(E)-4-Ethoxy-1,1,1-trifluoro-3-buten-2-one (stabilized with BHT)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

9 Aug.-7 Sept. 2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The study was conducted according to an internationally recognised method, and under GLP. However, the test item identification is incomplete (unspecified cis-trans isomery, therefore identified under the generic CAS 17129-06-5). Therefore validation applies with restrictions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Remarks:

The report mentions no deviation from the version of the guideline in place at that time, but some deviations from the study plan (see in the field "Any other information on materials and methods incl. tables").

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate from 2004-08-19

Type of assay:

bacterial reverse mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

liquid

Details on test material:

Appearance: liquid, yellow
Storage conditions: < -18°C, in the dark

Method

Target gene:

S. typhimurium strains: histidine requirement for growth
E. coli strains: tryptophan requirement for growth

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

- Source of S9: The batch of S9 was prepared according to Ames et al. (1975) and Maron and Ames (1983) as follows. Male Wistar rats (n =12; obtained from Charles River Deutschland , Sulzfeld, Germany) were injected intraperitoneally with a single dose of Aroclor 1254 (nominal dose of 500 mg/kg body weight) in soy bean oil (20% w/v). The rats were provided with tap water and the Institute's stock diet ad libitum. Five days after the injection of Aroclor 1254 the rats were killed by CO2 asphyxiation. The livers were removed aseptically and immediately put into a cold, sterile 0.15 M KCI solution. After washing in the KCI solution, the livers were weighed, cut into pieces and homogenized in 3 volumes of 0.15 M KCl solution in a Potter-Elvehjem apparatus with a Teflon pestle. The homogenate was centrifuged for 10 minutes at 9,000 g. The supematant, which is called S9, was collected and divided into small aliquots in sterile polypropylene vials. The vials were quickly frozen on dry ice and subsequently stored in a freezer at <-60 °C.
- Method of preparation of S9 mix : On the day of use, aliquots of S9 liver homogenate were thawed and mixed with a NADPH generating system. The S9-mix was kept on ice until use.
- Concentration or volume of S9 mix and S9 in the final culture medium: The final concentrations of the various ingredients in the S9-mix were: MgCl2 8 mM; KCI 33 mM; G-6-P 5 mM; NADP 4 mM; sodium phosphate 100 mM (pH 7.4), NaCI 46 mM, and S9 10 %. 0.5 mL of this S9-mix was used in the experiments with metabolic activation
- Quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): The S9 was checked for sterility. The protein and cytochrome P-450 content of the S9 fraction were determined according to the method published by Rutten et al. (1987). The protein content of the batch was 21.2 g/L. The cytochrome P450 content of the batch was 19.4 µmol/L. The batch contained 0.91 µmol cytochrome P450 per gram protein. The sterility check of the batch resulted in 0 colonies per 100 µL S9. The batch of S9 met all of the in-house quality criteria.

Test concentrations with justification for top dose:

According to OECD 471 guideline, cytoxicity and solubility are criteria to be taken into consideration when determining the highest test substance concentration. In this study, a preliminary test to assess the toxicity of the test substance was not performed; therefore, the toxicity investigation was incorporated in the first mutagenicity test.
First test: Five concentrations were tested, ranging from 62 to 5000 µg/plate. The highest concentration of 5 mg/plate was the guideline recommended maximum concentration for soluble non-cytotoxic substances. Highly toxic effects were observed in this first test. According to the guideline recommendation, substances that are cytotoxic already below 5 mg/plate should be tested up to a cytotoxic concentration. A second test was therefore performed at lowest concentrations, still including cytotoxic effects.
Second test: Six concentrations were tested, ranging from 16 to 500 µg/plate.

Vehicle / solvent:

- Vehicle used: DMSO.
- Justification for choice of vehicle: The absence of mutagenic effects of DMSO was demonstrated through assays performed in the testing laboratory (historical negative control data appended to the report covered assays conducted between May 2002 - March 2005).

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

ethylnitrosurea

other: 2-aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate.
- Number of independent experiments : 2 (see above in the field "Test concentrations with justification for top dose").

METHOD OF TREATMENT/ EXPOSURE:
- Test substance formulation: The plate-incorporation method was applied. DMSO was used as a vehicle. Just before use, the test substance was diluted at 50 mg/mL in the first test and 5 mg/mL in the second test (taking the purity of the test substance into account). A clear, light-yellow dilution was obtained in the first test and a clear, colourless dilution was obtained in the second test. In the first test, serial 3-fold dilutions in DMSO were prepared. Five concentrations were tested, ranging from 62 to 5000 µg/plate. In the second test serial 2-fold dilutions in DMSO were prepared. Six concentrations were tested, ranging from 16 to 500 µg/plate. The actual concentrations of the test substance in the test solutions were not determined. Therefore, the concentrations quoted in this report are nominal concentrations.
- Exposure conditions: To 2 mL molten top agar (containing 0.6 % agar, 0.5 % NaCI and 0.05 mM L-histidine.HCl/0.05 mM biotin for the S. typhimurium strains, and supplemented with 0.05 mM tryptophane for the E. coli WP2 uvrA strain), maintained at ca. 46 °C, were added subsequently: 0.1 ml of a fully grown culture of the appropriate strain, 0.1 ml of the test substance or of the negative or the positive control substance solution, and 0.5 ml S9-mix for the experiments with metabolic activation or 0.5 ml sodium phosphate 100 mM (pH 7.4) for the experiments without metabolic activation. The ingredients were thoroughly mixed and the mix was immediately poured onto minimal glucose agar plates (1.5 % agar in Vogel and Bonner medium E with 2 % glucose). The plates were incubated at ca. 37 °C for approximately 72 hours.

METHODS FOR MEASUREMENT OF CYTOTOXICITY :
Cytotoxicity is defined as a reduction (at least 50%) in the number of revertant colonies and/or a clearing of the background lawn of bacterial growth.

METHODS FOR MEASUREMENTS OF GENOTOXICIY :
The revertant bacteria his+ and trp+ were counted.
his+ = revertant S. typhimurium able to grow in the absence of histidine.
trp+ = revertant E. coli able to grow in the absence of tryptophan.

Evaluation criteria:

A test substance is considered to be positive if the mean number of revertant colonies on the test plates is concentration-related increased or if a reproducible two-fold or more increase is observed compared to that on the negative control plates. A test substance is considered to be negative if it produces neither a dose-related increase in the mean number of revertant colonies nor a reproducible positive response at any of the test points. Both numerical significance and biological relevance are considered together in the evaluation.

Statistics:

No statistical analysis was performed .

Results and discussion

Test results

Test results 1

Key result

Species / strain:

other: all strains (S. typhimurium + E. coli)

Remarks:

first test

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above 185 µg/plate: pinpoint colonies, (slightly) less dense background lawn of the bacterial growth and decrease in the mean number of revertants.

Vehicle controls validity:

valid

Remarks:

revertant colonies per plate within the acceptability ranges for negative control data.

Untreated negative controls validity:

other: not tested

True negative controls validity:

other: not tested

Positive controls validity:

valid

Remarks:

minimum mutation ratio (= number of induced revertants/number of control revertants) within the acceptability ranges for positive control data.

Test results 2

Key result

Species / strain:

other: all strains (S. typhimurium + E. coli)

Remarks:

second test

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above 250 µg/plate: pinpoint colonies, (slightly) less dense background lawn of the bacterial growth and decrease in the mean number of revertants.

Vehicle controls validity:

valid

Remarks:

revertant colonies per plate within the acceptability ranges for negative control data.

Untreated negative controls validity:

other: not tested

True negative controls validity:

other: not tested

Positive controls validity:

valid

Remarks:

minimum mutation ratio (= number of induced revertants/number of control revertants) within the acceptability ranges for positive control data.

Test results 3

Key result

Species / strain:

other: S. typhimurium TA 1537 and E. coli WP2 uvrA

Remarks:

second test

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 125 µg/plate: slightly less dense background lawn of the bacterial growth.

Vehicle controls validity:

valid

Remarks:

revertant colonies per plate within the acceptability ranges for negative control data.

Untreated negative controls validity:

other: not tested

True negative controls validity:

other: not tested

Positive controls validity:

valid

Remarks:

minimum mutation ratio (= number of induced revertants/number of control revertants) within the acceptability ranges for positive control data.

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

second test

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 125 µg/plate: slightly less dense background lawn of the bacterial growth.

Vehicle controls validity:

valid

Remarks:

revertant colonies per plate within the acceptability ranges for negative control data.

Untreated negative controls validity:

other: not tested

True negative controls validity:

other: not tested

Positive controls validity:

valid

Remarks:

minimum mutation ratio (= number of induced revertants/number of control revertants) within the acceptability ranges for positive control data.

Additional information on results:

STUDY RESULTS:
- Signs of toxicity:
* First test: ETFBO was toxic to all strains, in both the absence and presence of S9-mix, at and above 185 µg/plate, as was evidenced by pinpoint colonies, a (slightly) less dense background lawn of the bacterial growth and a decrease in the mean number of revertants compared to the negative control.
* Second test: ETFBO was slightly toxic to TA 1537 and WP2 uvrA, in the absence of S9-mix, and to TA 98, in both the absence and presence of S9-mix, at 125 µg/plate as was evidenced by a slightly less dense background lawn of the bacterial growth compared to the negative control. Additionally, the test substance ETFBO, was toxic to all strains, in both the absence and presence, at and above 250 µg/plate, as was evidenced by pinpoint colonies, a (slightly) less dense background lawn of the bacterial growth and a decrease in the mean number of revertants compared to the negative control.
- Signs of genotoxicity (first and second tests): In both the absence and presence of S9-mix in all strains, ETFBO did not cause a dose related increase in the mean number of revertant colonies appearing in the test plates compared to the background spontaneous reversion rate observed with the negative control. Except in the second test, in strain TA 1537, in the absence of S9-mix, ETFBO caused a 2.3-fold increase in the mean number of revertant colonies compared to the negative control, at the lowest concentration (16 µg/plate). However, the negative control is relatively low compared to the historical data and since a more than 2-fold increase was observed at only the lowest concentration of the test substance, this was considered to be not biological relevant.

HISTORICAL POSITIVE AND NEGATIVE CONTROL DATA
See in the field below "any other information on results incl. tables".

Any other information on results incl. tables

Number of revertants counted in the bacterial reverse mutation test with ETFBO:

Figures presented in the table below are average numbers of revertants per plate (standard deviations).

	TA 1535		TA 1537		TA 98		TA 100		WP2 uvrA
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
First test
Negative control	22 (6)	15 (4)	16 (5)	11 (6)	26 (5)	43 (13)	126 (19)	125 (10)	30 (10)	34 (8)
62 µg/plate	19 (5)	17 (3)	11 (6)	13 (3)	26 (4)	40 (9)	116 (20)	134 (19)	40 (9)	38 (4)
185 µg/plate	24^A (2)	21^A (3)	11^A (3)	13^A (7)	29^A (3)	36^A (8)	80^A (21)	77^A (9)	13^B (5)	20^A (3)
556 µg/plate	pp	pp	pp	pp	pp	pp	pp	pp	pp	pp
1667 µg/plate	pp	pp	pp	pp	pp	pp	pp	pp	pp	pp
5000 µg/plate	pp	pp	pp	pp	pp	pp	pp	pp	pp	pp
Positive control	605 (12)	356 (55)	1135 (448)	272 (19)	1499 (70)	708 (124)	738 (39)	1794 (111)	234 (32)	1812 (81)
Second test
Negative control	19 (3)	13 (2)	6 (1)	9 (4)	31 (8)	51 (4)	110 (15)	117 (8)	19 (5)	33 (9)
16 µg/plate	20 (3)	19 (4)	14 (2)	11 (1)	25 (3)	43 (12)	123 (5)	114 (11)	21 (6)	32 (5)
31.25 µg/plate	17 (1)	18 (2)	8 (2)	8 (1)	28 (3)	45 (6)	112 (16)	124 (10)	21 (5)	27 (4)
62.5 µg/plate	22 (3)	11 (3)	7 (3)	11 (3)	28 (6)	50 (3)	106 (15)	141 (20)	25 (2)	36 (9)
125 µg/plate	16 (5)	12 (0)	11^A (1)	13 (6)	25^A (4)	48^A (6)	108 (7)	116 (4)	20^A (12)	31 (9)
250 µg/plate	17^A (3)	17^A (4)	10^A (2)	10^A (4)	25^A (8)	41^A (5)	91^A (26)	113^A (20)	15^A (5)	23^A (1)
500 µg/plate	10^B (3)	16^B (7)	2^C (1)	1^C (2)	8^C (7)	27^C (10)	8^C (2)	12^B (4)	11^B (1)	17^B (3)
Positive control	655 (98)	309 (16)	1464 (49)	298 (46)	1472 (60)	779 (146)	808 (74)	1968 (59)	300 (43)	1382 (303)

pp = pinpoint colonies

^A= slightly less dense background lawn of bacterial growth

^B= less dense background lawn of bacterial growth

^C= less dense background lawn of bacterial growth and pinpoint colonies

Overview of historical positive control data from studies between May 2002 and March 2005:

Strain	Compound	Mutation ratio
		Mean	Standard deviation	Range	Number of assays
Without S9-mix
TA1535	NaN3 1µg/plate	31	37	13-297	(57)
TA1537	9-AA 80µg/plate	191	76	49-350	(57)
TA98	2-NF 2µg/plate	49	17	16-98	(66)
TA100	NaN3 1µg/plate	5	1	3-8	(60)
WP2 uvrA	ENU 100µg/plate	6	3	3-9	(55)
With S9-mix
TA1535	2-AA 2 µg/plate	28	9	11-61	(57)
TA1537	BP 4 µg/plate	13	5	6-35	(57)
TA98	2-AA 2µg/plate	24	11	9-80	(67)
TA100	2-AA 2µg/plate	15	3	8-22	(60)
WP2 uvrA	2-AA 80µg/plate	36	8	20-57	(55)

NaN3 = natrium azide

ENU = N-nitroso N-ethylurea

2-AA = 2-aminoanthracene

9-AA = 9-aminoacridine

BP = benzo(a)pyrene

2-NF = 2-nitrofluorene

Overview of historical negative control (DMSO) data from studies between May 2002 and March 2005:

Strain	Number of revertants
	Mean	Standard deviation	Number of assays
Without S9-mix
TA1535	26	5	20
TA1537	18	5	20
TA98	38	5	28
TA100	172	27	24
WP2 uvrA	38	7	20
With S9-mix
TA1535	20	5	20
TA1537	23	5	20
TA98	58	8	28
TA100	172	29	20
WP2 uvrA	42	6	20

 

Applicant's summary and conclusion

Conclusions:

The results obtained in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and in the Escherichia coli strain WP2 uvrA, in both the absence and the presence of the S9-mix, indicated that ETFBO was not mutagenic under the conditions employed in this study.

Executive summary:

The mutagenic activity was investigated in a GPL-compliant study performed according to OECD test guideline 471 (Bacterial Reverse Mutation Assay, Ames test).

The histidine-requiring Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and the tryptophan­requiring Escherichia coli strain WP2uvrA were tested in both the absence and presence of a Iiver fraction of Aroclor 1254-induced rats for metabolic activation (S9-mix). The test substance, ETFBO was diluted in dimethylsulphoxide (DMSO). Two independent tests were performed with all strains, in the absence and the presence of S9-mix, with the following concentrations of the test substance:

- First test: five concentrations ranging from 62 to 5000 µg/plate.

- Second test: six concentrations, ranging from 16 to 500 µg/plate.

Negative controls (DMSO) and positive controls were run simultaneously. All determinations were made in triplicate. The plates were incubated at ca. 37 °C for approximately 72 hours. Subsequently, the his+ and trp+ revertants were counted. A test substance is considered to be positive and thus to induce point mutations by base substitutions or frameshifts in the genome of either S. typhimurium and/or E. coli if the mean number of revertant colonies on the test plates is concentration-related increased or if a reproducible two-fold or more increase is observed compared to that on the negative control plates. A test substance is considered to be negative if it produces neither a dose-related increase in the mean number of revertant colonies nor a reproducible positive response at any of the test points. Cytotoxicity was also investigated and defined as a reduction (at least 50%) in the number of revertant colonies and/or a clearing of the background lawn of bacterial growth.

In the first test, ETFBO was toxic to all strains, in both the absence and presence of S9-mix, at and above 185 µg/plate, as was evidenced by pinpoint colonies, a (slightly) less dense background lawn of the bacterial growth and a decrease in the mean number of revertants compared to the negative control. In the second test, ETFBO was slightly toxic to TA 1537 and WP2 uvrA, in the absence of S9-mix, and to TA 98, in both the absence and presence of S9-mix, at 125 µg/plate as was evidenced by a slightly less dense background lawn of the bacterial growth compared to the negative control. Additionally, the test substance ETFBO, was toxic to all strains, in both the absence and presence, at and above 250 µg/plate, as was evidenced by pinpoint colonies, a (slightly) less dense background lawn of the bacterial growth and a decrease in the mean number of revertants compared to the negative control. In both tests, in the absence and presence of S9-mix in all strains, ETFBO did not cause a dose related increase in the mean number of revertant colonies appearing in the test plates compared to the background spontaneous reversion rate observed with the negative control. Except in the second test, in strain TA 1537, in the absence of S9-mix, ETFBO caused a 2.3-fold increase in the mean number of revertant colonies compared to the negative control, at the lowest concentration (16 µg/plate). However, the negative control is relatively low compared to the historical data and since a more than 2-fold increase was observed at only the lowest concentration of the test substance, this was considered to be not biological relevant. The mean number of his+ revertant colonies of the negative controls were within the acceptable range, and the positive controls gave the expected increase in the mean number of revertant colonies; the study was thus considered valid.

The results obtained in S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and in the E. coli strain WP2 uvrA, in both the absence and the presence of the S9-mix, indicated that ETFBO was not mutagenic under the conditions employed in this study.