	19, 11, 13
		10	9.3	2.1	0.6	11, 10, 7
		33	7.7	2.3	0.5	9, 9, 5
		100	1.0	0.0	0.1	1 B, 1 B, 1 B
	2-AA	5.0	6535.0	309.9	426.2	6886, 6299, 6420
TA 100	DMSO	-	105.3	12.9	-	96, 100, 120
	Test item	0.33	111.0	6.0	1.1	105, 117, 111
		1.0	112.7	14.0	1.1	117, 124, 97
		3.3	112.7	27.5	1.1	86, 141, 111
		10	100.3	14.6	1.0	90, 94, 117
		33	99.0	4.4	0.9	94, 102, 101
		100	0.0	0.0	0.0	0 B, 0 B, 0 B
	2-AA	5.0	5002.7	223.6	47.5	4745, 5118, 5145
TA 1537	DMSO	-	9.3	6.1	-	16, 4, 8
	Test item	0.33	13.7	4.0	1.5	16, 16, 9
		1.0	5.0	2.6	0.5	8, 4, 3
		3.3	5.3	2.9	0.6	7, 7, 2
		10	7.3	2.1	0.8	9, 8, 5
		33	5.3	0.6	0.6	6, 5, 5
		100	1.7	1.2	0.2	3 B, 1 B, 1 B
	2-AA	100	953.3	234.4	102.1	1224, 819, 817
TA 98	DMSO	-	24.0	0.0	-	24, 24, 24
	Test item	0.33	16.7	4.0	0.7	19, 19, 12
		1.0	15.7	4.9	0.7	19, 10, 18
		3.3	14.7	4.9	0.6	18, 9, 17
		10	22.7	3.8	0.9	27, 21, 20
		33	12.3	1.2	0.5	11, 13, 13
		100	0.0	0.0	0.0	0 B, 0 B, 0 B
	2-AA	10	958.3	17.1	39.9	947, 950, 978
E. coli	DMSO	-	21.3	6.5	-	28, 15, 21
	Test item	1.0	26.0	3.6	1.2	25, 23, 30
		3.3	26.7	7.4	1.3	24, 35, 21
		10	27.3	4.0	1.3	23, 28, 31
		33	18.7	2.9	0.9	17, 17, 22
		100	26.0	12.2	1.2	32, 34, 12
		333	9.3	6.8	0.4	17 B, 4 B, 7 B
	2-AA	5	655.3	0.6	30.7	655, 655, 656

HISTORICAL CONTROL DATA

Tab. 7: Historical negative control

Strain	S9 Mix	Vehicle	No. of Plates	No. of Values	Min	Max	Mean	SD
TA 1535	without	(All)	315	112	7	19	11	2.3
	with	(All)	318	112	6	21	11	2.6
TA 100	without	(All)	318	113	78	140	106	12.1
	with	(All)	327	113	72	135	107	11.2
TA 1537	without	(All)	309	112	4	12	8	1.7
	with	(All)	309	112	5	13	9	1.6
TA 98	without	(All)	318	113	12	27	18	2.9
	with	(All)	312	113	18	45	25	4.0
E. coli	without	(All)	306	113	19	38	26	4.2
	with	(All)	306	113	18	40	26	4.1

Tab. 8: Historical poositive control

Strain	S9 Mix	Positive control	No. of Plates	No. of Values	Min	Max	Mean	SD
TA 1535	Without	MNNG	252	88	2345	7317	4484	1042.2
	With	2-AA	252	88	50	334	193	53.3
TA 100	Without	MNNG	252	88	814	5801	3443	1004.7
	With	2-AA	258	88	413	2943	1563	492.8
TA 1537	Without	AAC	246	88	282	1497	809	237.1
	With	2-AA	252	88	41	254	108	36.8
TA 98	Without	NOPD	252	88	484	1239	986	141.1
	With	2-AA	249	88	161	2347	1098	379.9
E. coli	Without	4-NQO	246	88	176	1931	750	395.9
	With	2-AA	246	88	66	259	115	45.7

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions of this study, the test substance is not
mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the
absence and the presence of metabolic activation.

Executive summary:

The test substance was tested for its mutagenic potential based on the ability

to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella

typhimurium and Escherichia coli, in a reverse mutation assay.

Strains: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA

Dose range: 0.33 μg - 5000 μg/plate (SPT); 0.33 μg - 2500 μg/plate (PIT)

Test conditions: Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats).

Solubility: No precipitation of the test substance was observed with and without S9 mix.

Toxicity: A bacteriotoxic effect was observed depending on the strain and test conditions at and above 0.33 μg/plate

Mutagenicity: A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli

WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system (BASF SE, 2019)