3,4,5-trifluorophenol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item was not mutagenic in Salmonella typhimurium TA 98, TA 100, TA 102, TA 1535, TA 1537, and E. coli WP2 uvr A pKM 101 with or without addition of S9 mix (reference 7.6.1 -1). Furthermore, the substance is non-mutagenic in mammalian cells under conditions where the positive controls exerted potent mutagenic effects (reference 7.6.1-2).

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:

July 26, 2000 - January 10, 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21st July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29.Dec.1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

September 11, 1989

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

All these strains contain mutations in the histidine operon, thus imposing a requirement for histidine in the growth medium.

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

Due to migration, the value was transferred to one of the current document's attachments

Test concentrations with justification for top dose:

1st series: 5.00, 15.8, 50.0, 158, 500, 1580 and 5000 µg per plate
2nd series: 5.00, 15.8, 50.0, 158 and 500 µg per plate

The test material concentrations used were selected according to the EEC and OECD guidelines for this test system and the requirements of the Labor Ministry of Japan.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (test item and positive controls N-Ethyl-N'-nitro-N-nitrosoguanidine, cumene hydroperoxide, 2-Aminoanthracene, Benzo[a] pyrene) and Ethanol for positive control 9-Aminoanthracene

- Justification for choice of solvent/vehicle: Preferentially distilled water or dimethyl sulfoxide (DMSO), alternatively acetone or ethanol, are used as solvents. Analysis of the historical data of the laboratory and experience of other research groups (Maron et al. 1981) showed that the amounts of the selected solvents used have no influence on the number of spontaneous revertants of any strain. For this reason only the respective solvent control was used as the negative control in this study.

- Justification for percentage of solvent in the final culture medium: Since on the one hand organic solvents may have diverse effects on e.g. gene regulation and, on the other hand, high amounts of water (added as the solvent) will dilute the top agar, usually the maximum amount of solvent is limited to 100 µl per plate for water and 10 µL per plate for DMSO, ethanol, acetone or other organic solvents. Only the highest test material concentration may be plated with either 316 µL water or 31.6 µL organic solvent.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Daunomycin

Remarks:

without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cumene hydroperoxide

Remarks:

without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

with S9 mix

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
3 parallel plates were used for each concentration step of the test material and the positive controls. Twice as many solvent control plates were used for each bacterial strain.
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: Incubation of plates was performed at + 37 °C for 2 to 3 days.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

Evaluation criteria:

A test material is defined as non-mutagenic in this assay if
• "no" or "weak increases" occur in the first and second series of the main experiment. ("Weak increases" randomly occur due to experimental variation.)
A test material is defined as mutagenic in this assay if
• a dose-related (over at least two test material concentrations) increase in the number of revertants is induced, the maximal effect is a "clear increase", and the effects are reproduced at similar concentration levels in the same test system;
• "clear increases" occur at least at one test material concentration, higher concentrations show strong precipitation or cytotoxicity, and the effects are reproduced at the same concentration level in the same test system.
In all further cases, a third test series with the bacterial strain in question should be performed. If the criteria for a positive test result are not fulfilled in at least two out of the three series, the test material is defined as being non-mutagenic in this test 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:

not examined

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:

not examined

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:

not examined

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:

not examined

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:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: Precipitation of the test material on the agar plates occurred at concentrations > 500 µg/plate in the 1st test series.

STUDY RESULTS :
- Signs of toxicity :
toxicity to the bacteria was observed at concentrations > 500 or 1580 µg/plate depending upon the experimental conditions used.
- Mean number of revertant colonies per plate and standard deviation : see tables 1-4

Table 1: Results / Series No.: 1

Positive Controls: Individual and Mean Number of Revertant Colonies         

Strain	Positive Control	Concentr. [µg/plate]	+ /- S9-Mix #	Mean revertant colonies per plate	SD*	Individual revertant colonies per plate
TA 98	DAUN	2	-	643	196	869	538	521
TA 100	ENNG	5	-	904	41	866	947	899
TA 102	CUM	67	-	396	37	421	414	354
TA 1535	ENNG	10	-	332	12	329	321	345
TA 1537	9-AA	50	-	1683	331	1727	1990	1333
WP2	ENNG	5	-	1327	83	1264	1296	1421
TA 98	2-AA	1	+	322	19	338	301	326
TA 100	2-AA	1	+	537	25	564	532	514
TA 102	B (a) p	10	+	806	3	804	805	809
TA 1535	2-AA	1	+	151	17	152	134	167
TA 1537	2-AA	2	+	92	34	95	124	56
WP2	2-AA	10	+	1459	IB	1477	1441	1459

 

#        Test material incubated in the presence

(+)       or absence

(-)        of S9-Mix

*          Standard deviation

 

DAUN  Daunomycin

ENNG  N-Ethyl-N’-nitro-N-nitroso-guanidine

CUM    Cumene hydroperoxide

9-AA    9-Aminoacridine

2-AA    2-Aminoanthracene

B (a) p Benzo(a)pyrene

 

Table 2: Results/ Series No.: 2

Positive Controls: Individual and Mean Number of Revertant Colonies             

Strain	Positive Control	Concentr. [µg/plate]	+ /- S9-Mix #	Mean revertant colonies per plate	SD*	Individual revertant colonies per plate
TA 98	DAUN	2	-	93	18	73	97	108
TA 100	ENNG	5	-	520	39	565	500	494
TA 102	CUM	150	-	661	72	703	702	578
TA 1535	ENNG	10	-	187	11	194	192	174
TA 1537	9-AA	50	-	186	40	144	191	224
WP2	ENNG	5	-	1103	12	1117	1096	1097
TA 98	2-AA	10	+	329	150	434	396	158
TA 100	2-AA	10	+	333	30	302	336	361
TA 102	B (a) p	10	+	775	120	790	887	649
TA 1535	2-AA	2	+	94	11	107	87	649
TA 1537	2-AA	20	+	42	10	17	49	30
WP2	2-AA	10	+	933	81	861	1020	918

 

#        Test material incubated in the presence

(+)       or absence

(-)        of S9-Mix

*          Standard deviation

 

DAUN  Daunomycin

ENNG  N-Ethyl-N’-nitro-N-nitroso-guanidine

CUM    Cumene hydroperoxide

9-AA    9-Aminoacridine

2-AA    2-Aminoanthracene

B (a) p Benzo(a)pyrene

 

Table 3: Results / Series No.: 1

Summary of the Mean Number of Revertant Colonies              

Test material	Concentr. [µg/plate]	+ /- S9-Mix	Mean revertant colonies / plate
			TA 98	TA 100	TA 102	TA 1535	TA 1537	WP2
Solvent control Test item		-	14	97	269	12	4	128
	5	-	14	91	264	12	3	119
	15.8	-	17	96	268	12	6	127
	50	-	15	99	249	15	4	122
	158	-	16	112	242	12	7	114
	500 PE	-	8	73	189	11	2	53
	1580 PE	-	0	3	2	2	0	2
	5000 PE	-	0	3	0	2	0	2
Solvent control Test item		+	20	120	271	13	7	170
	5	+	25	121	277	20	9	164
	15.8	+	27	132	288	14	8	180
	50	+	26	142	278	13	9	167
	158	+	27	143	342	17	8	130
	500 PE	+	22	130	214	12	5	81
	1580 PE	+	2	4	2	3	0	3
	5000 PE	+	0	5	0	3	0	5
Postive controls	Name	-	DAUN	ENNG	CUM	ENNG	9-AA	ENNG
	Conc (µg/plate)		2	5	67	10	50	5
	Revert. / plate		343	904	396	332	1683	1327
	Name	+	2-AA	2-AA	B (a) p	2-AA	2-AA	2-AA
	Conc (µg/plate)		1	1	10	1	2	10
	Revert. / plate		322	537	806	151	92	1459

 

DAUN  Daunomycin

ENNG  N-Ethyl-N’-nitro-N-nitroso-guanidine

CUM    Cumene hydroperoxide

2-AA    2-Aminoanthracene

B (a) p Benzo(a)pyrene

9-AA    9-Aminoacridine

Table 4 Results / Series No.: 2

Summary of the Mean Number of Revertant Colonies              

Test material	Concentr. [µg/plate]	+ /- S9-Mix	Mean revertant colonies / plate
			TA 98	TA 100	TA 102	TA 1535	TA 1537	WP2
Solvent control Test item		-	15	93	218	11	5	130
	5	-	13	87	219	7	7	130
	15.8	-	12	92	220	11	8	127
	50	-	13	88	222	12	4	122
	158	-	16	88	221	15	7	119
	500	-	11	79	206	11	6	91
Solvent control Test item		+	25	140	275	18	6	134
	5	+	22	139	254	17	5	133
	15.8	+	20	153	226	20	7	151
	50	+	20	156	259	16	3	144
	158	+	26	176	266	18	6	128
	500	+	30	153	261	12	6	115
Postive controls	Name	-	DAUN	ENNG	CUM	ENNG	9-AA	ENNG
	Conc (µg/plate)		2	5	150	10	50	5
	Revert. / plate		93	520	661	187	186	1327
	Name	+	B (a) p	B (a) p	B (a) p	2-AA	B (a) p	2-AA
	Conc (µg/plate)		10	10	10	2	20	10
	Revert. / plate		329	333	775	94	42	1459

 

DAUN  Daunomycin

ENNG  N-Ethyl-N’-nitro-N-nitroso-guanidine

CUM    Cumene hydroperoxide

B (a) p Benzo(a)pyrene

9-AA    9-Aminoacridine

2-AA    2-Aminoanthracene

 

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 03, 2004 - January 26, 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test) (migrated information)

Version / remarks:

21st July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

8. June 2000

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: L5178Y TK(+/-) mouse lymphoma cells

For cell lines:
- Absence of Mycoplasma contamination: yes
- Methods for maintenance in cell culture: For the different experimental investigations, the cells were thawed rapidly, diluted in RPMI 10 and incubated in a humidified atmosphere of 5% v/v CO2 in air.
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Exposure medium:
RPMI- 5 (RPMI 1640 with 5% horse serum) 470 mL RPMI 1640 25 mL horse serum (heat-inactivated) 5 mL penicillin/streptomycin
- Culture medium:
RPMI-10 (RPMI 1640 with 10% heat-inactivated horse serum) 445 mL RPMI 1640 50 mL horse serum (heat-inactivated horse serum) 5 mL penicillin/streptomycin
- Survivor- and selection medium:
RPMI-20 (RPMI 1640 with 20% heat-inactivated horse serum) 395 mL RPMI 1640
100 mL horse serum {heat-inactivated horse serum) 5 mL penicillin/streptomycin

Metabolic activation:

with and without

Metabolic activation system:

Due to migration, the value was transferred to one of the current document's attachments

Test concentrations with justification for top dose:

without S9-Mix:
5.00, 15.8, 50.0, 158, 281 and 500 µg/ mL medium
with S9-Mix:
1.58, 5.00, 15.8,50.0, 158 and 500 µg / mL medium

The concentrations selected were modified and adapted in order to achieve a toxicity level recommended by OECD guideline 476. A dose-range finding test was performed to detect adequate concentrations.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Preferentially water (maximally 1%) or DMSO, acetone or ethanol (maximally 0.1%), are used as solvents.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with S9 mix

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration
Each treatment, in the absence or presence of S9 mix, was performed in duplicate (single cultures only used for positive control treatments).

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 10E7 cells in RPMI 5 medium
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 hours in the presence and 24 hours (1st series) or 3 hours (2nd series) in the absence of S9 mix at 37°C.

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): Cultures were maintained in flasks for 2 days (until day 3 of the experiment)
- Selective agent: At the end of the expression period (day 3), the cell densities in the selected cultures were adjusted to 1 x 10E4/mL. TFT (300 µg/mL) was diluted 100-fold into these suspensions to give a final concentration of 3 µg/mL. Plates were incubated until scorable (day 10 to day 14)
- Criteria for small (slow growing) and large (fast growing) colonies: At least for the negative and positive controls and, in case of a positive test material-induced effect, also for those cultures that showed the highest test material-induced effect, the mutation frequency is determined separately for small and large colonies in addition to the total mutation frequency.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative total growth (RTG)

Evaluation criteria:

The effects of the test material upon the mutation frequency are defined as
• "No effect" or "no increase" in the mutation frequency if the mean frequency of the parallel incubations of a given test material concentration is less than 2.0-fold above the mean of the actual negative controls or the mean mutation falls within the historical range of the negative controls.
• "Clear effect" or "clear increase" in the mutation frequency if the test material induces at least a 3.0-fold increase above the mean of the actual negative controls and the mean mutation frequency for a given test material concentration is at least 1.5-fold above the highest value of the historical negative controls.
• All other results are defined as a "weak effect" or a "weak increase" of the mutation frequency.

Test materials are assessed as negative or non-mutagenic in this test system if
• the assay is considered valid and
• no effect (no increase in the mutation frequency) occurs in the two experimental series performed or
• a weak effect (weak increase) occurs in one series and no effect (no increase) in the other series of experiments.
Test materials are assessed as positive or mutagenic in this test system if
• the assay is considered valid and
• a clear effect (clear increase in the mutation frequency) occurs at similar concentrations of the test material in the two experimental series performed, or
• a clear effect (clear increase) occurs in one series and a weak effect (weak increase) in the other series of experiments at identical concentrations, or
• weak effects (weak increases) occur dose-dependently (over at least two test material concentrations) and reproducibly at identical concentrations in the two experimental series performed.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: The test material did not lead to a relevant fluctuation in the pH of the cell culture medium.
- Data on osmolality: The test material did not lead to a relevant fluctuation in the osmolarity of the cell culture medium.
- Precipitation and time of the determination: No precipitation of the test material in the cell culture medium occurred

RANGE-FINDING/SCREENING STUDIES
In a preceding range finding test, the relative survival was determined after exposure to various test material concentrations ranging between 5.00 and 5000 µg/mL. A reduction in the relative survival of the cells occurred at concentrations > 158 or 1580 in the absence of S9 mix and 15.8 µg/mL in the presence of S9 mix, respectively.

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements: Toxicity to the cells was observed at concentrations > 5.00 or 500 µg/mL, depending upon the experimental condition.

- Genotoxicity results: see table 1

HISTORICAL CONTROL DATA : see table 2

Table 1: Summary of results

 

Without S9 mix (EZ.1571 / 1581)

1^stExperimental series
Test material [µg/mL]		RS^a (%)	RTG^b (%)	MF^c
Solvent	0	100	100	121
Test item	5.00	91.1	121	173
	15.8	83.5	111	151
	50.0	71.1	50.2	137
	158	1.37	0.0896	144
	281	0	-	-
	500	0	-	-
NQO	0.10	42.7	118	1050
	0.20	21.1	35.2	1140

 

2^ndExperimental series
Test material [µg/mL]		RS^a (%)	RTG^b (%)	MF^c
Solvent	0	100	100	89.6
Test item	15.8	89.8	100	120
	50.0	91.3	88.4	110
	158	77.2	75.8	122
	500	27.8	4.31	143
	889	0.622	-	-
	1580	-	-	-
NQO	0.10	86.7	31.2	792
	0.20	60.4	33.9	529

 

 

a:       Relative Survival (CE of cells after treatment)

b:        Relative Total Growth

c:        5-TFT Mutant Frequency per 10⁶viable cells

EZ:     Internal study number

NQO:  4-Nitroquinoline N-oxide

DMBA:           7,12-dimethylbenz[a]anthracene

- :       Lost due to toxicity of test material or not plated for selection of mutants

With S9 mix(EZ 1572/ 1582/ 1594)

1^stExperimental series
Test material [µg/mL]		RS^a (%)	RTG^b (%)	MF^c
Solvent	0	100	100	202
Test item	1.58	97.8	89.3	223
	5.00	82.3	61.2	289
	15.8	21.5	10.6	1110
	50.0	13.5	9.04	846
	158	5.56	2.09	1250
	500	0	-	-
DMBA	2.00	62.1	64.2	1160
	3.00	78.3	48.8	2000

 

2^nd. Experimental series
Test material [ug/mL]		RS^a (%)	RTG^b (%)	MF^c
Solvent	0	100	100	161
Test item	1.58	93.0	76.3	196
	5.00	91.4	35.6	344
	8.89	49.7	32.6	199
	15.8	23.2	19.8	256
	50.0	11.0	11.6	203
	158	2.02	0.165	250
DMBA	1.00	50.5	38.1	2120
	2.00	45.9	6.53	2970

 

3^rd. Experimental series
Test material [ug/mL]		RS^a (%)	RTG^b (%)	MF^c
Solvent	0	100	100	196
Test item	1.58	84.7	94.3	178
	2.81	64.8	79.0	190
	5.00	51.0	74.7	194
	8.89	30.7	36.3	256
	15.8	13.9	22.8	255
	28.1	20.8	23.1	269
DMBA	2.00	66.4	40.6	2460
	3.00	6.81	9.07	6640

 

a:       Relative Survival (CE of cells after treatment)

b:        Relative Total Growth

c:        5-TFT Mutant Frequency per 106 viable cells

EZ:     Internal study number

NQO:  4-Nitroquinoline N-oxide

DMBA:           7,12-dimethylbenz[a]anthracene

- :       Lost due to toxicity of test material or not plated for selection of mutants

Table 2: Historical data

Study number	Mean Mutation Frequency [x10-6]^a
	Solvent controls^b						Positive controls^c
	- S9 mix			+ S9 mix			- S9 mix			+ S9 mix
T14248	63	123		95	106		378	794		488	568
T14277	128	179		168	133		1440	755		955	963
T14278	184			94			718			429
T14325	118	158		172	219		498	684		445	816
T14327	199	79		148	86		1181	1490		672	413
T14506	88	103	166	123	112		746	843	571	673	675
T14646	178	246	289	233			1196	869	924	518
T14690	166	251		174	117		1920	1439		606	478
T14786	103	106		90	53		1263	1529		554	465
T14862	88	122		90	257		955	645		484	1448
T14905	219	131		143	246		760	1008		562	2046
T15101	147	141		121	120	233	541	857		576	1453	1230
T15141	222	159		157	152		749	537		1196	1440
T15152	132	183		186	114		465	473		696	632
T15358	156	153		179	170	202	883	1244		1548	2172	2152
T15359	146	129		121	215	137	443	683		1050	1741	2096
T15360	136	131		157	187		358	462		2400	955
T15597	71.8	84.1		104	96		486	2755		1098	805
T15549	82.5	91.4		102	88		431	661		463	608
T15575	179	97.8		87.9	55.6		614	315		836	669
T15630	93.9	89.3		177	78.7		1050	834		1320	12040
T15596	140	103		106	171		666	524		1700	1200
T15681	98	131		95.2	95.2		704	1110		5150	1560
T15682	113	136		122	245		957	471		1240	2470
T15766	219	157		301	230		970	369		1660	1530
Historical Controls
Mean±SD:	141±50			146±57			847±445			1160±810
Range:	53 - 301						315 - 2755			413 - 5150

a:       Mean values from 1 to 4 Individual cultures from 1 to 3 experimental series of studies performed in the laboratory using the described protocol

b;       All solvent controls from different exposure times are pooled for each study

c:       NQO (0.10 to 0.25 µg/mL) in the absence and BaP (1.25 to 5.0 µg/mL) or DMBA (1.0 to 3.0 µg/mL) in the presence of S9 mix

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test

A study according OECD TG 471 was performed to test for the mutagenic potential of the substance using Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA pkM101. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed. In the two series with S9 mix, 10 % and 30 % S9 in the S9 mix were used in the 1st and 2nd series, respectively.

The substance was dissolved in dimethyl sulfoxide and tested at concentrations ranging from 5.0 to 5000 µg/plate. Precipitation of the test material on the agar plates occurred at concentrations > 500 µg/plate. Toxicity to the bacteria was observed at concentrations > 500 or 1580 µg/plate depending upon the experimental conditions used.

Daunomycin, N-ethyl-N'-nitro-N-nitrosoguanidine, 9-aminoacridine and cumene hydroperoxide served as strain specific positive control compounds in the absence of S9 mix. 2-Aminoanthracene and benzo[a]pyrene were used for testing the bacteria and the activity of the S9 mix. Each treatment with the substances used as positive controls led to a clear increase in revertant colonies, thus showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.

With and without addition of S9 mix as the external metabolizing system, the substance was not mutagenic under the experimental conditions described (reference 7.6.1-1).

In vitro gene mutation in mammalian cells

The substance was tested for its ability to induce mutations at the TK locus (5-trifluorothymidine resistance) in mouse lymphoma cells according to OECD TG 476. The study consisted of two or three independent experimental series, conducted in the absence and presence of an exogenous metabolizing system (S9 mix from livers of rats pre-treated with Aroclor 1254). The exposure times were 3 and 24 hours in the absence and 3 hours in the presence of S9 mix, respectively.

Concentrations ranging from 1.58 to 1580 µg/mL were tested in the absence or presence of S9 mix. No Precipitation of the test material in the incubation medium was observed. Clear cytotoxic effects, i.e. a relevant decrease in either the % relative survival or % total growth of the test cells, occurred at concentrations > 5.00 or < 500 µg/mL, depending upon the experimental condition used. The doses tested were selected to determine viability and mutagenicity (5-trifluorothymidine (TFT) resistance) 2 days after treatment.

Negative (solvent) and positive control treatments were included in each mutation experiment in the absence and presence of S9 mix. Mutant frequencies in negative control cultures fell within normal ranges, and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline N-oxide (without S9 mix) and 7,12-dimethylbenz[a]anthracene (with S9 mix). Therefore, the study was accepted as valid.

No biologically relevant increases in mutant frequency were observed following treatment with the test item in the three experimental series performed.

It is therefore concluded that the substance is non-mutagenic in this test system under conditions where the positive controls exerted potent mutagenic effects (reference 7.6.1-2).

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Thus, the test item is considered not to be classified for genotoxicity under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) 2019/521.