Ammonium undecafluorohexanoate

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:

03 -13 Mar 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in 1997

Deviations:

yes

Remarks:

missing DMSO (solvent for positive controls) control

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Ammonium undecafluorohexanoate

EC Number:

244-479-6

EC Name:

Ammonium undecafluorohexanoate

Cas Number:

21615-47-4

Molecular formula:

C6H4F11NO2

IUPAC Name:

2,2,3,3,4,4,5,5,6,6,6-Undecafluorohexanoic acid, ammonium salt (1:1)

Method

Target gene:

his operon (for S. typhimurium strains)
trp operon (for E. coli strain)

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:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital (one time at 30 mg/kg bw and three times at 60 mg/kg bw) and 5,6-benzoflavone (one time at 80 mg/kg bw)

Test concentrations with justification for top dose:

The test substance was corrected by the purity (50%) before preparation of dosing solutions.

Experiment 1: 4.88, 19.5, 78.1, 313, 1250 and 5000 µg/mL
Experiment 2: 313, 625, 1250, 2500 and 5000 µg/mL

Vehicle / solvent:

- Vehicle/solvent used: destilled water (test substance and NaN3), DMSO (AF-2, ICR-191 and 2AA)
- Justification for choice of solvent/vehicle: The solvent was chosen based on a good solubililty and stability (no change in color, exothermic reaction nor gas generation at room temperature within 4 h) at 50 mg/mL .

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: 2-Aminoanthracene (2AA); 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamine (AF-2); ICR-191

Remarks:

Efficasy of S9-mix was characterised with benzo(a)pyrene and 9,10-dimethylanthracene (mutagens that requires metabolic activation by microsomal enzymes).

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation
- Cell density at seeding:
Experiment 1: 2.9, 3.2, 6.9, 4.5 and 3.7 x10E9 cells/mL for TA100, TA1535, WP2uvrA, TA98 and TA1535, respectively
Experiment 2: 2.5, 3.2, 6.6, 4.7 and 3.4 x10E9 cells/mL for TA100, TA1535, WP2uvrA, TA98 and TA1535, respectively

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: Triplicates each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn observed using a stereomicroscope

- OTHER: All plates were counted with a colony analyzer (CA-1 lD, SYSTEM SCIENCE). Square correction and miscounting correction were conducted when counting with the colony analyzer.

Evaluation criteria:

The test substance was judged to be positive when the number of revertant colonies increased to twice or more than that in the negative control and when the responses were dose-related and/or reproducible.

Statistics:

Mean values and standard deviations were calculated.

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:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test substance was observed in either the presence or absence of S9 mix.

HISTORICAL CONTROL DATA : The test results showed that the numbers of revertant colonies in the negative and positive controls were within the range of the historical data at the testing facility.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Bacterial growth inhibition was not observed at any test condition.

Any other information on results incl. tables

Table 1. Results of experiment 1 (pre-incubation method)

	Number of revertant colonies per plate
S9-Mix	Without
Test substance (µg/plate)	TA 100	TA 1535	WP2 uvr A	TA 98	TA1537
SC	133 ± 10	13 ± 3	32 ± 7	16 ± 2	16 ± 3
4.88	136 ± 17	9 ± 5	34 ± 8	15 ± 4	16 ± 1
19.5	119 ± 7	14 ± 4	37 ± 8	15 ± 2	18 ± 8
78.1	130 ± 15	9 ± 2	39 ± 2	17 ± 3	19 ± 7
313	136 ± 13	10 ± 5	36 ± 6	17 ± 3	16 ± 10
1250	149 ± 4	10 ± 4	31 ± 9	21 ± 4	16 ± 3
5000	132 ± 20	16 ± 6	34 ± 7	18 ± 7	15 ± 3
Positive Control	AF-2	NaN3	AF-2	AF-2	ICR-191
Dose (µg/plate)	0.01	0.5	0.01	0.1	0.5
Number of revertant colonies/plate	899 ± 70	361 ± 31	164 ± 8	478 ± 27	720 ± 42
S9-Mix	With
Test substance (µg/plate)	TA 100	TA 1535	WP2 uvr A	TA 98	TA 1537
SC	139 ± 17	10 ± 2	29 ± 3	24 ± 3	20 ± 10
4.88	119 ± 9	15 ± 6	33 ± 7	24 ± 5	20 ± 3
19.5	134 ± 12	14 ± 3	35 ± 11	24 ± 5	20 ± 3
78.1	135 ± 16	6 ± 2	37 ± 10	31 ± 15	18 ± 3
313	126 ± 8	12 ± 1	34 ± 4	27 ± 5	17 ± 7
1250	117 ± 10	7 ± 1	32 ± 6	20 ± 1	22 ± 10
5000	113 ± 17	8 ± 4	40 ± 2	26 ± 6	22 ± 5
Positive Control	2AA	2AA	2AA	2AA	2AA
Dose (µg/plate)	1	2	10	0.5	2
Number of revertant colonies/plate	1517 ± 53	368 ± 11	627 ± 56	506 ± 26	250 ± 35
SC = Solvent Control (distilled water) NaN3: sodium acide; 2AA: 2-Aminoanthracene; AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamine

Table 2. Results of experiment 2 (pre-incubation method)

	Number of revertant colonies per plate
S9-Mix	Without
Test substance (µg/plate)	TA 100	TA 1535	WP2 uvr A	TA 98	TA1537
SC	131 ± 12	13 ± 5	38 ± 4	18 ± 6	22 ± 2
313	123 ± 26	12 ± 1	30 ± 8	26 ± 8	19 ± 2
625	144 ± 23	14 ± 3	34 ± 8	21 ± 3	19 ± 8
1250	142 ± 20	9 ± 2	34 ± 10	19 ± 4	22 ± 3
2500	143 ± 2	9 ± 5	29 ± 8	20 ± 5	18 ± 2
5000	133 ± 20	10 ± 2	30 ± 8	17 ± 5	17 ± 5
Positive Control	AF-2	NaN3	AF-2	AF-2	ICR-191
Dose (µg/plate)	0.01	0.5	0.01	0.1	0.5
Number of revertant colonies/plate	969 ± 45	324 ± 32	157 ± 5	575 ± 39	658 ± 55
S9-Mix	With
Test substance (µg/plate)	TA 100	TA 1535	WP2 uvr A	TA 98	TA 1537
SC	144 ± 8	11 ± 3	36 ± 7	29 ± 11	25 ± 0
313	109 ± 13	8 ± 5	31 ± 8	28 ± 4	27 ± 7
625	123 ± 18	9 ± 3	33 ± 10	30 ± 10	27 ± 6
1250	128 ± 4	11 ± 6	39 ± 8	21 ± 7	27 ± 7
2500	114 ± 5	8 ± 2	37 ± 3	29 ± 4	21 ± 6
5000	114 ± 22	9 ± 6	35 ± 8	26 ± 5	26 ± 14
Positive Control	2AA	2AA	2AA	2AA	2AA
Dose (µg/plate)	1	2	10	0.5	2
Number of revertant colonies/plate	1757 ± 91	385 ± 35	585 ± 42	485 ± 29	274 ± 35
SC = Solvent Control (distilled water) NaN3: sodium acide; 2AA: 2-Aminoanthracene; AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamine

Table 3. Histrorical negative control data (mean ± 3S.D.)

	-S9 mix					+S9mix
	TA100	TA1535	WP2uvrA	TA98	TA1537	TA100	TA1535	WP2uvrA	TA98	TA1537
Mean S.D.	119 25	11 4	32 10	20 6	12 6	124 23	11 4	31 10	29 8	14 6
UpperLimit Lower Limit	194 44	23 1	62 2	38 2	30 1	193 55	23 1	61 1	53 5	32 1

Table 4. Historical positive control data (mean± 3S.D.)

	-S9 mix					+S9mix
	TA100	TA1535	WP2uvrA	TA98	TA1537	TA100	TA1535	WP2uvrA	TA98	TA1537
Chemical	AF-2	NaN3	AF-2	AF-2	ICR-191	2AA	2AA	2AA	2AA	2AA
Dose(µg/plate)	0.01	0.5	0.01	0.1	0.5	1	2	10	0.5	2
Mean S.D.	854 104	312 57	112 21	498 54	635 103	1561 194	292 35	379 101	397 40	207 39
Upper Limit Lower Limit	1166 542	483 141	175 49	660 336	944 326	2143 979	397 187	682 76	517 277	324 90

AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

NaN3: Sodiumazide

2AA.: 2-Aminoanthracene

Test period: From September, 2016 to February, 2017

Applicant's summary and conclusion

Conclusions:

Under the conditions of the conducted test the substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 98, TA 100 and WP2 uvrA) tested with and without metabolic activation.