4,4'-[2,2,2-trifluoro-1-(trifluoromethyl)ethylidene]diphenol; bisphenol AF

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:

28 July - 28 September 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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-[2,2,2-trifluoro-1-(trifluoromethyl)ethylidene]diphenol

EC Number:

216-036-7

EC Name:

4,4'-[2,2,2-trifluoro-1-(trifluoromethyl)ethylidene]diphenol

Cas Number:

1478-61-1

Molecular formula:

C15H16O2

IUPAC Name:

4-[1,1,1,3,3,3-hexafluoro-2-(4-hydroxyphenyl)propan-2-yl]phenol

Test material form:

solid

Details on test material:

Storage: ambient (5 °C – 30 °C), dark, dry

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Stability under test conditions: unstable after repeated contact to air and light
- Solubility and stability of the test substance in the solvent/vehicle: Fully miscible in DMSO (dimethyl sulfoxide) and assumed stable.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: the test item was dissolved in in dimethyl sulphoxide by mixing on a vortex mixer on the day of each experiment. Formulated test item was used within 1 hour of preparation.
- Preliminary purification step (if any): No
- Final dilution of a dissolved solid, stock liquid or gel: Concentration of stock solution = 50 mg/mL.
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material): n/a

OTHER SPECIFICS: No

Method

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

Pre-experiment tester strains TA 98 and TA 100: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
Main Experiment I:0.100, 0.316, 1.00, 3.16, 10.0, 31.6, 100 and 316 µg/plate, in addition1000 µg/plate only for TA 102 (+/-S9)
Main Experiment II: 0.100, 0.316, 1.00, 3.16, 10.0, 31.6, 100 and 316 µg/plate (+/-S9)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: guideline recommended

Controls

Untreated negative controls:

yes

Remarks:

(A. dest., Eurofins Munich, Lot No. 170627,170710)

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene, 4-nitro-o-phenylene-diamine

Details on test system and experimental conditions:

Mutation Experiment:

4,4‘-[2,2,2-trifluoro-1-(trifluoromethyl)ethylidene]diphenol was tested for mutation (and toxicity) in five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102), using plate incorporation test (experiment I) and the pre-incubation test (experimentII), using triplicate plates for test article, vehicle and positive controls with and without metabolic activation.
For the plate incorporation test, the following materials were mixed in a test tube and poured over the surface of a minimal agar plate:
• 100 μL test solution at each dose level, solvent control negative control or reference mutagen solution (positive control)
• 500 μL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without S9)
• 100 μL bacteria suspension (cf. Preparation of Bacteria, pre-culture of the strain)
• 2000 μL
• overlay agar.
For the pre-incubation method 100 μL of the test item preparation was pre-incubated with the tester strains (100 μL) and sterile buffer or the metabolic activation system (500 μL) for 60 min at 37 °C prior to adding the overlay agar (2000 μL) and pouring onto the surface of a minimal agar plate.
For each strain and dose level, including the controls, three plates were used.
After solidification, the plates were inverted and incubated at 37 °C for at least 48 h in the dark.
Data recording and cytotoxicity evaluation:
The colonies were counted using a ProtoCOL counter (Meintrup DWS Laborgeräte GmbH). If precipitation of the test item precluded automatic counting the revertant colonies were counted by hand. In addition, tester strains with a low spontaneous mutation frequency like TA 1535 and TA 1537 were counted manually.
Cytotoxicity can be detected by a clearing or rather diminution of the background lawn (indicated as "N" or "B", respectively in the result tables) or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.

Evaluation criteria:

A test item is considered as mutagenic if:
- A clear and dose-related increase in the number of revertants occurs and/or
- A biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.

A biologically relevant increase is described as follows:
- If in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- If in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control

Statistics:

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

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

Positive controls validity:

valid

Any other information on results incl. tables

Table 1. Experiment I (Plate-incorporation Test) Results

Tester Strain: TA98

Treatment	Dose/plate		REVERTANT COLONIES PER PLATE						MUTATION FACTOR
			Without activation (-S9)			With activation (+S9)
			Counts	Mean	SD	Counts	Mean	SD	-S9	+S9
A. dest.			31 18 33	27	8.1	25 28 29	27	2.1	0.9	1
DMSO			34 27 28	30	3.8	35 21 27	28	7.0	1.0	1.0
Test Item		0.100 μg	27 19 39	28	10.1	33 38 39	37	3.2	1.0	1.3
Test Item		0.316 μg	33 30 33	32	1.2	37 34 37	36	1.7	1.1	1.3
Test Item		1.00 μg	34 26 22	27	6.1	33 31 37	33	1.5	0.9	1.2
Test Item		3.16 μg	24 24 26	25	1.2	31 29 35	32	3.1	0.8	1.1
Test Item		10.0 μg	32 22 21	25	6.1	33 33 29	32	2.3	0.8	1.1
Test Item		31.6 μg	32 16 32	27	92	24 44 24	31	11.5	0.9	1.1
Test Item		100 μg	20 B 20 B 13 B	18	4.0	26 22 26	25	2.3	0.6	09
Test Item		316 μg	10 B 1 B 0 B	4	5.5	0 B 0 B 0 B	0	0.0	0.1	0.0
4-NOPD		10 μg	249 340 288	292	45.7	/ / /	/	/	9.9	/
2-AA		2.5 μg	/ / /	/	/	2002 1486 1563	1684	278.4	/	60.9

Taster Strain: TA100

Treatment	Dose/plate		REVERTANT COLONIES PER PLATE						MUTATION FACTOR
			Without activation (-S9)			With activation (+S9)
			Counts	Mean	SD	Counts	Mean	SD	-S9	+S9
A. dest.			106 87 103	99	10.2	91 77 86	85	7.1	1.1	1.0
DMSO			105 80 83	89	13.7	83 99 82	88	9.5	1.0	1.0
Test Item		0.100 μg	85 72 110	89	19.3	87 73 82	81	7.1	1.0	0.9
Test Item		0.316 μg	71 78 71	73	4.0	101 89 99	96	6.4	0.8	1.1
Test Item		1.00 μg	102 102 72	92	17.3	86 83 87	85	2.1	1.0	1.0
Test Item		3.16 μg	95 79 96	90	9.5	86 83 87	84	8.7	1.0	1.0
Test Item		10.0 μg	93 76 82	84	8.6	73 85 92	83	9.6	0.9	0.9
Test Item		31.6 μg	89 91 96	92	3.6	87 91 105	94	9.5	1.0	1.1
Test Item		100 μg	0B 0B 0B	0	0.0	83 B 60 B 54 B	66	15.3	0.0	0.7
Test Item		316 μg	0 N 0 N 0 N	0	0.0	0 B 0 B 0 B	0	0.0	0.0	0.0
NaN3		10 μg	644 772 792	736	80.3	/ / /	/	/	8.2	/
2-AA		2.5 μg	/ / /	/	/	1051 1576 1314	1314	262.5	/	14.9

ester Strain: TA1535

Treatment	Dose/plate		REVERTANT COLONIES PER PLATE						MUTATION FACTOR
			Without activation (-S9)			With activation (+S9)
			Counts	Mean	SD	Counts	Mean	SD	-S9	+S9
A. dest.			24 21 21	22	1.7	28 22 20	23	4.2	1.2	1.2
DMSO			15 22 18	18	3.5	20 18 20	19	1.2	1.0	1.0
Test Item		0.100 μg	19 24 22	22	2.5	17 15 20	17	2.5	1.2	0.9
Test Item		0.316 μg	17 29 18	21	6.7	18 20 19	19	1.0	1.2	1.0
Test Item		1.00 μg	21 26 23	23	2.5	19 26 21	22	3.6	1.3	1.1
Test Item		3.16 μg	21 18 23	21	2.5	15 25 16	19	5.5	1.1	1.0
Test Item		10.0 μg	17 21 21	20	2.3	24 25 17	22	4.4	1.1	1.1
Test Item		31.6 μg	24 12 21	19	6.2	29 10 13	21	9.7	1.0	1.1
Test Item		100 μg	0B 0B 0B	0	0.0	7 B 3 B 2 B	4	2.6	0.0	0.2
Test Item		316 μg	0 N 0 N 0 N	0	0.0	0 B 0 B 0 B	0	0.0	0.0	0.0
NaN3		10 μg	716 825 749	763	55.9	/ / /	/	/	41.6	/
2-AA		2.5 μg	/ / /	/	/	129 92 158	126	33.1	/	65

Tester Strain: TA3537

Treatment	Dose/plate		REVERTANT COLONIES PER PLATE						MUTATION FACTOR
			Without activation (-S9)			With activation (+S9)
			Counts	Mean	SD	Counts	Mean	SD	-S9	+S9
A. dest.			12 12 13	12	0.6	9 8 9	9	0.6	1.0	1.1
DMSO			11 14 11	12	1.7	7 7 10	8	1.7	1.0	1.0
Test Item		0.100 μg	12 12 11	12	0.6	10 11 10	10	0.6	1.0	1.3
Test Item		0.316 μg	13 11 12	12	1.0	9 9 9	9	0.0	1.0	1.1
Test Item		1.00 μg	12 10 14	12	2.0	8 6 9	8	1.5	1.0	1.0
Test Item		3.16 μg	11 10 12	11	1.0	8 11 7	9	2.1	0.9	1.1
Test Item		10.0 μg	11 10 12	11	1.0	12 10 7	10	2.5	0.9	1.2
Test Item		31.6 μg	13 12 9	11	2.1	9 8 8	8	0.6	0.9	1.0
Test Item		100 μg	5B 7B 4B	5	1.5	4 B 2 B 1 B	2	1.5	0.4	0.3
Test Item		316 μg	1B 2B 1B	1	0.6	0 B 0 B 0 B	0	0.0	0.1	0.0
4-NOPD		10 μg	79 71 84	78	6.6	/ / /	/	/	6.5	/
2-AA		2.5 μg	/ / /	/	/	222 245 229	232	11.8	/	29.0

Tester Strain: TA102

Treatment	Dose/plate		REVERTANT COLONIES PER PLATE						MUTATION FACTOR
			Without activation (-S9)			With activation (+S9)
			Counts	Mean	SD	Counts	Mean	SD	-S9	+S9
A. dest.			241 248 223	237	12.9	297 261 258	272	21.7	1.3	1.3
DMSO			204 192 167	188	18.9	214 219 193	209	13.8	1.0	1.0
Test Item		0.100 μg	224 211 242	226	15.6	272 277 244	264	17.8	1.2	1.3
Test Item		0.316 μg	234 232 195	220	22.0	330 324 344	333	10.3	1.2	1.6
Test Item		1.00 μg	233 202 244	226	21.8	308 284 320	304	18.3	1.2	1.5
Test Item		3.16 μg	218 257 230	235	20.0	276 292 331	300	28.3	1.3	1.4
Test Item		10.0 μg	189 194 169	184	13.2	280 292 209	260	44.9	1.0	1.2
Test Item		31.6 μg	211 192 234	212	21.0	282 345 347	325	37.0	1.1	1.6
Test Item		100 μg	117 B 66 B 84 B	89	25.9	206 256 266	243	32.1	1.1	1.6
Test Item		316 μg	0 B 0 B 0 B	0	0.0	206 235 216	219	14.7	0.0	1.0
Test Item		1000 μg	0 B 0 B 0 B	0	0.0	74 B 53 B 52	60	12.4	0.0	1.0
MMS		1 μL	1355 1884 1837	1692	292.8	/ / /	/	/	9.0	/
2-AA		2.5 μg	/ / /	/	/	713 779 789	760	41.3	/	3.6

Table 2: Experiment II(Pre-incubation Test) Results II

Tester Strain: TA98

Treatment	Dose/plate		REVERTANT COLONIES PER PLATE						MUTATION FACTOR
			Without activation (-S9)			With activation (+S9)
			Counts	Mean	SD	Counts	Mean	SD	-S9	+S9
A. dest.			27 24 24	25	1.7	24 31 22	26	4.7	1.1	1.1
DMSO			28 18 24	23	5.0	24 26 18	23	4.2	1.0	1.0
Test Item		0.100 μg	27 26 27	27	0.6	37 21 21	26	9.2	1.1	1.2
Test Item		0.316 μg	21 18 25	21	3.5	27 24 21	24	3.0	0.9	1.1
Test Item		1.00 μg	19 24 25	23	3.2	19 23 19	20	2.3	1.0	0.9
Test Item		3.16 μg	20 21 18	20	1.5	18 23 18	20	2.9	0.8	0.9
Test Item		10.0 μg	22 21 28	24	3.8	27 17 27	24	5.8	1.0	1.0
Test Item		31.6 μg	25 28 23	25	2.5	25 24 23	24	1.0	1.1	1.1
Test Item		100 μg	0B 0B 0B	0	0.0	2 B 5 B 7 B	5	2.5	0.0	0.2
Test Item		316 μg	0 N 0 N 0 N	0	0.0	0 B 0 B 0 B	0	0.0	0.0	0.0
4-NOPD		10 μg	250 288 248	262	22.5	/ / /	/	/	11.2	/
2-AA		2.5 μg	/ / /	/	/	340 598 554	497	138.0	/	21.9

Tester Strain: TA100

Treatment	Dose/plate		REVERTANT COLONIES PER PLATE						MUTATION FACTOR
			Without activation (-S9)			With activation (+S9)
			Counts	Mean	SD	Counts	Mean	SD	-S9	+S9
A. dest.			87 96 88	90	4.9	113 89 99	100	12.1	1.1	1.1
DMSO			70 84 85	80	8.4	100 78 87	88	11.1	1.0	1.0
Test Item		0.100 μg	90 93 92	92	1.5	99 76 89	88	11.5	1.2	1.0
Test Item		0.316 μg	109 81 76	89	17.8	79 86 71	79	7.5	1.1	0.9
Test Item		1.00 μg	104 98 85	96	9.7	74 78 69	74	4.5	1.2	0.8
Test Item		3.16 μg	97 75 91	88	11.4	73 79 106	86	17.6	1.1	1.0
Test Item		10.0 μg	65 97 75	79	16.4	83 83 75	80	4.6	1.0	0.9
Test Item		31.6 μg	51 56 53	53	2.5	105 85 86	92	11.3	0.7	1.0
Test Item		100 μg	0B 0B 0B	0	0.0	51 B 45 B 52 B	49	3.8	0.0	0.6
Test Item		316 μg	0 N 0 N 0 N	0	0.0	0 N 0 N 0 N	0	0.0	0.0	0.0
NaN3		10 μg	516 553 797	622	152.7	/ / /	/	/	7.8	/
2-AA		2.5 μg	/ / /	/	/	1060 666 850	859	197.1	/	9.7

Tester Strain: TA1535

Treatment	Dose/plate		REVERTANT COLONIES PER PLATE						MUTATION FACTOR
			Without activation (-S9)			With activation (+S9)
			Counts	Mean	SD	Counts	Mean	SD	-S9	+S9
A. dest.			12 14 13	13	1.0	15 15 16	15	0.6	1.0	1.0
DMSO			15 12 14	14	1.5	17 13 16	15	2.1	1.0	1.0
Test Item		0.100 μg	12 14 16	14	1.5	13 15 14	14	1.0	1.0	0.9
Test Item		0.316 μg	14 14 12	13	1.2	15 12 16	14	2.1	1.0	0.9
Test Item		1.00 μg	13 15 15	14	1.2	17 15 16	16	1.0	1.0	1.0
Test Item		3.16 μg	16 14 17	16	1.5	18 16 15	16	1.5	1.1	1.1
Test Item		10.0 μg	13 15 15	14	1.2	15 16 17	16	1.0	1.0	1.0
Test Item		31.6 μg	17 13 13	14	2.3	18 15 16	16	1.5	1.0	1.1
Test Item		100 μg	1 B 2 B 3 B	2	0.0	2 B 5 B 4 B	4	1.5	0.1	0.2
Test Item		316 μg	0 N 0 N 0 N	0	0.0	0 B 0 B 0 B	0	0.0	0.0	0.0
NaN3		10 μg	919 923 915	919	4.0	/ / /	/	/	67.2	/
2-AA		2.5 μg	/ / /	/	/	56 79 53	63	14.2	/	4.1

Tester Strain: TA1537

Treatment	Dose/plate		REVERTANT COLONIES PER PLATE						MUTATION FACTOR
			Without activation (-S9)			With activation (+S9)
			Counts	Mean	SD	Counts	Mean	SD	-S9	+S9
A. dest.			10 8 10	9	1.2	10 8 11	10	1.5	1.1	0.9
DMSO			9 7 9	8	1.2	11 9 11	10	1.2	1.0	1.0
Test Item		0.100 μg	8 11 9	9	1.5	8 9 9	9	0.6	1.1	0.8
Test Item		0.316 μg	9 8 11	9	1.5	7 7 11	8	2.3	1.1	0.8
Test Item		1.00 μg	8 7 9	8	1.0	8 9 7	8	1.0	1.0	0.8
Test Item		3.16 μg	11 8 8	9	1.7	14 0 9	8	7.1	1.1	0.9
Test Item		10.0 μg	9 10 9	9	0.6	9 12 8	10	2.1	1.1	0.9
Test Item		31.6 μg	8 8 11	9	1.7	8 9 11	9	1.5	1.1	09
Test Item		100 μg	1B 2B 2B	2	0.6	2 B 3 B 2 B	2	0.6	0.2	0.2
Test Item		316 μg	0 N 0 N 0 N	0	0.0	0 N 0 N 0 N	0	0.0	0.0	0.0
4-NOPD		40 μg	78 84 89	84	5.5	/ / /	/	/	10.0	/
2-AA		2.5 μg	/ / /	/	/	250 277 218	248	29.5	/	24.0

Tester Strain: TA102

Treatment	Dose/plate		REVERTANT COLONIES PER PLATE						MUTATION FACTOR
			Without activation (-S9)			With activation (+S9)
			Counts	Mean	SD	Counts	Mean	SD	-S9	+S9
A. dest.			365 347 321	344	22.1	454 421 425	433	18.0	1.2	1.2
DMSO			307 263 297	289	23.12	383 361 325	373	11.1	1.0	1.0
Test Item		0.100 μg	257 301 315	291	30.4	371 354 344	350	23.3	1.0	0.9
Test Item		0.316 μg	307 316 314	312	4.7	357 355 344	352	7.0	1.1	0.9
Test Item		1.00 μg	256 302 317	292	31.8	365 249 206	273	82.2	1.0	0.7
Test Item		3.16 μg	343 343 317	338	8.1	303 249 206	345	37.1	1.2	0.9
Test Item		10.0 μg	284 277 299	286	9.6	346 364 360	357	9.5	1.0	1.0
Test Item		31.6 μg	336 285 235	285	50.5	353 361 350	355	5.7	1.0	1.0
Test Item		100 μg	166 B 133 B 118 B	139	24.6	372 354 352	359	11.0	0.5	1.0
Test Item		316 μg	31 N 32 N 17 N	27	8.4	126 B 280 B 280 B	229	88.9	0.1	0.6
MMS		1 μL	1471 1507 1559	1512	44.2	/ / /	/	/	5.3	/
2-AA		10 μg	/ / /	/	/	1021 804 840	888	116.3	/	2.4

SD:      Standard-deviation                     P:        Precipitation
B:        Background lawn reduced           C:        Contamination
N:        No background lawn

Mutation Factor = Mean revertants (test item)/Mean revertants (vehicle control)

Applicant's summary and conclusion

Conclusions:

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

Executive summary:

OECD 471 (2017) - In a reverse gene mutation assay in bacteria, strains TA98, TA100, TA102, TA1535 and TA1537 of S. typhimurium were exposed to 4,4'- [2,2,2 -trifluoro-1 -(trifluoromethyl)ethylidene]diphenol in DMSO at concentrations of 0.100, 0.316, 1.00, 3.16, 10.0, 31.6, 100 and 316 µg/plate, in addition1000 µg/plate only for TA 102 (+/-S9) and 0.100, 0.316, 1.00, 3.16, 10.0, 31.6, 100 and 316 µg/plate (+/-S9).

 

The test item was tested up to cytotoxic concentrations. All positive controls induced the appropriate responses in the corresponding strains.

 

There was no evidence of induced mutant colonies over background in the test item treated colonies.

 

This study was classified as acceptable and it satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.