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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

• Bacterial Reverse Gene Mutation Assay: Negative; OECD 471; Anon., 2017


• Comet Assay: Negative; EPA OPPTS 870.5300; Fic et al., 2013


• In Vitro Mammalian Cell Micronucleus Test: Positive; OECD 487; Pfeiffer et al., 1997


• In Vitro Mammalian Cell Transformation Assay: Positive; SHE Cell Transformation Assay; Tsutsui et al., 2000

Link to relevant study records

Reference

Reference 1

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

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

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

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

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.

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

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

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

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

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

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)

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.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

Microtubule (MT) polymerization assay using Chinese hamster V79 cells

Deviations:

no

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Not reported
- Stability under test conditions: Not reported
- Solubility and stability of the test substance in the solvent/vehicle: Treatment solutions prepared in DMSO (dimethylsulfoxide) and further diluted in DMEM (Dulbecco's Modfied Eagle's Medium).
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not reported

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test item dissolved in DMSO and DMEM prior to application.
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: Not reported
- Final preparation of a solid: Not reported

FORM AS APPLIED IN THE TEST (if different from that of starting material): Applied as a liquid.

OTHER SPECIFICS: n/a

Test concentrations with justification for top dose:

25, 50, 100 and 200 µM.
No justification given for top dose.

Vehicle / solvent:

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

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

After treatment with BPAF (50µM), the mitotic spindle of all metaphase cells was no longer visible and diffuse tubulin stains were surrounded by chromosomes in an irregular arrangement.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

BPAF incubation (at 50 µM) led to an average of 6.9 % dead cells (V79). Cell growth was lowered to 22.5 % of the control value.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Inhibition of microtubule assembly

BPAF was studied in the cell-free microtubule (MT) polymerization assay with colchicine (COL) as a positive control. In the conventional assay, microtubule proteins (MTP) in assembly buffer are first incubated with various concentrations of the test compound at 35 ºC for 20 min. GTP is then added to start the formation of MT which is measured by the increase in absorbance at 350 nm due to turbidity. The assembled MT are subsequently depolymerized by lowering the temperature to 48 ºC, followed by a second polymerization step at 35 ºC without adding additional GTP. The inhibition of MT assembly caused by COL is reflected by the difference in absorbance between the control and the COL-containing incubation. When BPAF was studied in the MT assay, it was noted that it caused an increase in absorbance during the first incubation time prior to the addition of GTP. This increase continued during depolymerization and is due to a partial denaturation of MTP known as aggregation. In order to determine the inhibition of MT polymerization in the presence of this aggregation, the depolymerization of assembled MT was measured. The data obtained for BPAF was an active MT inhibitor with an approximate EC values of 30 µM. In contrast to COL with an EC of about 3.5 µM, BPAF showed a rather steep decline in its concentration effect curve. In a further experiment, the effect of BPAF and COL on pre-formed MT was studied. MT were first assembled and the test compounds added later. COL did not interact with the preformed MT, as they depolymerized completely upon cooling to 48 ºC. In contrast, BPAF appeared to aggregate MTP even in assembled MT, as depolymerization was absent at a concentration of 100 µM.

Electron microscopy of microtubules

The morphology of the MT obtained in the polymerization assay under various conditions was analyzed by electron microscopy (EM). Unpolymerized MTP did not yield any structures in EM independent of its treatment, i.e., whether it was native or aggregated. Native MTP polymerized in the absence of an interfering substance gave rise to long cylindrical MT which disappeared completely at 48 ºC. MT obtained in the presence of BPAF at concentrations causing half-maximal depolymerization gave rise to abnormal MT in addition to some normal MT which disassembled at 48 ºC. Most MT displayed an altered morphology characterized by spiral structures and did not disappear upon cooling.

Staining of the cytoplasmic microtubule complex in V79 cells

In order to confirm the ability of BPAF to affect MT, the effect of the test item on the cytoplasmic microtubule complex CMTC of cultured Chinese hamster V79 cells was studied. Interphase cells treated with BPAF caused a significant reduction of the CMTC in virtually every cell, leading to a condensed staining with short fibers around the cell nucleus. When metaphase cells were stained with anti-a-tubulin antibodies to see the MT and also with DAPI to visualize chromatin, results were as follows: in control cells, the mitotic spindle originating from the two poles was clearly visible, and chromosomes were located in the metaphase plate. After treatment with BPAF (50µM), the mitotic spindle of all metaphase cells was no longer visible and diffuse tubulin stains were surrounded by chromosomes in an irregular arrangement. The observed effects on the CMTC and mitotic spindle occurred at concentrations without detectable cytotoxicity for the cells. When the cell culture medium containing the test compound was removed after 6 h and the cells were incubated for another 6 h with fresh medium and subsequently stained, both interphase and metaphase cells appeared normal in all cases. Therefore, the effect on tubulin caused by BPAF is reversible.

Cytotoxicity in V79 cells

In order to get information about the cytotoxicity of BPAF, V79 cells were incubated for 6 h at a concentration of 50 µM. Subsequently, cells were stained with trypan blue and the viable (unstained) and dead (blue cells) counted. As a comparison, untreated cultures contained 1.6% dead cells. The same value was found after 6 h incubation with DMSO 0.1 %. BPAF incubation led to an average of 6.9 % dead cells. Cell growth, i.e., the number of cells after treatment as compared to untreated controls, was lowered to 22.5 % by BPAF.

Metaphase arrest in V79 cells

Cells were incubated with various concentrations of the test compound for 6 h. Following fixation and staining with DAPI, the number of metaphase cells per 1000 cells was scored. BPAF arrested cells in metaphase in a concentration-dependent manner. No significant difference in the number of arrested cells was noted between the various incubation times, implying that the test item lead to a prolongation of the metaphase period but not to a complete block of the cell cycle.

To study the reversibility of metaphase arresting and the transition into anaphase, cells were first incubated with BPAF for 6 h. Medium was then removed and the incubation continued in fresh medium without the test compound for 60 min. At various time points during the second incubation period, the numbers of metaphase and anaphase cells were determined. It was note that the number of metaphase cells had declined by 50 % after about 20 min. The peak concentration of anaphase cells occurred at 30 min.

Induction of micronuclei in V79 cells

In three independent experiments, V79 cells were incubated for 6 h with the BPAF at concentrations which did not cause gross cytotoxicity, and subsequently kept for 3 h in fresh medium. The number of micronucleated cells was then determined using CREST antikinetochore antibodies to distinguish MN containing whole chromosomes/ chromatids from MN containing chromosomal fragments. A clear induction of MN was observed with BPAF. The induced MN were all CREST positive, indicating an aneugenic effect. About 90 % of the micronucleated cells had one MN, and 60–70 % of the MN displayed one CREST signal. In addition to cells with one or two MN, there were some cells with an apparently fragmented nucleus in cultures treated with the test item. These multimicronucleated cells accounted for about 1 % of the total cell number.

Conclusions:

BPAF, at a concentration of 50 µM, was observed to inhibit the cell-free assembly of microtubules; disrupt the cytoplasmicmicrotubule complex and mitotic spindle of Syrian hamster embryos and V79 cells; and induce mitotic arrest and CREST-positive in micronuclei in V79 cells.

Executive summary:

Investigations into the aneuploidogenic potential of BPAF was investigated in Syrian hamster embryo and V79 cells by assaying its interference with the cell-free assembly of microtubules (MT); disruption of the cytoplasmic MT complex in cultured Chinese hamster V79 cells; disruption of the mitotic spindle and induction of metaphase arrest in V79 cells; and induction of micronuclei (MN) in V79 cells.

 

The cells were exposed at concentrations without gross cytotoxicity, i.e. BPAF active at all endpoints tested. The presensce of Kinetochores was also assessed by staining with CREST antibodies.

 

Bisphenol AF demonstrated clear interaction with MTP and disruption of MT in vitro. The test item also caused reversible disruption of the cytoplasmic mircrotuble complex (CMTC) and mitotic spindle. reversible mitotic arrest at metaphases to anaphase was also demonstrated. Further more, induction of micronuclei (MN) was also observed in V79 cells after 6 hour exposure and all were CREST-positive indicating the presences of kinetochores hence implying the presence of whole chromatids/chromosomes. A longer exposure resulted in an increased multimicronucleated cells (MMNC) formation.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2000

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

other: In Vitro Carcinogenicity: Syrian Hamster Embryo (SHE) Cell Transformation Assay

Version / remarks:

Draft guideline, February 2013

Deviations:

no

GLP compliance:

no

Type of assay:

in vitro mammalian cell transformation assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Not reported
- Stability under test conditions: Not reported
- Solubility and stability of the test substance in the solvent/vehicle: Stock solution prepared by dissolving in DMSO (dimethylsulfoxide) at 30 mM.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not reported

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Dissolved in DMSO to form a stock solution.
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: Stock solution was prepared at 30 mM.
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material): Applied as a liquid.

OTHER SPECIFICS: n/a

Target gene:

n/a

Species / strain / cell type:

other: Syrian Hamster Embryo (SHE) cells

Details on mammalian cell type (if applicable):

SHE cell cultures were established from 13-day-gestation fetuses and grown as previously described (Barrett et al., 1978;Tsutsui, et al., 1983).

Test concentrations with justification for top dose:

12.5, 25 and 50 µM.
No justification given for top concentration.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: No justification reported.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Key result

Species / strain:

mammalian cell line, other: Syrian Hamster Embryo (SHE) cells

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

statistically significant increases in the percentage of aneuploid metaphases with chromosome losses and gain

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

The concentration-dependent effects on cellular growth of treatment with BPAF for 1–3 days were examined. In cultures treated at either 12.5 or 25 µM, little effect on cellular growth was observed. However, growth was reduced dramatically by treatment at 50µM.

Frequencies of both morphological transformations and Oua and TGr mutations in SHE cells following treatment with BPAF for 48 hr were evaluated. In both experiments, a comparable standard was used; a single concentration of BP-A (100 µM), which induces a maximum level of morphological transformation of SHE cells (Tsutsui et al., 1998). BPAF could only be tested at up to 50 µM due to toxicity. Although not statistically significant, transformation frequencies in cells treated with at this concentration were increased over the control level. In particular, the frequency in cells treated with BPAF at 50 µM was comparable to that in cells treated with BP-A at 100 µM.

Treatment of SHE cells with BPAF for 48 hr failed to induce gene mutations at either the Na⁺/K⁺ATPase locus or the hprt locus. By contrast, cells treated with the positive control B[a]P at 4 µM for 48 hr underwent mutations at the both loci indicating that the test result was reliable. When SHE cells were treated with BPAF for 6, 24 or 48 hr at the same concentrations used for transformation experiments, no statistically significant increases in the frequency of chromosomal aberrations were observed. Treatment of cells with BPAF for 48 hr resulted in statistically significant increases in the percentage of aneuploid metaphases with chromosome losses and gains in the near-diploid range. Statistically significant increases in the percentage of heteroploid cells with a tetraploid or near-tetraploid number of chromosomes were observed in treated cells.

Conclusions:

The test item did not induce gene mutation at 2 loci or chromosomal aberrations in SHE cells however, aneuploid cells with near-diploid number of chromosomes were increased. Both chromosome losses and gains were induced, suggesting a non-disjunctional mechanism.

Executive summary:

The ability of BPAF (bisphenol-AF) to induce cellular transformation and genetic effects in SHE cells were examined at 3 test concetrations; 12.5, 25 and 50 µM.

 

Cellular growth was inhibited by BPAF in a concentration-related manner where minimal reduction in growth was observed at 12.5 and 25 µM and a significant reduction at 50 µM. Morphological transformation of SHE cells was induced by BPAF. The test item did not induce gene mutations at the Na⁺/K⁺ATPase locus or the hprt locus, or chromosomal aberrations in SHE cells. By contrast, aneuploidy induction in the near-diploid range was exhibited by BPAF.

 

The results indicate BPAF can exhibit transforming activity in SHE cells and that aneuploidy induction may be a causal mechanism of the transforming activity.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

In vitro Comet Assay using Hepg2 cells

Deviations:

not specified

GLP compliance:

no

Remarks:

Published study not conducted to GLP

Type of assay:

comet assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Not reported
- Stability under test conditions: Not reported
- Solubility and stability of the test substance in the solvent/vehicle: Test item dissolved in DMSO (dimethylsulfoxide)
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not reported

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test item dissolved in DMSO
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: Test item dissolved in DMSO
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material): applied as a liquid

OTHER SPECIFICS: n/a

Species / strain / cell type:

mammalian cell line, other: Human HepG2 cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Leiden University Medical Centre, Department of Toxicogenetics, Leiden, The Netherlands
- Suitability of cells: Not reported
- Cell cycle length, doubling time or proliferation index: Not reported
- Sex, age and number of blood donors if applicable: Not reported
- Whether whole blood or separated lymphocytes were used if applicable: Not reported
- Number of passages if applicable: 3 and 10
- Methods for maintenance in cell culture if applicable: Grown in William’s medium E containing 15 % FBS, 2 mmol L-1 L-glutamine, and 100 U mL-1 penicillin/streptomycin in 5 % CO2 at 37 °C. The cells were used at passages between 3 and 10. For sub-cultivation, the cells were trypsinised, washed with phosphatebuffered saline (PBS, pH 7.4), centrifuged at 100 g for 5 min, and separated by pressing the suspensions through a syringe
- Modal number of chromosomes: Not reported
- Normal (negative control) cell cycle time: Not reported

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: William’s medium E containing 15 % FBS, 2 mmol L-1 L-glutamine, and 100 U mL-1 penicillin/ streptomycin in 5 % CO2 at 37 °C.
- Properly maintained: Cannot be determined.
- Periodically checked for Mycoplasma contamination: Not reported
- Periodically checked for karyotype stability: Not reported
- Periodically 'cleansed' against high spontaneous background: Not reported

Additional strain / cell type characteristics:

not specified

Metabolic activation:

not applicable

Metabolic activation system:

n/a

Test concentrations with justification for top dose:

100 mMol/L-1

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Not provided although commonly used in OECD guideline studies.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

0.1 % DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

30 µmol/L

Positive control substance:

benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 40,000 cells/well

DURATION
- Preincubation period: Overnight
- Exposure duration: 4 h and 24 h
- Expression time (cells in growth medium): 1 h
- Selection time (if incubation with a selection agent): n/a
- Fixation time (start of exposure up to fixation or harvest of cells): 15 mins

SELECTION AGENT (mutation assays): n/a

SPINDLE INHIBITOR (cytogenetic assays): 300 mmol L-1 NaOH, 1 mmol L-1 EDTA, pH 13

STAIN (for cytogenetic assays): 400 mmol L-1 Tris buffer (pH 7.5) with EtBr

NUMBER OF REPLICATIONS: 50

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Electrophoresed at 25 V (300 mA) for 20 min, neutralised and stained.

NUMBER OF CELLS EVALUATED: 50 cells randomly selected

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): n/a

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: Not reported

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: DNA strand breaks
- Any supplementary information relevant to cytotoxicity: no

OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: no
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): no

- OTHER: n/a

Statistics:

The statistical analyses were performed using GraphPad Prism 5 software (GraphPad Software, Inc., San Diego, CA, USA). A one-way analysis of variance (one-way ANOVA) was used to analyse the differences in tail intensity between treatments within each experiment. Dunnett’s multiple comparison test was used to compare sample groups with control. Fifty cells were analysed per experimental point in each of at least two independent experimental cultures.

Key result

Species / strain:

mammalian cell line, other: HEPG2

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Test item did not induce a significant increase in DNA strand breaks at any of the tested concentrations after 4 h or 24 h at concentration of 0.1, 1 and 10 µmol/L.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cell viability was reduced by 50 % at 50 μmol/L and by 70 % at 100 μmol/L

Vehicle controls validity:

valid

Positive controls validity:

valid

BPAF reduced cell viability by 50 % at 50 μmol/Land by 70 % at 100 μmol/L. The comet assay was performed at levels that would not induce cytotoxicity (0.1, 1 and 10 µmol/L)

 

As DNA lesions detected with the comet assay may be transient due to the effective DNA repair, both short-term (4-hour) and long-term (24-hour) exposure to BPAF was recorded.

 

BPAF did not induce a significant increase in DNA strand breaks at any of the tested concentrations (0.1, 1 and 10 µmol/L).

Conclusions:

BPAF did not induce a significant increase in DNA strand breaks at any of the tested concentrations (0.1, 1 and 10 µmol/L).

Cytotoxicity was observed at higher tested concentrations (50 and 70 % reduction in cell viability at 50 and 100 µmol/L, respectively).

Executive summary:

Potential genotoxicity of Bisphenol AF was determined in a Comet assay as described by Singh et al 1988 using human hepatoma cell line (HepG2) at non-cytotoxic concentrations (0.1 μmol/L to 10 μmol/L) after 4-hour and 24-hour exposure.

 

Statistical analyses were performed using GraphPad Prism 5 software. The results of Bisphenol treated cells are compared with those of solvent control cells.

 

BPAF reduced cell viability by 50 % at 50 μmol L-1 and by 70 % at 100 μmol L-1  but did not induce a significant increase in DNA strand breaks.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

• Micronucleus test (OECD 474, 2016): negative up to 1400 mg/kg/day (a dose deemed to have exceeded the MTD).

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 August to 09 November 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.

Reason / purpose for cross-reference:

reference to same study

Remarks:

Formulation Validation report

Reason / purpose for cross-reference:

reference to same study

Remarks:

BioAnalytical phase study report

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

Adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at 15-25°C, protected from light
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Formulations were then stirred on a magnetic stirrer to homogenise and aliqoutted.
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: Not stated
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: Not stated
- Reactivity of the test material with the incubation material used (e.g. plastic ware): Not stated

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final concentration of a dissolved solid, stock liquid or gel: 350, 700, 1400 mg/kg/day

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Hsd:Sprague DawleySD

Details on species / strain selection:

The rat has been selected because there is a large volume of background data in this species. There is also significant further data for this species on the test item being investigated.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo, Blackthorn, UK.
- Sex: DRF phase had 3 males and 3 females per group. Main study phase had 6 males per group
- Age at study initiation: 7-8 weeks
- Weight at study initiation: DRF; 216-235 g (males) or 173-183 g (females) on the first day of dosing. Main study; 220-268 g on the first day of dosing.
- Assigned to test groups randomly: Yes.
- Fasting period before study: No
- Housing: Animals were housed in wire topped, solid bottomed cages, with three animals of the same sex per cage.
- Diet: 5LF2 EU Rodent Diet available ad libitum. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
- Water: Municipal tap water, available ad libitum in water bottles.
- Acclimation period: at least 5 days before commencement of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature: 19 - 25 °C
- Humidity: 40 - 70 %:
- Air changes: Fifteen or more air changes per hour:
- Photoperiod: 12 hours light and 12 hours dark (except during designated procedures)

IN-LIFE DATES: From: 22 Aug 2022 To: 22 Sep 2022

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Arachis Oil
- Justification for choice of solvent/vehicle: Selected as shown to be suitable in previous in vivo studies using the test material and a solubility test
- Concentration of test material in vehicle: 35 - 140 mg/mL (based on dose level of 350 - 1400 mg/kg/day and dose volume of 10 mL/kg).
- Amount of vehicle (if gavage or dermal): Dose Volume of 10 mL/kg.

Details on exposure:

The test material was formulated in Arachis Oil. Test material concentrations were stirred on a magnetic stirrer to homogenise and then aliquoted. All formulations used for animal dosing were protected from light and stored at 15-25°C when not required for dosing. All formulations were used within 2 days of preparation.

Duration of treatment / exposure:

24 hours

Frequency of treatment:

Two administrations, at 0 and 24 hours

Post exposure period:

day 1: Prior to dose, immediate, 1, 2 and 4 hours post dose; day 2: Prior to dose, immediate, 1, 2 and 4 hours post dose*; day 3: Prior to necropsy
(* End of day observations also performed on group 4 animals for welfare reasons.)

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle control group (arachis oil)

Dose / conc.:

350 mg/kg bw/day

Remarks:

Test. material

Dose / conc.:

700 mg/kg bw/day

Remarks:

Test. material

Dose / conc.:

1 400 mg/kg bw/day

Remarks:

Test. material

Dose / conc.:

20 mg/kg bw/day

Remarks:

Positive control (Cyclophosphamide, CPA)

No. of animals per sex per dose:

DRF phase had 3 males and 3 females per group. Main study phase had 6 animals per group.

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
-Justification for choice of solvent/vehicle: Based on guideline.
Three positive control slides from animals dosed with 20 mg/kg bw cyclophosphamide (prepared under Labcorp study 8487955, Dreher, 2022) were stained and analysed alongside the study slides.

Tissues and cell types examined:

Bone Marrow. Initially the relative proportions of polychromatic erythrocytes (PCE), seen as bright orange enucleate cells, and normochromatic erythrocytes (NCE), seen as smaller dark green enucleate cells, were determined until a total of at least 500 cells (PCE plus NCE) had been analysed. Then at least 4000 PCE/animal were examined for the presence of MN.

Details of tissue and slide preparation:

Bone marrow was sampled 48hours after dosing. One femur was removed and cleaned of adherent tissue and the ends removed from the shanks. The bone was flushed with approximately 2 mL of fetal calf serum. The cell suspension was collected and centrifuged at 200 g for 5 min at 15-25°C.

The supernatant was aspirated and discarded. A further 3 mL of foetal bovine serum was added to the tubes followed by gentle resuspension of the cell pellet. The cells were pelleted again and the supernatant aspirated to leave one or two drops and the cell pellet. The pellet was mixed into this small volume of serum in each tube by using a Pasteur pipette, and from each tube one drop of suspension was placed on the end of each of three uniquely labelled slides. A smear was made from the drop by drawing the end of a clean slide along the labelled slide. The slides were immediately stained for 5 minutes in 12.5 µg/mL acridine orange made up in 0.1 M phosphate buffer pH 7.4. Slides were rinsed in phosphate buffer, then dried and stored protected from light at room temperature prior to analysis. Unstained slides were air-dried and initially stored at <-10°C with desiccant.

Evaluation criteria:

A micronucleus test is considered acceptable if it meets the following criteria:
1. Cells must be of normal cell morphology
2. Areas where erythrocytes overlap are ignored
3. A MN must be round or oval in shape
4. A cell containing more than one MN is scored as a single micronucleated cell
5. MN which are refractive, improperly stained or not in the focal plane of the cell are judged to be artefacts and are not scored.

Statistics:

MN PCE in the test article treated groups (Groups 2 to 4) and from the positive control slides were compared against the vehicle control group (Group 1) using ranks of the data via the Wilcoxon Rank Sum Test. The Terpstra-Jonckheere test was conducted to evaluate dose response
A test material is considered positive in the micronucleus test if all of the following criteria are met:
1. A statistically significant increase in the frequency of MN PCE occurred at one or more dose levels
2. The incidence and distribution of MN PCE exceeded the laboratory’s historical vehicle control data
3. A dose-response trend in the proportion of MN PCE was observed.
Positive results in the micronucleus test indicate that a test chemical induces micronuclei, which are the result of chromosomal damage or damage to the mitotic apparatus in the erythroblasts of the test species.

The test article was considered negative in this assay if none of the above criteria were met and bone marrow exposure was confirmed.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF THE DOSE FORMULATION ANALYSIS:

Accuracy:
The concentrations analysed in the dose formulation samples were in agreement with target concentrations (i.e. mean sample concentration results were within or equal to 85%-115%). No test article was detected in the vehicle sample.

RESULTS OF RANGE-FINDING STUDY

- Dose range: 1400 mg/kg bw administered to 3 male and 3 female animals
- Clinical signs of toxicity in test animals: None
- Evidence of cytotoxicity in tissue analysed: N/A
- Rationale for exposure: In a previous single oral dose acute toxicity study the LD50 value of the test item exceeded 2000 mg/kg/day. Clinical signs of toxicity included hunched posture, lethargy, chromodacryorrhoea, rales and piloerection.
- Harvest times: N/A
- High dose with and without activation: N/A
- Other: N/A

RESULTS OF THE BIOANALYSIS PHASE STUDY:

The results of the bioanalysis phase confirm that animals dosed at 350, 700 and 1400 mg/kg/day were systemically exposed to BIS-AF. The highest concentration of test article in blood:water (50:50 v:v) observed in all animals was 1 hour post dose on Day 2 and concentrations generally depleted over time. However, for 2 animals dosed at 350 mg/kg/day (R0107 and R0109) it should be noted that concentrations were lower at the 4 hour post dose time point compared to the 8 hour post dose time point but animal to animal variation can be expected. The accuracy of these results were confirmed during ISR and as such are accepted as valid. There was no test article contamination in the vehicle samples.

RESULTS OF DEFINITIVE STUDY

- Induction of micronuclei (for Micronucleus assay):
No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of test material treated animals compared to the vehicle treated animals.
The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all vehicle control animals was within the historical vehicle control ranges.
Although the MTD was exceeded at 1400 mg/kg (mortality in two animals) the individual micronucleus frequencies of the four remaining animals were comparable with those seen in animals dosed with the vehicle control, such that there was clearly no evidence of micronucleus induction at this dose level.
Cyclophosphamide, the positive control material, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. In addition, the number of micronucleated polychromatic erythrocytes found in the positive control animals was within the historical positive control ranges. Hence, all criteria for an acceptable assay were met.

- Ratio of PCE (for Micronucleus assay):
The animals of the groups, which were treated with test material showed no decrease in the ratio of polychromatic erythrocytes, which indicated a lack of toxic effects of this test material on the erythropoiesis. The animals of the groups treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic erythrocytes, demonstrating toxic effects on erythropoiesis.

- Appropriateness of dose levels and route:
Dose level and route of exposure were selected according to test guideline and guided by a DRF study

Results Summary Table

 

Mean Number of Micronucleated Polychromatic Erythrocytes and Polychromatic Erythrocytes

 

Group	Treatment	Number of Animals	Dose (mg/kg body weight)	Percentage of polychromatic erythrocytes (mean and S.D.)		Percentage of micronucleated polychromatic erythrocytes (mean and S.D.)
	MALES			Mean	SD	Mean	SD
1	Vehicle Control	6	0	43.97	3.56	0.10	0.07
2	Test Material	6	350	45.47	4.49	0.05	0.02
3	Test Material	6	700	48.43	7.78	0.11	0.04
4	Test Material	6	1400	39.25	5.98	0.09	0.07
6	CP	^(1)	20	53.80	3.65	2.84	0.63

Vehicle control = Arachis oil

CP = Cyclophosphamide.

(1)  CPA positive control results were generated using slides from Labcorp study 8487955

(2)  Significantly different from corresponding control group (Wilcoxon Rank Sum test, P>0.05).

Conclusions:

The test item did not induce micronuclei in the polychromatic erythrocytes of the bone marrow when tested up to 1400 mg/kg/day (a dose deemed to have exceeded the MTD), under the experimental conditions employed.
NB. Awaiting final report pending BioAnalytical phase reporting completion. See attached letter.

Executive summary:

Study Objective 

The objective of this study was to evaluate the potential of the test item to induce micronuclei (MN) in the polychromatic erythrocytes (PCE) of the bone marrow of treated rats.

 

Study Design

The Sprague Dawley rat was the species and strain of choice because it is a readily available rodent which is commonly used for genotoxicity testing, with documented susceptibility to a wide range of toxic materials. Moreover, historical control background data has been generated with this strain.

 

The study procedures described in this report were based on the most recent OECD (29 July 2016) guideline.

 

The test material was a white powder. The test material was prepared in Arachis oil.

 

Based on the results of the dose-range finding study, test concentrations of 1400 mg/kg/day for male animals was selected as maximum dose for the main test due to clinical toxicity signs observed in a previous single oral dose acute toxicity study at concentrations of 2000 mg/kg/day. Based on this information an initial dose of 1400 mg/kg/day was administered in a Range-Finder Experiment. This dose was considered a suitable estimate of the maximum tolerated dose (MTD) Since there were no substantial differences in toxicity between sexes only males were used in the main study.

 

Results of the analyses demonstrated that the achieved concentrations were within 100±15% (with an RSD of ≤10%) of the nominal test article concentrations. The formulations were therefore considered acceptable. No test article was detected in the vehicle sample.

In the main study male animals were dosed two times by oral gavage with vehicle or with 350, 700 and 1400 mg test material per kg body weight at 0 and 24 hours. A positive control group from a separate study was used in this study. In total 4 treatment groups were used, each consisting of 6 animals.

 

In addition, blood for bioanalysis of the test material in plasma was collected from TK animals from all dose groups.

 

In those animals used for bioanalysis (toxicokinetic animals), blood was sampled 0.5, 1, 2 and 4 h after the second dose of either vehicle or the test material. Vehicle dosed animals showed levels below the lower limit of quantification in the plasma. All test material dosed animals showed increased levels of the test material in the plasma, confirming systemic exposure.

 

Approximately 48 hours the animals were sacrificed and bone marrow smears were prepared for micronucleus analysis.

 

Study Results

The results of the bioanalysis phase confirm that animals dosed at 350, 700 and 1400 mg/kg/day were systemically exposed to BIS-AF. 

 

Animals treated with the test material at all doses exhibited group mean %PCE that were similar to the concurrent vehicle control group and which fell within the laboratory’s historical vehicle control data, thus confirming there was no evidence of test article related bone marrow toxicity.

 

Animals treated with the test material at all doses exhibited MN PCE frequencies that were similar to the concurrent vehicle control group and that fell within laboratory's historical vehicle control data. There were no statistically significant increases in micronucleus frequency for any of the groups receiving the test article, compared to the concurrent vehicle control and no evidence of a linear trend.

 

Although the MTD was exceeded at 1400 mg/kg (mortality in two animals) the individual micronucleus frequencies of the four remaining animals were comparable with those seen in animals dosed with the vehicle control, such that there was clearly no evidence of micronucleus induction at this dose level. Given this and the lack of any bone marrow toxicity, together with negative findings at both 350 and 700 mg/kg/day (low and mid dose groups) it is considered to be of sufficient assurance that the micronucleus data were unaffected by toxicity-related artefacts (mortality) and therefore an accurate assessment of micronucleus induction has been achieved from 350 to 1400 mg/kg.

 

 

Conclusions

In conclusion, the test material did not induce micronuclei in the polychromatic erythrocytes of the bone marrow when tested up to 1400 mg/kg/day (a dose deemed to have exceeded the MTD), under the experimental conditions employed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Summary of genetic toxicity data

 

Table 7.6/1 : Summary of genotoxicity data:

Test n°	Test Guideline / Reliability	Focus	Strains / cells tested	Metabolic activation	Test concentration	Outcome
1 (Anon, 2017)	Ames Test (OECD 471, no GLP claimed) Key, rel.1	Gene mutation	S. typhimurium TA 1535, TA 1537, TA 98, TA 100, TA 102	-S9 +S9	Tested up to 1000 µg/plate	-S9: not mutagenic + S9: not mutagenic
2 (Fic, 2013)	In vitro Comet test: (equiv. EPA OPPTS 870.5300, no GLP claimed) WoE, rel.2	Gene mutation	Hepg2 cells	N/A	0.1, 1, 10, 50 and 100 µmol/L	Not genotoxic
3 (Pfeiffer, 1997)	In vitro micronucleus test: (equiv. OECD 487, no GLP claimed) Key, rel.2	Clastogenicity / Aneugenicity	Chinese hamster V79 cells	N/A	25, 50, 100 and 200 µM	Positive
4 (Tsutsui, 2000)	In vitro gene mutation test (equiv. OECD 473, no GLP claimed,) WoE, rel. 2	Gene mutation	Syrian hamster embryo (SHE) cells	N/A	12.5, 25 and 50 µM	Positive
5 (Parker, 2022)	In vivo micronucleus test (OECD 474, GLP) Key, rel.1	Clastogenicity / Aneugenicity	Male rats (Sprague-Dawley) Bone Marrow	N/A	Up to 1400 mg/kg bw/day (a dose deemed to have exceeded the MTD)	Not clastogenic

 

Genetic toxicity in vitro

The genotoxic potential of the substance (BPAF) has been investigated in several in vitro studies performed according to or comparable to OECD guidelines.

 

In vitro gene mutation study in bacteria (Test n° 1)

In a reverse gene mutation assay in bacteria, strains TA98, TA100, TA102, TA1535 and TA1537of S. typhimurium were exposed to the test item in DMSO at concentrations of 0.100, 0.316, 1.00, 3.16, 10.0, 31.6, 100 and 316 µg/plate, in addition 1000 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). No mutagenicity was observed when the test item was tested up to cytotoxic concentrations and positive controls induced the appropriate responses in the corresponding strains. 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. The test item was considered to be non-mutagenic under the conditions of the test.

 

In vitro gene mutation study (Comet test) in mammalian cells (Test n° 2)

Potential genotoxicity of BPAF was determined in a Comet assay using human hepatoma cell line (HepG2) at non-cytotoxic concentrations (0.1 μmol/L to 10 μmol/L) after 4-hour and 24-hour exposure. Statistical analyses were performed using GraphPad Prism 5 software. The results of Bisphenol treated cells are compared with those of solvent control cells.

BPAF reduced cell viability by 50 % at 50 μmol L-1 and by 70 % at 100 μmol L-1,  but did not induce a significant increase in DNA strand breaks when tested at levels which did not induce cytotoxicity. BPAF did not induce a significant increase in DNA strand breaks at any of the tested concentrations (0.1, 1 and 10 µmol/L). The test item is considered to be not genotoxic under the conditions of the test.

 

In vitro cytogenicity/micronucleus assay (Test n° 3)

The cytogenic / aneuploidogenic potential of the test item was investigated in a mammalian cell cytogenicity/micronucleus assay using Chinese hamster V79 cells. The presensce of Kinetochores was also assessed by staining with CREST antibodies.

V79 cells were spread on slides and exposed to the test item at a concentration without gross cytotoxicity (50 µM) for 6 hours. Investigations were included into aneuploidogenic potential via i) assaying interference with the cell-free assembly of microtubules (MT); ii) disruption of the cytoplasmic MT complex; iii) disruption of the mitotic spindle and induction of metaphase arrest; and iv) induction of micronuclei (MN).

BPAF demonstrated clear interaction with microtubule proteing and disruption of MT in vitro. IT also caused reversible disruption of the cytoplasmic mircrotuble complex (CMTC) and mitotic spindle. reversible mitotic arrest at metaphases to anaphase was also demonstrated. Induction of micronuclei (MN) was observed following test material exposure. All were CREST-positive, indicating the presences of kinetochores and as such, the presence of whole chromatids/chromosomes. A longer exposure resulted in increased multimicronucleated cells (MMNC) formation.

BPAF, at a concentration of 50 µM, was observed to inhibit the cell-free assembly of microtubules; disrupt the cytoplasmicmicrotubule complex and mitotic spindle of Syrian hamster embryos and V79 cells; and induce mitotic arrest and CREST-positive in micronuclei in V79 cells.

 

Gene mutation assay in vitro (Test n° 4)

This study is used in a Weight-of-Evidence alongside test n° 2 for appraisal of gene mutation in vitro. The ability of test item to induce cellular transformation and genetic effects in SHE cells was examined at 3 test concentrations; 12.5, 25 and 50 µM. Cellular growth was inhibited by BPAF in a concentration-related manner where minimal reduction in growth was observed at 12.5 and 25 µM and a significant reduction at 50 µM. Morphological transformation of SHE cells was induced by BPAF.

The test item did not induce gene mutations at the Na+/K+ ATPase locus or the hprt locus, or chromosomal aberrations in SHE cells. By contrast, aneuploidy induction in the near-diploid range was exhibited. The results indicate BPAF can exhibit transforming activity in SHE cells and that aneuploidy induction may be a causal mechanism of the transforming activity.

 

In accordance with the REACH mutagenicity testing strategy (ECHA R7a, 2017, R7.7-1), positive results in in vitro mammalian cell mutagenicity testing, adequately conducted somatic cell in vivo testing is required to ascertain if this potential can be expressed in vivo.  Following a Testing Proposal Decision, an in vivo mammalian erythrocyte micronucleus test (test methods: OECD 474) was performed.

 

Micronucleus test in vivo (Test n° 5)

In the mammalian in vivo micronucleus test (OECD 474), the potential cytogenicity of the test material was investigated when administered to rats by measuring for an increase in the number of micronucleated polychromatic erythrocytes per 4000 polychromatic erythrocytes in rat bone marrow in treated v. control animals.

Based on the results of the dose-range finding study in male and female Sprague Dawley rats, a maximum dose of 1400 mg/kg/day was selected as a suitable estimate of the maximum tolerated dose (MTD). Since there were no substantial differences in toxicity between sexes only males were used in the main study. In the main study, male animals (6 per dose group) were dosed two times by oral gavage with vehicle or with 350, 700 and 1400 mg test material per kg body weight at 0 and 24 hours. A positive Cyclophosphamide-dosed control group from a separate study was used in this study.

In addition, in order to confirm exposure of bone marrow (OECD 474, 29 Jul 2016, p. 40), blood for bioanalysis of the test material in plasma was collected from TK animals from all groups. In those animals used for bioanalysis (toxicokinetic animals), blood was sampled 0.5, 1, 2 and 4 hours after the second dose of either vehicle or the test material. Vehicle dosed animals showed levels below the lower limit of quantification in the plasma. All test material dosed animals showed increased levels of the test material in the plasma, confirming systemic exposure.

Approximately 48 hours the animals were sacrificed, and bone marrow smears were prepared for micronucleus analysis.

No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with the test material compared to the vehicle treated animalsand which fell within the laboratories historical vehicle control data, indicating a lack of toxic effects of this test material on erythropoiesis. All acceptability criteria were met. The test item is considered not to be clastogenic in this in vivo micronucleus test.

 

Weight-of-Evidence Evaluation

Five genetic toxicity studies are available of sufficient quality and reliability to assess the respective endpoints: four in vitro studies and one in vivo studies (table 7.6/1). A smmary of their conclusions is presented within table 7.6/2.

 

Table 7.6/2: summary of study outcomes according to Annex and key mechanism

	Gene Mutation	Cytogenicity
in vitro:	Ames (OECD 471, 2017, WoE rel. 1): negative	-
in vitro:	Gene mutation (equiv. EPA OPPTS 870.5300, 2013, rel.2, WoE): negative	In Vitro Mammalian Cell Micronucleus Test (OECD 487, 1997, WoE rel. 2): positive In Vitro Mammalian Cell Transformation Assay (equiv. OECD 473, 2000, WoE rel. 2): positive
in vivo:	-	Cytogenicity (OECD 474, 2022, rel. 1, key): negative
Outcome:	No concern for gene mutation	No concern for cytogenicity (clastogen or aneugen)

 

Gene mutation: 

Both bacterial and mammalian cell gene mutation assays in vitro reported negative outcomes. Therefore, no concern for gene mutation was identified for BPAF and no further testing was required.

 

Cytogenicity:

Two mammalian cell micronucleus assays in vitro (equiv. OECD 473 and OECD 487), reported positive results, triggering the requirement for further investigation in vivo, in accordance with the ECHA Integrated Testing Strategy for mutagenicity (ECHA R7a, Figure R.7.7-1). Subsequently, an in vivo mammalian erythrocyte micronucleus test (test method: OECD 474) was performed. Under the experimental conditions described, the substance was not found to be clastogenic in the bone marrow micronucleus test of male rats.

Conclusion: Based on WoE and expert judgement following the in vivo erythrocyte micronucleus test, there is no concern for cytogenicity for BPAF.

 

Conclusion:

Following weight-of-evidence analysis and application of expert judgement, it is concluded that no self-classification is proposed regarding mutagenicity according to the CLP and to the GHS.

 

Justification for classification or non-classification

Harmonised classification

 

The substance has no harmonised classification according to the Regulation (EC) No. 1272/2008 (CLP). 

 

Self Classification

 

Based on information available no additional self-classification is proposed regarding mutagenicity according to the CLP and to the GHS.