Ethyl [2-(4-phenoxyphenoxy)ethyl]carbamate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 Oct 1988 to 03 Nov 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl [2-(4-phenoxyphenoxy)ethyl]carbamate

EC Number:

276-696-7

EC Name:

Ethyl [2-(4-phenoxyphenoxy)ethyl]carbamate

Cas Number:

72490-01-8

Molecular formula:

C17 H19 N O4

IUPAC Name:

ethyl [2-(4-phenoxyphenoxy)ethyl]carbamate

Method

Target gene:

his- (S. typhimurium)

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 97

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9 : Phenobarbital/β-Naphthoflavone induced rat liver S9
- Method of preparation of S9 mix : Male albino rats were killed by decapitation and the livers were removed aseptically. The livers of were chopped and mixed with homogenizer with addition of a volume of ice cold 0.15 M potassium chloride equivalent to 3 times the weight of the livers. The homogenate was pooled and centrifuged at 4°C for 10 minutes at 9000 g. The supernatant fluid was collected and dispensed in 2 mL cryotubes. The tubes were rapidly cooled and kept in the deep freezer at -70°C until use. At the end of the preparation about 1 mL of the S9 fraction was dispensed on complete medium plates and incubated at 37°C for 2 days. Absence of colony growth verified the sterility of the S9 fraction
- Concentration or volume of S9 mix and S9 in the final culture medium: The composition of the S9 mixture was as followed: Potassium chloride 0.165 M, magnesium chloride 0.04 M (both 0.2 mL per mix), sodium phosphate buffered saline 0.2 M, pH 7.4 (0.5 mL per mL mix), NADP (Boehringer, 3.2 mg per mL mix), glucose-6-phosphate (Boehringer, 1.53 mg per mL mix) and S9 fraction (0.1 mL per mL mix).
- Quality controls of S9: In order to test the activity of the S9 mix used in the experiments positive controls requiring metabolic activation were tested concurrently. 2--AAF and 2-Aminoanthr. were tested with and without metabolic activation to examine the activity of the S9-mix.

Test concentrations with justification for top dose:

Ames standard assay (experiment 1 and 2): 15.8, 50, 158, 500 and 1580 µg/plate
Liquid pre-incubation assay (experiment 3 and 5): 10, 31.6, 100, 316 and 1000 µg/plate

Vehicle / solvent:

- Solvent used: DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

sodium azide

mitomycin C

other: 2-Aminoanthracene, ICR 191

Details on test system and experimental conditions:

PRE- EXPERIMENT FOR TOXICITY
Toxicity of the test substance was determined in a preliminary toxicity assay by evaluating the growth on NB- and/or minimal- medium (determination of the growth of the background lawn and/or frequency of spontaneous revertants). Each test substance dose, as well as the appropriate solvent control, was evaluated in duplicate in strain TA 100.

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation) for experiment 1 and 2. A liquid pre-incubation assay was performed for experiment 3 and 5.

EXPERIMENTAL PERFORMANCE
Ames test:
Test tubes containing 2 mL of 0.6% agar medium autoclaved and a prewarmed water bath at 42°C to 45°C. Following solutions were added in order:
- 0.2 mL of the histidine/biotin mixture corresponding to 21 µg L-histidine and 24.4 µg biotin.
- 0.1 mL of the test substance at different concentrations. The negative control tubes contained 0.1 mL of the solvent and the positive control ones received 0.05 mL of the different reference substances which were thawed shortly before use.
- 0.1 mL of the overnight cultures of the Salmonella typhimurium strains.
- 0.5 mL of the S9 mixture where metabolic activation was needed. The S9 mixture was replaced by 0.5 mL sodium phosphate buffered saline 0.2 M, pH 7.4 in the treatment without metabolic activation.
The contents of the tubes were mixed and poured immediately ontotable 1 Vogel-Bonner minimal agar plates. Four replicate plates for the test substance and negative control or two replicate plates for the positive controls were incubated at 37°C, upside down, for 2 days.

Liquid preincubation assay:
In the modified procedure 0.1 mL of the test compound solutions and of the solvent or 0.05 mL of the reference substances thawed shortly before use, 0.5 mL of sodium phosphate buffered saline 0.2 M, pH 7.4 or 0.5 mL of the 89 mixture and 0.1 mL of the NB cultures of the bacterial tester strains were incubated for 30 minutes at 37°C under shaking. 2.2 mL soft-agar supplemented with 21 µg L-histidine and 24.4 µg biotin was added afterwards and the tubes were poured on Vogel—Bonner minimal agar plates. Four replicate plates for the test compound and negative control or two replicate plates for the positive controls were incubated at 37°C, upside down, for 2 days.

DATA REPORTING
The determination of the number of colonies growing on the plates was made by hand. The colonies of the positive controls were generally evaluated using a New Brunswick Inc Biotran III Automatic colony counter. No correction factor was determined. Representative plates were examined microscopically for microcolony growth and the absence of a confluent lawn of bacteria. A reduced background lawn was reported as BR and the absence of bacterial lawn as B0. The inhibition of the growth was attributed to toxic effects by the substance

Evaluation criteria:

There is as yet no generally accepted statistical treatment of Ames test data. In most tests the results are either clearly positive or clearly negative. A positive result is defined reproducible, dose- related increase in the number of his+ revertants. The increase should reach at least a doubling of the number of spontaneous revertants for Salmonella typhimurium strains TA 1535, TA 1537, TA 1538 and TA 98. For strains TA 97, TA 100 and TA 102 a 1.5- fold increase over control values might be indicative of a mutagenic effect provided the negative control values fall within the historical control data. Other investigators have set higher limits for a mutagenic response (factor 3 and 2 for the respective groups of strains). These rules of thumb have a questionable scientific foundation (Claxton et al. 1987) and biological relevance should always be taken into account. Negative result is defined as the absence of a reproducible increase in the number of his+ revertant colonies.

Results and discussion

Test results

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 97, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

STUDY RESULTS
- Some signs of toxic activity (reduced background growth) was seen in the preincubation assay. In strain TA 1538 the negative control (-S9) was rather low so that a doubling of the mutant frequency was found at one intermediate test concentrations. This was however, clearly due to low spontaneous value and cannot be taken as test substance related. Accordingly no increase was found in the preincubation assay.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: Precipitation on the plates was observed at the dose level of 1580 μg/plate
- Water solubility: The test compound was insoluble in water. Stock solutions in DMSO were prepared before each experiment. Upon addition of the DMSO solutions to the aqueous medium a milky suspension was visible at concentrations of 158 pg/plate and above. At 1.58 mg/plate small droplets remained in the soft agar during the two day incubation period. Therefore, it was decided to use 1.58 mg/plate as the highest dose in the standard assay and 1 mg/plate in the preincubation assay where during the liquid incubation period the test concentration is higher than in the plate assay due to the smaller volume.

Any other information on results incl. tables

Table 1. Salmonella mutagenicity test (Ames standard assay). Mean values and standard deviations.

Experiment no	1	1	1	1	1	1
Activation	(-)S9	(+)S9	(-)S9	(+)S9	(-)S9	(+)S9
Strain	TA 1535	TA 1535	TA 1537	TA 1537	TA 1538	TA 1538
Concentration in µg/plate
0.00	11 ± 2	12 ± 3	13 ± 3	13 ± 4	8 ± 1	22 ± 6
15.80	11 ± 2	12 ± 3	13 ± 3	13 ± 4	8 ±  1	22 ± 8
50.00	13 ± 5	8 ±  3	10 ± 6	13 ± 5	12 ± 5	21 ± 5
158.00	15 ± 6	7 ± 4	14 ± 6	9 ± 3	14 ± 3	24 ± 3
500.00	9 ± 2	8 ± 4	11 ± 1	8 ± 3	17 ± 8	28 ± 4
1580	10 ± 3	12 ± 2	8 ± 4	10 ± 3	13 ± 6	17 ± 2

Experiment no	2	2	2	2	2	2	2	2
Activation	(-)S9	(+)S9	(-)S9	(+)S9	(-)S9	(+)S9	(-)S9	(+)S9
Strain	TA 97	TA 97	TA 98	TA 98	TA 100	TA 100	TA 102	TA 102
Concentration in µg/plate
0.00	150 ± 14	178 ± 18	23 ± 5	29 ± 2	96 ± 9	95 ± 6	295 ± 21	283 ± 10
15.80	156 ± 5	180 ± 15	25 ± 3	33 ± 2	88 ± 8	103 ± 11	288 ± 29	298 ± 16
50.00	167 ± 8	166 ± 8	33 ± 4	29 ± 7	81 ± 11	100 ± 8	296 ± 32	273 ± 9
158.00	160 ± 9	178 ± 12	29 ± 2	26 ± 5	106 ± 11	95 ± 8	255 ± 32	292 ± 15
500.00	151 ± 8	165 ± 8	24 ± 8	23 ± 8	102 ± 8	92 ± 8	166 ± 8	236 ± 8
1580	153 ± 8	181 ± 8	32 ± 8	26 ± 8	97 ± 8	87 ± 8	114 ± 8	160 ± 8

Table 2. Salmonella mutagenicity test (Liquid preincubation assay). Mean values and standard deviations

Experiment no	3	3	3	3	3	3
Activation	(-)S9	(+)S9	(-)S9	(+)S9	(-)S9	(+)S9
Strain	TA 1535	TA 1535	TA 1537	TA 1537	TA 1538	TA 1538
Concentration in µg/plate
0.00	6 ± 3	10 ± 1	12 ± 2	11 ± 5	19 ± 7	24 ± 5
10.00	9 ± 3	10 ± 2	14 ± 1	12 ± 3	13 ± 3	26 ± 5
31.60	8 ± 2	10 ± 2	12 ± 5	14 ± 1	16 ± 1	24 ± 2
100.00	10 ± 2	8 ± 2	6 ± 3	13 ± 4	6 ± 2 t	25 ± 6
316.00	10 ± 2	9 ± 5	2 ± 1 t	12 ± 3	5 ± 3 t	29 ± 10
1000.00	9 ± 3	8 ± 1	2 ± 1 t	7 ± 2 t	4 ± 1	10 ± 3 t

.

Experiment no	5	5	5	5	5	5	5	5
Activation	(-)S9	(+)S9	(-)S9	(+)S9	(-)S9	(+)S9	(-)S9	(+)S9
Strain	TA 97	TA 97	TA 98	TA 98	TA 100	TA 100	TA 102	TA 102
Concentration in µg/plate
0.00	167 ± 7	± 214 16	± 20 6	26 ± 5	106 ± 12	105 ± 7	179 ±15	248 ±18
10.00	174 ± 10	± 203 23	± 21 3	25 ± 8	116 ± 6	106 ± 3	196 ±16	265 ±25
31.60	165 ± 7	226 ± 20	19 ±5	28 ± 8	116 ± 15	112 ± 9	177 ±8	247 ±22
100.00	165 ± 9	221 ± 12	14 ±5	26 ± 5	54 ± 10 t	106 ± 6	163 ± 15	239 ± 8
316.00	155 ± 6	221 ± 17	13 ±3	23 ± 5	40 ± 4 t	105 ± 12	108 ± 12	137 ± 16
1000.00	160 ± 6	233 ± 14	13 ±3 t	20 ± 3	42 ± 4 t	81 ± 10	111 ± 15	118 ± 7

t : Toxic effect

Applicant's summary and conclusion

Conclusions:

The test substance is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Executive summary:

The test substance was examined for mutagenic activity in the standard plate incorporation as well as in the preincubation versions of the Ames test, performed equivalent to OECD TG 471 and following GLP principles. The following concentrations were used for the Ames standard assay: 15.8, 50, 158, 500 and 1580 µg/plate and the following concentrations for the liquid preincubation assay: 10, 31.6, 100, 316 and 1000 µg/plate. The test substance was dissolved in DMSO. The test substance was tested in all strains currently in general use Salmonella typhimurium TA 1535, TA 1537, TA 1538, 97, TA 98, TA 100 and 102 in presence and absence of a metabolic activation system prepared from the livers of phenobarbital/ß-naphthoflavone-induced rats. Responsiveness of the strains and activity of the metabolic enzymes were verified by including appropriate positive controls in each experiment. Appropriate reference mutagens (sodium azide, 2-aminoanthracene, ICR 191, 2-acetylaminofluorene and mitomycin C) were used as positive controls. Precipitation on the plates was observed at the dose level of 1580 μg/plate in the standard assay and 1000 µg/plate for the preincubation assay.

Results showed that the test substance did not cause an increase of the number of revertant colonies under these test conditions. Some signs of toxic activity (reduced background growth) was seen in the preincubation assay. In strain TA 1538 the negative control (-S9) was rather low so that a doubling of the mutant frequency was found at one intermediate test concentrations. This was however, clearly due to low spontaneous value and cannot be taken as test substance related. Accordingly no increase was found in the preincubation assay.

In conclusion, the test substance is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.