Iodonium, bis[4-(1,1-dimethylethyl)phenyl]-, hexafluorophosphate(1-) (1:1)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro: Negative Ames test (OECD 471) with S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA, with and without metabolic activation.

Link to relevant study records

Reference

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:

17 - 30 Nov 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Konpoan No. 287 – Environment Protection Agency

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Eisei No. 127 – Ministry of Health and Welfare

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Heisei 09/10/31 Kikyoku No. 2 – Ministry of Economy, Trade and Industry

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon (S. typhimurium) and trp operon (E. coli)

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers rats induced with 80 mg/kg bw (i.p.) phenobarbital and peroral administrations of β-naphthoflavone, each on 3 consecutive days

Test concentrations with justification for top dose:

Pre-experiment (experiment I): 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation
Experiment II: 0.1#, 0.3, 1, 3, 10, 33, 100, 333 and 1000# µg/plate with and without metabolic activation
#this additional concentrations were used for treatments in the absence of S9 only

Vehicle / solvent:

- Vehicle/solvent used: DMSO (100 µL)
- Justification for choice of solvent/vehicle: the solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide (10 µg/plate, -S9, TA 100 and TA 1535); 4-nitro-o-phenylene-diamine (10 or 50 µg/plate: TA 98 or TA 1537, -S9); methyl methane sulfonate (3 µL/plate, -S9, WP2 uvrA); 2-aminoanthracene (2.5 and 10 µg/plate: all strains, +S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION 1: in agar (plate incorporation) for the pre-experiment (experiment I)

DURATION
- Exposure duration: 48 h

METHOD OF APPLICATION 2: preincubation method for experiment II

DURATION
- Preincubation period: 1 h
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: inspection of the bacterial background lawn and count of the number of revertants

Evaluation criteria:

The test substance was considered to be mutagenic if the following criteria were met:
1) the assay was acceptable (regular background growth in the negative and solvent controls; spontaneous reversion rates in the negative and solvent controls are in the normal ranges of historical data; the positive control chemicals induced significant increases in mutant colony frequencies)
2) the increase in the number of revertants of the test strains compared to the colony counts of the solvent controls was biologically relevant (2-fold increases over controls for TA 98, TA 100, and WP2 uvrA and 3-fold increases over controls for TA 1535 and TA 1537)
3) the data sets showed a biologically relevant dose correlation (thresholds for the fold increase in the number of revertants compared to controls were exceeded at more than one concentration)
4) increases exceeding the threshold at only one concentration were considered biologically relevant, if they were reproduced in an independent second experiment
5) dose dependent increases in the number of revertant colonies below the threshold were regarded as an indication of a mutagenic potential if reproduced in an independent second experiment (such increases were not considered biologically relevant, if colony counts of the treated samples remained within the historical range of the negative and solvent controls)

Statistics:

Mean values and standard deviations for revertant colonies were calculated.

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:

cytotoxicity

Remarks:

plate incorporation (- and +S9): ≥ 33 µg/plate (TA 1537), ≥ 100 µg/plate (TA 1535, TA 98 and TA 100); preincubation (- and +S9): ≥ 10 µg/plate (TA 1537), ≥ 100 µg/plate (TA 1535, TA 98 and TA 100)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

plate incorporation (+ and -S9): ≥ 333 µg/plate; preincubation (-S9): ≥ 100 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: in experiment 1 (plate incorporation), precipitation of the test substance was observed on all incubated plates treated at 2500 and 5000 µg/plate (maximum concentration). In experiment 2 (preincubation), precipitation of the test substance was observed on plates treated at 1000 µg/plate (maximum concentration). The undissolved particles had no influence on data recording.

RANGE-FINDING/SCREENING STUDIES:
Experiment I served as pre-experiment for the assessment of toxicity, using final concentrations of the test substance at 3-5000 µg/plate with or without metabolic activation, plus negative, solvent and positive controls. Following these treatments, strong toxic effects were seen as shown by thinning of the background bacterial lawn and a reduction in the number of spontaneous revertants at higher concentrations. Thus, the maximum concentrations chosen for incubation in the absence or presence of metabolic activation were 333 or 1000 µg/plate, respectively.

COMPARISON WITH HISTORICAL CONTROL DATA:
The mean numbers of spontaneous revertants in the negative, solvent and positive controls of the strains used were all within the normal historical ranges.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the plate incorporation assay (experiment I), cytotoxicity (reduced background lawn and reduced number of revertants) was observed in all strains tested, ranging from 33 up to 5000 µg/plate with or without metabolic activation, respectively. In the preincubation assay (experiment II), cytotoxicity (reduced background lawn and reduced number of revertants) occurred in all strains of S. typhimurium at concentrations ranging from 10 to 333 µg/plate in the absence or 33 to 1000 µg/plate in the presence of metabolic activation. In the E. coli strain WP2 uvrA, cytotoxicity was observed at concentrations ≥ 333 µg/plate in the plate incorporation experiment with and without metabolic activation. In the E. coli strain WP2 uvrA, cytotoxicity was observed at concentrations ≥ 333 µg/plate in the plate incorporation experiment with and without metabolic activation. In contrast, cytotoxicity was only seen without metabolic activation in the preincubation experiment at concentrations ≥ 100 µg/plate.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1. Test results of experiment I (plate incorporation)

PF-6: Bacterial Reverse Mutation Assay, mean revertant colonies/plate (n=3 ± SD)
EXPERIMENT I (plate incorporation)
S9-Mix	Without
Concentration (per plate)	TA 1535	TA1537	TA98	TA100	E.coli WP2 uvrA
SC	13 ± 5	21 ± 5	34 ± 2	104 ± 6	51 ± 10
NC	15 ± 5	24 ± 2	31 ± 8	121 ± 3	50 ± 4
PF-6
3 µg	14 ± 5	22 ± 2	26 ± 6	90 ± 9	42 ± 9
10 µg	12 ± 6	24 ± 4	31 ± 8	104 ± 10	42 ± 5
33 µg	10 ± 4	13 ± 2M, R	27 ± 5	92 ± 13	52 ± 14
100 µg	1 ± 1R	1 ± 1M, R	5 ± 2M, R	12 ± 3M,R	31 ± 3
333 µg	1 ± 1R	1 ± 1M, R	1 ± 1M, R	1 ± 1M, R	2 ± 1R
1000 µg	0 ± 0R	0 ± 0M, R	0 ± 0M, R	0 ± 0M, R	0 ± 1R
2500 µg	0 ± 0R, P	0 ± 0P, M, R	0 ± 0P, M, R	0 ± 0P, M, R	0 ± 0P, R
5000 µg	0 ± 0R, P	0 ± 0P, M, R	0 ± 0P, M, R	0 ± 0P, M, R	0 ± 0P, R
PC
NaN3 (10 µg)	1810 ± 21	-	-	1972 ± 33	-
4NOPD (10 µg)	-	-	342 ± 17	-	-
4NOPD (50 µg)	-	87 ± 15	-	-	-
MMS (3 µL)	-	-	-	-	1270 ± 74
S9-Mix	With
Concentration (per plate)	TA 1535	TA1537	TA98	TA100	E.coli WP2 uvrA
NC	20 ± 5	24 ± 3	37 ± 9	122 ± 8	61 ± 6
SC	20 ± 2	24 ± 5	54 ± 8	110 ± 11	60 ± 5
PF-6
3 µg	22 ± 1	17 ± 2	38 ± 4	120 ± 23	54 ± 10
10 µg	22 ± 2	26 ± 4	44 ± 7	119 ± 7	50 ± 11
33 µg	19 ± 5	35 ± 7	36 ± 3	145 ± 19	53 ± 3
100 µg	8 ± 2	7 ± 2M, R	28 ± 1	116 ± 21	59 ± 5
333 µg	1 ± 1R, M	1 ± 1M, R	2 ± 1M, R	8 ± 1R	8 ± 3M, R
1000 µg	1 ± 1R, M	0 ± 0M, R	0 ± 0M, R	1 ± 1R	0 ± 0M, R
2500 µg	0 ± 0P, R, M	0 ± 0P, M, R	0 ± 0P, M, R	0 ± 0P, M, R	0 ± 0P, M, R
5000 µg	0 ± 0P, R, M	0 ± 0P, M, R	0 ± 0P, M, R	0 ± 0P, M, R	0 ± 0P, M, R
PC
2AA (2.5 µg)	415 ± 22	348 ± 10	2109 ± 253	2728 ± 35	-
2AA (10 µg)	-	-	-	-	237 ± 16
NC = Negative Control; SC = Solvent control; PC = Positive control substances; SD = standard deviation; NaN3 = sodium azide; 4NOPD = 4-nitro-o-phenylene-diamine; MMS = methyl methane sulfonate; 2AA = 2-aminoanthracene M = Manual count; P = Precipitate; R = Reduced background growth

Table 2. Test results of experiment II (pre-incubation)

PF-6: Bacterial Reverse Mutation Assay, mean revertant colonies/plate (n=3 ± SD)
EXPERIMENT II (pre-incubation)
S9-Mix	Without
Concentration (per plate)	TA 1535	TA1537	TA98	TA100	E.coli WP2 uvrA
SC	15 ± 6	14 ± 3	28 ± 4	115 ± 7	39 ± 6
NC	11 ± 4	11 ± 6	34 ± 5	137 ± 19	35 ± 0
PF-6
0.1 µg	14 ± 2	13 ± 1	31 ± 6	112 ± 1	33 ± 8
0.3 µg	14 ± 7	15 ± 3	26 ± 6	93 ± 15	39 ± 6
1 µg	13 ± 2	16 ± 2	30 ± 9	106 ± 14	33 ± 3
3 µg	15 ± 3	16 ± 2	33 ± 4	111 ± 5	35 ± 6
10 µg	17 ± 3	8 ± 1M, R	27 ± 6	99 ± 14	42 ± 4
33 µg	12 ± 2	6 ± 1M, R	24 ± 4	74 ± 6	45 ± 2
100 µg	1 ± 1M, R	0 ± 0M, R	6 ± 2M, R	15 ± 3M, R	8 ± 2M, R
333 µg	0 ± 0M, R	0 ± 0M, R	0 ± 0M, R	0 ± 0M, R	0 ± 1M, R
PC
NaN3 (10 µg)	1981 ± 77	-	-	2264 ± 48	-
4NOPD (10 µg)	-	-	353 ± 8	-	-
4NOPD (50 µg)	-	96 ± 18	-	-	-
MMS (3 µL)	-	-	-	-	537 ± 34
S9-Mix	With
Concentration (per plate)	TA 1535	TA1537	TA98	TA100	E.coli WP2 uvrA
NC	20 ± 4	23 ± 4	41 ± 7	118 ± 22	53 ± 9
SC	25 ± 6	22 ± 0	42 ± 10	134 ± 13	42 ± 5
PF-6
0.3 µg	22 ± 2	21 ± 1	47 ± 3	114 ± 15	49 ± 8
1 µg	22 ± 1	23 ± 4	39 ± 10	119 ± 1	50 ± 12
3 µg	21 ± 4	18 ± 3	44 ± 10	126 ± 9	49 ± 7
10 µg	23 ± 3	25 ± 2	40 ± 4	119 ± 4	50 ± 5
33 µg	13 ± 1	20 ± 3M, R	38 ± 9	104 ± 22	54 ± 15
100 µg	10 ± 4	3 ± 1M, R	26 ± 7	40 ± 9M, R	51 ± 6
333 µg	0 ± 0R	0 ± 0M, R	0 ± 0R	0 ± 0M, R	4 ± 1
1000 µg	0 ± 0R, P	0 ± 0M, R, P	0 ± 0RP	0 ± 0P, M, R	0 ± 0P
PC
2AA (2.5 µg)	351 ± 21	181 ± 18	1677 ± 121	2089 ± 153	-
2AA (10 µg)	-	-	-	-	203 ± 20
NC = Negative Control; SC = Solvent control; PC = Positive control substances; SD = standard deviation; NaN3 = sodium azide; 4NOPD = 4-nitro-o-phenylene-diamine; MMS = methyl methane sulfonate; 2AA = 2-aminoan-thracene M = Manual count; P = Precipitate; R = Reduced background growth

Conclusions:

Interpretation of results (migrated information):
negative

The test substance did not induce mutations in the bacterial mutation tests in the absence and presence of metabolic activation in the selected strains of S. typhimurium (TA 98, TA 100, TA 1535 and TA 1537) and E. coli (WP2 uvrA).

Executive summary:

A bacterial gene mutation assay (Ames test) was conducted with PF6 according to OCED guideline 471 and under conditions of GLP. The assay included two series of experiments, both performed in the absence and in the presence of S9 mix. In the first experiment, the direct plate incorporation procedure was conducted with Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537 and Escherichia coli WP2 uvrA at concentrations ranging from 3 to 5000 µg/plate for a period of 48 h. In the second experiment, concentrations ranging from 0.1 to 333 µg/plate without S9 mix and 0.3 to 1000 µg/plate with S9 mix were analysed using the same strains, but with modification of the study design (with pre-incubation period of 1 h). Cytotoxicity was observed in both independent experiments, as shown by a reduction in background growth at ≥ 10 µg/plate in S. typhimurium and at ≥ 100 µg/plate in E. coli. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains with and without metabolic activation. The positive and negative controls included showed the expected results in each experiment. Under the conditions of both experiments, the test substance did not induce mutations in the bacterial mutation assay in the absence and presence of metabolic activation in the selected strains of S. typhimurium (TA 98, TA 100, TA 1535 and TA 1537) and E.coli WP2 uvrA.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

A bacterial gene mutation assay (Ames test) was conducted with PF6 according to OCED guideline 471 and under conditions of GLP (Sokolowski, 2011). The assay included two series of experiments, both performed in the absence and in the presence of S9 mix. In the first experiment, the direct plate incorporation procedure was conducted with Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537 and Escherichia coli WP2 uvrA at concentrations ranging from 3 to 5000 µg/plate for a period of 48 h. In the second experiment, concentrations ranging from 0.1 to 333 µg/plate without S9 mix and 0.3 to 1000 µg/plate with S9 mix were analysed using the same strains, but with modification of the study design (with pre-incubation period of 1 h). Cytotoxicity was observed in both independent experiments, as shown by a reduction in background growth at ≥ 10 µg/plate in S. typhimurium and at ≥ 100 µg/plate in E. coli. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains with and without metabolic activation. The positive and negative controls included showed the expected results in each experiment. Under the conditions of both experiments, the test substance did not induce mutations in the bacterial mutation assay in the absence and presence of metabolic activation in the selected strains of S. typhimurium (TA 98, TA 100, TA 1535 and TA 1537) and E.coli WP2 uvrA.

Justification for classification or non-classification

The available data on genetic toxicity of PF-6 do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.