Alkali metal tetrakis (perfluoro-alcoholato)metallate

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:

May - July 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21.7.1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

solid

Details on test material:

Test material was sublimed for purification.

Specific details on test material used for the study:

Name: AM(pfa)4
Appearance: white powder

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Species / strain 2

Species / strain / cell type:

E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

- S9-mix: Phosphate buffer 22.5 mL, 0.1M NADP-solution 1.0 mL, 1M G6P-solution 0.125 mL, Salt solution 0.5 mL and rat liver S9 1.0 mL
- S9: S9 was obtained by Trinova Biochem GmbH, Gießen, Batch nos. 4180, 4069; produced from the livers of male Sprague-Dawley rats which were treated with Phenobarbital/5,6-Benzoflavone.

Test concentrations with justification for top dose:

See information on materials and methods incl. tables

Vehicle / solvent:

In a non-GLP pre-test, the solubility of the test item was tested in a concentration of 50 g/L in demineralized (demin.) water and dimethyl sulfoxide (DMSO). The solid test item was soluble in demin. water. Based on the results of the non-GLP pre-test, demin. water was chosen as vehicle, because the test item was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: for positive control substances per strain, with and without metaboloic activation see overview in material and methods

Details on test system and experimental conditions:

See information on materials and methods incl. tables

Rationale for test conditions:

See information on materials and methods incl. tables

Evaluation criteria:

See information on materials and methods incl. tables

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 6

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

In all experiments, no precipitation of the test item AM(pfa)4 was observed at any of the tested concentrations up to 5000 µg/plate.
The tested strains showed different behaviour after incubation with the test item. As the test item showed toxic effects towards all bacterial strains and at least five non-toxic concentrations are required for evaluation, a repetition of experiment 1 was performed (exp. 1b) for all bacterial strains.

S. typhimurium TA98: In experiment 1, the test item showed toxicity towards the strain TA98 at the item concentrations 5000; 1500 and 500 µg/plate with metabolic activation and at 5000; 1500, 500 and 150 µg/plate without metabolic activation. In experiment 1b, a clear reduction in the colony count was observed at 500 µg/plate with metabolic activation and at 150 µg/plate without metabolic activation. In experiment 2, toxicity was observed at the concentration 150 µg/plate without metabolic activation. The bacterial background lawn was not affected in all experimental conditions.

S. typhimurium TA100: In experiment 1, the test item showed toxicity towards the strain TA100 at the test item concentrations 5000 and 1500 µg/plate with and without metabolic activation and at 500 µg/plate without metabolic activation. In experiment 1b with metabolic activation, the number of revertant colonies was reduced at a test item concentration of 1500 µg/plate. At 500 µg/plate, no colonies were visible for TA100 without metabolic activation. In experiment 2, no colonies were visible on the plates and the background lawn was only slightly visible at a concentration of 1500 µg/plate with metabolic activation. At 500 and 250 µg/plate no colonies and no/reduced background lawn was observed for TA100 without metabolic activation.

S. typhimurium TA102: In experiment 1, the test item showed toxicity towards the strain TA102 as no colony growth was visible at a test item concentration of 5000 µg/plate and 1500 µg/plate with and without metabolic activation. Also, no colonies were observed at 500 µg/plate without metabolic activation. In experiment 1b, no colony growth was visible for TA102 at a test item concentration of 1500 µg/plate with metabolic activation. In experiment 2, no colonies were visible on the plates and the background lawn was only slightly visible at 1500 µg/plate with metabolic activation. In the experimental part without metabolic activation, no colonies and reduced background lawn were observed at a test item concentration of 500 µg/plate.

S. typhimurium TA1535: In experiment 1, the test item showed toxicity towards the strain TA1535 at a test item concentration of 5000 and 1500 µg/plate with and without metabolic activation. No colonies were observed at 500 µg/plate without metabolic activation. At 150 µg/plate, the number of revertant colonies was clearly reduced for TA1535 without metabolic activation. In experiment 1b, signs of toxicity could be observed at a test item concentration of 1500 µg/plate with metabolic activation and at 150 µg/plate without metabolic activation. In experiment 2, no colony growth and a reduction of the bacterial background lawn were visible at a test item concentration of 1500 µg/plate with metabolic activation. Toxic effects were also observed at 150 µg/plate without metabolic activation.

S. typhimurium TA1537: In experiment 1, the test item showed toxicity towards the strain TA1537 at a test item concentration of 5000 and 1500 µg/plate with and without metabolic activation and at 500 µg/plate without metabolic activation. In experiment 1b, signs of toxicity were visible at a test item concentration of 1500 and 500 µg/plate with metabolic activation and at 500 and 150 µg/plate without metabolic activation. In experiment 2, toxic effects and absence of the bacterial background lawn were observed at 500 µg/plate with metabolic activation and also at a test item concentration of 150 µg/plate without metabolic activation.

E. coli WP2: In experiment 1, the test item showed toxicity towards the strain E. coli WP2 at a test item concentration of 5000 µg/plate and 1500 µg/plate with and without metabolic activation. The number of revertant colonies was also clearly reduced at 500 µg/plate without metabolic activation. In experiment 1b, at a test item concentration of 1500 µg/plate toxicity could be observed with metabolic activation. On the plates with a test item concentration of 500 µg/plate, a clear reduction in the colony count was observed without metabolic activation. In experiment 2, signs of toxicity were observed at 1500 µg test item/plate with metabolic activation. At 500 µg/plate without metabolic activation, neither bacterial background lawn nor colony growth were found.

In summary, the test item showed toxic effects towards all bacterial strains, but a sufficient number (at least five, according to OECD 471) of non-toxic concentrations could be evaluated for mutagenicity in the plate incorporation and pre-incubation method. Based on the results of this study it is concluded that AM(pfa)4 is not mutagenic in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 and E. coli WP2 in the presence and absence of metabolic activation under the experimental conditions in this study.

Any other information on results incl. tables

Experiment 1 - Confirmation of the Criteria and Validity

All strains met the criterion of at least 10⁹ bacteria/mL (correlating to 100 colonies/plate after dilution), and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory. All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and nearly all were within the historical control data ranges. The positive control 2-amino anthracene of E. coli with metabolic activation was too low but showed a clear mutagenic effect.

 

Solubility and Toxicity

In the first experiment, the test item showed no precipitates, and the bacterial background lawn was visible and not affected on the plates in all tested concentrations. At a test item concentration of 5000 µg/plate and 1500 µg/plate, no colony growth was visible for all tested strains, except from TA1535 with metabolic activation at 1500 µg/plate, where a mean value of one colony was counted on the replicates.

The number of revertant colonies was clearly reduced at 500 µg/plate for S. typhimurium TA98 with metabolic activation and E. coli WP2 without metabolic activation. No colonies were observed for TA98, TA100, TA102, TA1535 and TA1537 all without metabolic activation. For E. coli WP2 with metabolic activation, no relevant decrease could be observed at a test item concentration of 500 µg/plate.

At the fourth test item concentration (150 µg/plate) the number of revertant colonies was relevantly reduced for TA1535 without metabolic activation and no colony growth could be observed for TA98 without metabolic activation. For all other strains at the concentration 150 µg/plate and for all strains at a test item concentration of 50 µg/plate, no relevant decrease in colony numbers and therefore no toxic effects towards the bacteria strains could be observed.

 

Mutagenicity

No significant or concentration-related increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. According to the guideline OECD 471, five non-toxic concentrations must be evaluated for mutagenicity. Based on the toxicity results, this experiment was repeated under the same conditions with additional lower concentrations (see experiment 1b).

The mean revertant values of experiment 1 are shown in Table 3.1.

 

Table 3.1 Mean Revertants Experiment 1

Strain		S. typhimurium										E. coli WP2
		TA98		TA100		TA102		TA1535		TA1537
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	19	23	57	58	349	360	10	8	6	6	33	39
	sd	2.0	1.5	4.4	2.6	9.2	0.0	1.5	0.6	0.6	1.2	2.1	3.2
DMSO	Mean	19	20	56	57	355	363	10	10	5	6	39	36
	sd	1.2	2.5	3.5	1.5	4.6	9.2	1.0	1.0	0.6	1.2	2.6	4.4
Positive Controls*	Mean	s.g.	124	333	s.g.	728	s.g.	237	180	163	249	s.g.	164
	sd	n.c.	10.6	24.4	n.c.	16.0	n.c.	8.3	12.0	8.3	10.1	n.c.	4.0
	f(I)	>2	6.20	5.84	>2	2.05	>2	23.70	18.00	32.60	41.50	>2	4.56
5000 µg/plate	Mean	0	0	0	0	0	0	0	0	0	0	0	0
	sd	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
	f(I)	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
1500 µg/plate	Mean	0	0	0	0	0	0	0	1	0	0	0	0
	sd	0.0	0.0	0.0	0.0	0.0	0.0	0.0	1.0	0.0	0.0	0.0	0.0
	f(I)	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.13	0.00	0.00	0.00	0.00
500 µg/plate	Mean	0	11	0	63	0	336	0	5	0	5	6	37
	sd	0.0	2.5	0.6	3.2	0.0	13.9	0.0	1.0	0.0	1.0	3.1	3.2
	f(I)	0.00	0.48	0.00	1.09	0.00	0.93	0.00	0.63	0.00	0.83	0.18	0.95
150 µg/plate	Mean	0	20	37	69	347	344	2	9	4	5	39	42
	sd	0.0	0.6	4.5	6.8	20.1	8.0	1.5	1.0	1.5	0.6	1.5	2.0
	f(I)	0.00	0.87	0.65	1.19	0.99	0.96	0.20	1.13	0.67	0.83	1.18	1.08
50 µg/plate	Mean	17	21	68	55	352	339	9	9	5	6	37	34
	sd	0.0	3.6	2.0	3.6	8.0	18.5	1.0	1.7	0.0	1.2	2.6	2.6
	f(I)	0.89	0.91	1.19	0.95	1.01	0.94	0.90	1.13	0.83	1.00	1.12	0.87

sd = standard deviation ±

* Different positive controls were used, see chapter 6.2.4, page 16

s.g. = strong growth, too strong for counting of revertants

n.c. = not calculable

f(I) = increase factor, calculation see chapter 7.4, page 24

 

Experiment 1b - Confirmation of the Criteria and Validity

All strains met the criterion of at least 10⁹ bacteria/mL (correlating to 100 colonies/plate after dilution), and no inconsistencies were found in the sterility control. Nearly all determined values for the spontaneous revertants of the vehicle and negative controls were in the normal range of the test laboratory. The values of the solvent controls DMSO and demin. water of TA1535 with metabolic activation were slightly too low. All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and all were within the historical control data ranges.

 

Solubility and Toxicity

In experiment 1b, the test item showed no precipitates, and the bacterial background lawn was visible and not affected on the plates in all tested concentrations. At a test item concentration of 1500 µg/plate, no colony growth was visible for S. tyhimurium TA102 and TA1537 and E. coli WP2 with metabolic activation. For S. tyhimurium TA100 and TA1535 with metabolic activation, the number of revertant colonies was reduced. On the plates with a test item concentration of 500 µg/plate no colonies were visible for S. typhimurium TA100 and TA1537 without metabolic activation and a clear reduction in the colony count was observed for S. typhimurium TA98 and TA1537 with metabolic activation and E. coli WP2 without metabolic activation. At 150 µg test item/plate no colony growth was observed for S. typhimurium TA1537 without metabolic activation and the number of colonies was clearly reduced for S. typhimurium TA98 and TA1535 without metabolic activation. For all other strains at the concentration 150 µg test item/plate and for all strains at the lower test item concentrations, no relevant decrease in colony numbers and therefore no toxic effects towards the bacteria strains could be observed.

 

Mutagenicity

A sufficient number of non-toxic test item concentrations could be evaluated. No significant or concentration-related increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed.

For TA1535 the f(I) value was increased to 2.25 at the test item concentrations 500; 50 and 5 µg/plate and to 2.50 at the test item concentrations 150; 15 and 1.5 µg/plate. But in this experiment the number of spontaneous revertants of demin. water was slightly lower than the historical data, the threshold of an at least 3-fold increase was not reached, the increase was not dose-related over the tested range and did not exceed the historical control data range of demin. water. Therefore, the test item is stated as not mutagenic under the conditions of this experiment. To verify this result, a further experiment with the pre-incubation method (exp. 2) was performed.

The mean revertant values of experiment 1b are shown in Table 3.1b.

 

Table 3.1b: Mean Revertants Experiment 1b

Strain		S. typhimurium										E. coli WP2
		TA98		TA100		TA102		TA1535		TA1537
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	15	16	73	69	339	328	6	4	7	8	33	35
	sd	0.6	0.6	6.1	2.3	32.3	8.0	0.0	1.5	2.1	2.3	2.6	2.6
DMSO	Mean	17	18	73	73	341	325	7	5	7	5	31	38
	sd	1.0	1.7	6.1	9.2	12.2	20.1	3.0	1.0	2.3	1.2	1.5	3.1
Positive Controls*	Mean	s.g.	97	389	s.g.	816	837	360	288	107	224	s.g.	493
	sd	n.c.	6.1	25.7	n.c.	34.9	33.3	34.9	16.0	9.2	20.0	n.c.	24.4
	f(I)	> 2	5.39	5.33	> 2	2.39	2.58	60.00	57.60	15.29	44.80	> 2	12.97
1500 µg/plate	Mean	--	--	--	1	--	0	--	1	--	0	--	0
	sd	--	--	--	1.5	--	0.0	--	0.0	--	0.0	--	0.0
	f(I)	--	--	--	0.01	--	0.00	--	0.25	--	0.00	--	0.00
500 µg/plate	Mean	--	4	0	80	245	309	--	9	0	2	11	32
	sd	--	0.6	0.0	4.0	30.3	12.2	--	1.0	0.0	1.5	1.0	3.8
	f(I)	--	0.25	0.00	1.16	0.72	0.94	--	2.25	0.00	0.25	0.33	0.91
150 µg/plate	Mean	3	16	80	80	328	323	1	10	0	6	28	33
	sd	0.6	2.0	4.0	6.9	48.0	12.2	1.2	0.6	0.0	2.1	0.6	2.6
	f(I)	0.20	1.00	1.10	1.16	0.97	0.98	0.17	2.50	0.00	0.75	0.85	0.94
50 µg/plate	Mean	14	16	84	76	344	333	6	9	5	4	30	35
	sd	2.0	0.6	4.0	0.0	28.8	20.1	2.5	0.0	1.5	0.6	2.6	3.1
	f(I)	0.93	1.00	1.15	1.10	1.01	1.02	1.00	2.25	0.71	0.50	0.91	1.00
15 µg/plate	Mean	15	19	83	84	309	371	10	10	5	5	28	31
	sd	0.6	0.6	6.1	0.0	16.7	16.7	1.0	1.2	1.0	2.6	2.1	1.5
	f(I)	1.00	1.19	1.14	1.22	0.91	1.13	1.67	2.50	0.71	0.63	0.85	0.89
5 µg/plate	Mean	14	15	77	76	352	315	10	9	5	7	33	31
	sd	0.6	1.0	2.3	0.0	16.0	4.6	0.6	0.6	0.6	1.2	1.7	0.0
	f(I)	0.93	0.94	1.05	1.10	1.04	0.96	1.67	2.25	0.71	0.88	1.00	0.89
1.5 µg/plate	Mean	15	17	76	84	344	349	10	10	6	5	31	35
	sd	0.6	2.6	4.0	4.0	34.9	30.3	1.2	0.6	0.0	1.7	1.0	3.0
	f(I)	1.00	1.06	1.04	1.22	1.01	1.06	1.67	2.50	0.86	0.63	0.94	1.00
0.5 µg/plate	Mean	14	16	76	--	328	--	10	--	8	--	34	--
	sd	0.0	1.2	0.0	--	32.0	--	0.0	--	2.0	--	1.2	--
	f(I)	0.93	1.00	1.04	--	0.97	--	1.67	--	1.14	--	1.03	--
0.15 µg/plate	Mean	15	--	--	--	--	--	10	--	--	--	--	--
	sd	1.2	--	--	--	--	--	0.6	--	--	--	--	--
	f(I)	1.00	--	--	--	--	--	1.67	--	--	--	--	--

sd= standard deviation ±

* Different positive controls were used, see chapter 6.2.4, page 16

s.g. = strong growth, too strong for counting of revertants

n.c. = not calculable

f(I) = increase factor, calculation see chapter 7.4, page 24

-- = not tested

 

Experiment 2 - Confirmation of the Criteria and Validity

All strains met the criterion of at least 10⁹ bacteria/mL (correlating to 100 colonies/plate after dilution), and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the vehicle and negative controls were in the normal range of the test laboratory. All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and all were within the historical control data ranges.

 

Solubility and Toxicity

In experiment 2, the test item showed no precipitates on the plates in all tested concentrations. For the strains with a starting concentration of 1500 µg/plate (S. typhimurium TA100, TA102 and TA1535 and E. coli WP2 with metabolic activation) no colonies were visible on the plates and the background lawn was only slightly visible. For E. coli WP2 the number of revertants was decreased and the background lawn was not affected. In the lower concentrations, no signs of toxicity could be observed. In the approach with highest test item concentration 500 µg/plate (S. typhimurium TA98 and TA1537 with S9, TA100 and TA102 without S9, E. coli WP2 without S9), no colonies and absent or reduced background lawn was observed for S. typhimurium TA100, TA102 and E. coli WP2 without metabolic activation. For S. typhimurium TA1537 with metabolic activation the background lawn was absent, and the colony number clearly reduced. At 250 µg/plate, no colonies were counted for S. typhimurium TA100 without metabolic activation and the background lawn was slightly visible. In the lower concentrations and for S. typhimurium TA98 with metabolic activation, no signs of toxicity could be observed. For the strains with a starting concentration of 150 µg/plate (S. typhimurium TA98, TA1535 and TA1537 without S9), no colonies and the absence of the bacterial background lawn were observed for S. typhimurium TA1537 without metabolic activation. For the strains TA98 and TA1535 without metabolic activation the bacterial background lawn was not affected but the number of revertant colonies was clearly reduced. In the lower concentrations, no clear signs of toxicity could be observed. The mean number of revertants are shown in Table 3.2.

 

Mutagenicity

No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found. Therefore, the test item is stated as not mutagenic under the conditions of this experiment.

 

Due to toxic effects in the experiments 1 and 1b, different concentrations were tested for the individual strains – see also in the following tables.

Table 3.2a: Mean Revertants Experiment 2, S. thyphimurium TA100 (+S9), TA102 (+S9), TA1535 (+S9), E. coli WP2 (+S9)

Strain		S. typhimurium										E. coli WP2
		TA98		TA100		TA102		TA1535		TA1537
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	13	18	71	72	357	352	11	7	6	7	39	44
	sd	2.6	0.6	5.5	3.2	30.3	27.7	2.5	1.2	0.6	1.2	3.5	2.1
DMSO	Mean	15	16	57	59	331	328	8	7	8	8	40	40
	sd	0.6	0.6	2.3	3.2	9.2	21.2	0.6	1.0	1.0	0.6	3.2	3.0
Positive Controls*	Mean	s.g.	81	461	s.g.	811	875	291	131	121	155	s.g.	s.g.
	sd	n.c.	2.3	37.8	n.c.	16.7	40.3	12.2	8.3	6.1	6.1	n.c.	n.c.
	f(I)	> 2	5.06	6.49	> 2	2.45	2.67	26.45	18.71	15.13	19.38	> 2	> 2
1500 µg/plate	Mean	--	--	--	0	--	0	--	0	--	--	--	8
	sd	--	--	--	0.0	--	0.0	--	0.0	--	--	--	1.0
	f(I)	--	--	--	0.00	--	0.00	--	0.00	--	--	--	0.18
750 µg/plate	Mean	--	--	--	58	--	352	--	7	--	--	--	42
	sd	--	--	--	3.6	--	24.0	--	2.3	--	--	--	3.5
	f(I)	--	--	--	0.81	--	1.00	--	1.00	--	--	--	0.95
375 µg/plate	Mean	--	--	--	59	--	333	--	6	--	--	--	46
	sd	--	--	--	3.1	--	20.1	--	0.6	--	--	--	4.4
	f(I)	--	--	--	0.82	--	0.95	--	0.86	--	--	--	1.05
187.5 µg/plate	Mean	--	--	--	64	--	331	--	6	--	--	--	47
	sd	--	--	--	8.2	--	32.3	--	1.0	--	--	--	4.0
	f(I)	--	--	--	0.89	--	0.94	--	0.86	--	--	--	1.07
93.8 µg/plate	Mean	--	--	--	61	--	349	--	8	--	--	--	48
	sd	--	--	--	4.6	--	37.8	--	1.5	--	--	--	2.5
	f(I)	--	--	--	0.85	--	0.99	--	1.14	--	--	--	1.09
46.9 µg/plate	Mean	--	--	--	56	--	323	--	6	--	--	--	37
	sd	--	--	--	2.0	--	12.2	--	1.2	--	--	--	3.8
	f(I)	--	--	--	0.78	--	0.92	--	0.86	--	--	--	0.84
23.4 µg/plate	Mean	--	--	--	59	--	339	--	9	--	--	--	47
	sd	--	--	--	5.5	--	9.2	--	2.6	--	--	--	4.4
	f(I)	--	--	--	0.82	--	0.96	--	1.29	--	--	--	1.07

sd = standard deviation ±

* Different positive controls were used, see chapter 6.2.4, page 16

s.g. = strong growth, too strong for counting of revertants

n.c. = not calculable

f(I) = increase factor, calculation see chapter 7.4, page 24

-- = not tested

 

Table 3.2b: Mean Revertants Experiment 2, S. thyphimurium TA98 (+S9), TA100 (-S9), TA102(-S9), TA1537 (+S9), E. coli WP2 (-S9):

Strain		S. typhimurium										E. coli WP2
		TA98		TA100		TA102		TA1535		TA1537
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	13	18	71	72	357	352	11	7	6	7	39	44
	sd	2.6	0.6	5.5	3.2	30.3	27.7	2.5	1.2	0.6	1.2	3.5	2.1
DMSO	Mean	15	16	57	59	331	328	8	7	8	8	40	40
	sd	0.6	0.6	2.3	3.2	9.2	21.2	0.6	1.0	1.0	0.6	3.2	3.0
Positive Controls*	Mean	s.g.	81	461	s.g.	811	875	291	131	121	155	s.g.	s.g.
	sd	n.c.	2.3	37.8	n.c.	16.7	40.3	12.2	8.3	6.1	6.1	n.c.	n.c.
	f(I)	> 2	5.06	6.49	> 2	2.45	2.67	26.45	18.71	15.13	19.38	> 2	> 2
500 µg/plate	Mean	--	14	0	--	0	--	--	--	--	1	0	--
	sd	--	0.6	0.0	--	0.0	--	--	--	--	1.0	0.0	--
	f(I)	--	0.78	0.00	--	0.00	--	--	--	--	0.14	0.00	--
250 µg/plate	Mean	--	13	0	--	325	--	--	--	--	7	19	--
	sd	--	1.2	0.0	--	20.1	--	--	--	--	1.5	0.6	--
	f(I)	--	0.72	0.00	--	0.91	--	--	--	--	1.00	0.49	--
125 µg/plate	Mean	--	16	62	--	344	--	--	--	--	7	30	--
	sd	--	1.0	2.9	--	21.2	--	--	--	--	0.6	0.6	--
	f(I)	--	0.89	0.87	--	0.96	--	--	--	--	1.00	0.77	--
62.5 µg/plate	Mean	--	18	60	--	323	--	--	--	--	6	32	--
	sd	--	1.7	7.6	--	20.1	--	--	--	--	0.0	0.6	--
	f(I)	--	1.00	0.85	--	0.90	--	--	--	--	0.86	0.82	--
31.3 µg/plate	Mean	--	16	66	--	347	--	--	--	--	5	35	--
	sd	--	1.5	8.1	--	44.1	--	--	--	--	0.0	0.6	--
	f(I)	--	0.89	0.93	--	0.97	--	--	--	--	0.71	0.90	--
15.6 µg/plate	Mean	--	18	70	--	339	--	--	--	--	5	41	--
	sd	--	2.3	9.0	--	16.7	--	--	--	--	0.6	0.6	--
	f(I)	--	1.00	0.99	--	0.95	--	--	--	--	0.71	1.05	--
7.8 µg/plate	Mean	--	18	73	--	352	--	--	--	--	6	41	--
	sd	--	3.2	4.2	--	28.8	--	--	--	--	1.0	3.1	--
	f(I)	--	1.00	1.03	--	0.99	--	--	--	--	0.86	1.05	--

sd = standard deviation ±

* Different positive controls were used, see chapter 6.2.4, page 16

s.g. = strong growth, too strong for counting of revertants

n.c. = not calculable

f(I) = increase factor, calculation see chapter 7.4, page 24

-- = not tested

 

Table 3.2c: Mean Revertants Experiment 2, TA98 (-S9), TA1535 (-S9), TA1537 (-S9)

Strain		S. typhimurium										E. coli WP2
		TA98		TA100		TA102		TA1535		TA1537
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	13	18	71	72	357	352	11	7	6	7	39	44
	sd	2.6	0.6	5.5	3.2	30.3	27.7	2.5	1.2	0.6	1.2	3.5	2.1
DMSO	Mean	15	16	57	59	331	328	8	7	8	8	40	40
	sd	0.6	0.6	2.3	3.2	9.2	21.2	0.6	1.0	1.0	0.6	3.2	3.0
Positive Controls*	Mean	s.g.	81	461	s.g.	811	875	291	131	121	155	s.g.	s.g.
	sd	n.c.	2.3	37.8	n.c.	16.7	40.3	12.2	8.3	6.1	6.1	n.c.	n.c.
	f(I)	> 2	5.06	6.49	> 2	2.45	2.67	26.45	18.71	15.13	19.38	> 2	> 2
150 µg/plate	Mean	2	--	--	--	--	--	5	--	0	--
	sd	0.6	--	--	--	--	--	2.1	--	0.0	--
	f(I)	0.15	--	--	--	--	--	0.45	--	0.00	--
75 µg/plate	Mean	12	--	--	--	--	--	6	--	4	--
	sd	1.5	--	--	--	--	--	1.0	--	1.2	--
	f(I)	0.92	--	--	--	--	--	0.55	--	0.67	--
37.5 µg/plate	Mean	12	--	--	--	--	--	7	--	6	--
	sd	0.0	--	--	--	--	--	1.5	--	1.0	--
	f(I)	0.92	--	--	--	--	--	0.64	--	1.00	--
18.8 µg/plate	Mean	13	--	--	--	--	--	7	--	6	--
	sd	1.0	--	--	--	--	--	1.2	--	1.0	--
	f(I)	1.00	--	--	--	--	--	0.64	--	1.00	--
9.4 µg/plate	Mean	14	--	--	--	--	--	6	--	7	--
	sd	1.0	--	--	--	--	--	0.6	--	0.0	--
	f(I)	1.08	--	--	--	--	--	0.55	--	1.17	--
4.7 µg/plate	Mean	14	--	--	--	--	--	6	--	8	--
	sd	1.2	--	--	--	--	--	0.6	--	0.6	--
	f(I)	1.08	--	--	--	--	--	0.55	--	1.33	--
2.3 µg/plate	Mean	14	--	--	--	--	--	8	--	6	--
	sd	0.0	--	--	--	--	--	2.1	--	1.5	--
	f(I)	1.08	--	--	--	--	--	0.73	--	1.00	--

sd =standard deviation ±

* Different positive controls were used, see chapter 6.2.4, page 16

s.g. = strong growth, too strong for counting of revertants

n.c. = not calculable

f(I) = increase factor, calculation see chapter 7.4, page 24

-- = not tested

 

Mutagenicity of Test item

The study was performed with the plate incorporation (experiments 1 and 1b) and the pre-incubation method (experiment 2) in the absence and presence of a metabolic activation system (S9). Under these conditions, the influence of the test item on bacterial test strains was evaluated in three experiments (1, 1b and 2). The test item AM(pfa)4 showed no relevant increase in the number of revertants in the Salmonella typhimurium test strains TA98, TA100, TA102, TA1535 and TA1537 and E. coli WP2 in all evaluated experiments at the respective evaluated concentrations.

Based on the results of this study it is concluded that AM(pfa)4 is not mutagenic in the Salmonella typhimurium test strains TA98, TA100, TA102, TA1535 and TA1537 and E. coli WP2 in the absence and presence of metabolic activation under the experimental conditions of the present study.

 

VALIDITY

Nearly all 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. All positive controls showed f(I) values > 2 or > 3 (strain specific threshold, see above) which demonstrated the mutagenic potential of the diagnostic mutagens. The positive control 2-amino anthracene of E. coli WP2 with metabolic activation was lower than historical controls in experiment 1 but showed a clear mutagenic effect. The values of the solvent controls DMSO and demin. water of TA1535 with metabolic activation were slightly too low in experiment 1b, but within an acceptable range. The confirmation tests of the genotype performed by Trinova BioChem GmbH did not show any irregularities. The control of the titre was above the demanded value of 10⁹ bacteria/mL. In the sterility control no growth of bacteria could be detected.

Since all criteria for acceptability have been met, the study is considered valid.

Applicant's summary and conclusion

Conclusions:

AM(pfa)4 is not mutagenic in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 and E. coli WP2 in the presence and absence of metabolic activation under the experimental conditions in this study.

Executive summary:

The study procedures described in this report were based on the most recent Guideline OECD 471 and EU Method B.13/14. The test item AM(pfa)4 was tested in the Salmonella typhimurium reverse mutation assay with five strains of Salmonella typhimurium (TA98, TA100, TA102, TA1535 and TA1537) and E. coli WP2. The test was performed in three experiments in the presence and absence of metabolic activation, with +S9 standing for the presence of a metabolic activation, and -S9 standing for absence of metabolic activation. Three valid experiments were performed. The initial experiment (exp. 1) had to be repeated due to an insufficient number of analysable concentrations due to cytotoxic effects.

 

Experiment 1: In the first experiment, the test item (dissolved in demin. water) was tested up to concentrations of 5000 µg/plate in the absence and presence of S9 mix in the strains S. typhimurium TA98, TA100, TA102, TA1535 and TA1537 and E. coli WP2 using the plate incorporation method. The test item showed no precipitates on the plates and bacterial background lawn was not reduced at any of the concentrations. The test item showed signs of toxicity towards all bacteria strains in both the presence and the absence of metabolic activation. Due to an insufficient number of analysable non-toxic concentrations, no evaluation of experiment 1 is was possible.

 

Experiment 1b: Based on the toxicity results of the experiment 1, the experiment was repeated under the same conditions with additional lower concentrations as follows:

- TA98 (-S9) and TA1535 (-S9): 150; 50; 15; 5; 1.5; 0.5; 0.15 µg/plate

- TA98 (+S9), TA100 (-S9), TA102 (-S9), TA1537 (-S9) and E. coli WP2 (-S9): 500; 150; 50; 15; 5; 1.5; 0.5 µg/plate

- TA100 (+S9), TA102 (+S9), TA1535 (+S9), TA1537 (+S9) and E. coli WP2 (+S9): 1500; 500; 150; 50; 15; 5; 1.5 µg/plate

The test item showed no precipitates on the plates at any of the concentrations. The test item showed signs of toxicity towards nearly all bacteria strains in both the presence and the absence of metabolic activation, but a sufficient number of non-toxic test item concentrations could be evaluated for mutagenicity. The results of this experiment showed that none of the tested concentrations showed an increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

 

Experiment 2: Based on the results of the first experiments (exp. 1; exp. 1b), the test item was tested up to the following concentrations:

- TA100 (+S9), TA102 (+S9), TA1535 (+S9) and E. coli WP2 (+S9): 1500; 750; 375; 187.5; 93.8; 46.9; 23.4 µg/plate

- TA98 (+S9), TA100 (-S9), TA102(-S9), TA1537 (+S9) and E. coli WP2 (-S9): 500; 250; 125; 62.5; 31.3; 15.6; 7.8 µg/plate

- TA98 (-S9), TA1535 (-S9) and TA1537 (-S9): 150; 75; 37.5; 18.8; 9.4; 4.7, 2.3 µg/plate

The test item showed no precipitates on the plates at any of the test item concentrations. The test item showed signs of toxicity towards nearly all bacteria strains in both the presence and the absence of metabolic activation, but a sufficient number of non-toxic test item concentrations could be evaluated for mutagenicity. The results of these experiments showed that the test item caused no significant or dose-related increase in the number of revertants in all bacteria strains compared to the solvent control, in both the presence and absence of metabolic activation.

 

Conclusion: Based on the results of this study it is concluded that AM(pfa)4 is not mutagenic in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 and E. coli WP2 in the presence and absence of metabolic activation under the experimental conditions in this study.