Allyl phenoxyacetate

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:

29 July - 04 Sep 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Allyl phenoxyacetate

EC Number:

231-335-2

EC Name:

Allyl phenoxyacetate

Cas Number:

7493-74-5

Molecular formula:

C11H12O3

IUPAC Name:

allyl phenoxyacetate

Method

Target gene:

his operon (for S. typhimurium strains)
trp operon (for E. coli strain)

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:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone

Test concentrations with justification for top dose:

Pre-Experiment
3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation.

The pre-experiment is reported as Experiment I.

Experiment II
without metabolic activation:
1, 3, 10, 33, 100, 333, 1000 and 2500 µg/plate (TA1535, TA1537, TA98 and TA100)
33, 100, 333, 1000, 2500 and 5000 µg/plate (WP2 uvrA)

with metabolic activation:
3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate (TA1535, TA1537, TA98 and TA100)
33, 100, 333, 1000, 2500 and 5000 µg/plate (WP2 uvrA)

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Controls

Untreated negative controls:

yes

Remarks:

untreated controls

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene (2-AA); 4-nitro-o-phenylene-diamine (4-NOPD)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) (Experiment I); preincubation (Experiment II)

DURATION
- Preincubation period: 1 h
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

Mean values and standard deviation were calculated.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in all Salmonella strains with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item was observed in the overlay agar in the test tubes. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 2500 to 5000 µg/plate in the presence of S9 mix in both experiments. The undissolved particles had no influence on the data recording.

RANGE-FINDING/SCREENING STUDIES: The pre-experiment is reported as Experiment I.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. Only in Experiment II reduced background growth was observed in the absence of S9 mix at 2500 µg/plate in strain TA 100 and at 5000 µg/plate in strain WP2 uvrA. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all Salmonella strains with and without metabolic activation. Please refer to table 3.

Any other information on results incl. tables

Table 1. Test results of main test 1 (plate incorporation).

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA1535	TA100	WP2 uvrA	TA1537	TA98
-	0 (DMSO)	9 ± 1	138± 8	44 ± 2	9 ± 2	28± 7
-	0	11 ± 2	164± 6	38 ± 5	14 ± 6	25± 4
-	3	11 ± 4	150± 7	52 ± 3	9 ± 3	22± 3
-	10	10 ± 4	134± 12	38 ± 10	11 ± 4	20 ± 2
-	33	9 ± 3	156± 21	45 ± 8	10 ± 2	26± 8
-	100	8 ± 2	151± 10	44 ± 9	11 ± 4	21± 8
-	333	6 ± 1	126± 11	43 ± 2	6 ± 1	20± 3
-	1000	3 ± 1	43± 11	38 ± 7	2 ± 1	10± 4
-	2500	1 ± 1	28± 10	43 ± 4	2 ± 1	7± 0
-	5000	0 ± 0	7± 2	29 ± 2	0 ± 0	4± 2
Positive controls, –S9	Name	NaN3	NaN3	MMS	4-NOPD	4-NOPD
	Concentrations (μg/plate)	10	10	2.0 µL	50	10
	Mean No. of colonies/plate (average of 3 ± SD)	1288 ± 46	2161± 203	926 ± 110	81 ± 8	299± 19
+	0 (DMSO)	13 ± 6	112± 17	43 ± 9	8 ± 2	26± 4
+	0	12 ± 3	151± 8	54 ± 4	10 ± 3	32± 11
+	3	12 ± 5	109± 16	43 ± 7	10 ± 3	32± 3
+	10	12 ± 4	100± 16	44 ± 2	9 ± 4	25± 3
+	33	6 ± 2	117± 7	47 ± 12	8 ± 2	25± 8
+	100	7 ± 1	115± 8	63 ± 8	7 ± 1	32± 4
+	333	7 ± 2	115± 6	58 ± 4	9 ± 2	27± 6
+	1000	4 ± 1	94± 13	51 ±19	7 ± 1	14± 2
+	2500	0 ± 0^P	8± 1^P	49 ± 8^P,M	1 ± 1^P	1± 2^P
+	5000	0 ± 0^P	1± 1^P	44 ± 5^P,M	0 ± 0^P	0± 0^P
Positive controls, +S9	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentrations (μg/plate)	2.5	2.5	10	2.5	2.5
	Mean No. of colonies/plate (average of 3 ± SD)	335 ± 38	3122± 296	321 ± 34	155 ± 26	3640± 482

NaN3: Sodium azide

4-NOPD: 4-nitro-o-phenylene-diamine

MMS: Methylmethanesulfonate

2-AA: 2-aminoanthracene

M: Manual count

P: Precipitate

 

Table 2. Test results of main test 2 (preincubation).

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA1535	TA100	WP2 uvrA	TA1537	TA98
-	0 (DMSO)	13 ± 4	128± 15	37 ± 14	10 ± 3	33± 6
-	0	13 ± 3	173± 10	34 ± 8	13 ± 1	31± 5
-	1	10 ± 4	111± 2	-	10 ± 3	25± 6
-	3	11 ± 4	142± 27	-	6 ± 1	30± 2
-	10	13 ± 5	134± 27	-	9 ± 3	32± 3
-	33	11 ± 1	139± 10	41 ± 2	8 ± 3	37± 9
-	100	10 ± 4	122± 18	34 ± 1	5 ± 3	23± 1
-	333	13 ± 2	109± 9	42 ± 6	6 ± 1	32± 10
-	1000	3 ± 1	26± 15	36 ± 5	6 ± 3	12± 3
-	2500	4 ± 3	18± 4^R	36 ± 5	6 ± 1	2± 3
-	5000	-	-	24 ± 7^M,R	-	-
Positive controls, –S9	Name	NaN3	NaN3	MMS	4-NOPD	4-NOPD
	Concentrations (μg/plate)	10	10	2.0 µL	50	10
	Mean No. of colonies/plate (average of 3 ± SD)	1200 ± 44	2482± 103	547 ± 18	73 ± 17	419± 19
+	0 (DMSO)	14 ± 4	113± 8	56 ± 7	9 ± 1	42± 2
+	0	13 ± 4	159± 14	58 ± 6	8 ± 1	43± 9
+	3	9 ± 2	100± 2	-	12 ± 3	35± 6
+	10	14 ± 1	102± 9	-	10 ± 5	43± 7
+	33	11 ± 5	105± 6	48 ± 7	7 ± 1	41± 6
+	100	12 ± 4	92± 5	47 ± 3	11 ± 3	30± 3
+	333	10 ± 2	87± 8	58 ± 12	10 ± 2	37± 4
+	1000	8 ± 1	66± 3	65 ± 10	11 ± 3	19± 4
+	2500	6 ± 1^P	42± 11^P	85 ± 14^P	2 ± 2^P	5± 4^P
+	5000	6 ± 3^P	0± 0^P	86 ± 21^P	0 ± 0^P	0± 0^P
Positive controls, +S9	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentrations (μg/plate)	2.5	2.5	10	2.5	2.5
	Mean No. of colonies/plate (average of 3 ± SD)	334 ± 23	3303± 232	333 ± 2	148 ± 17	4081± 283

NaN3: Sodium azide

2-AA: 2-aminoanthracene

4-NOPD: 4-nitro-o-phenylene-diamine

MMS: Methylmethanesulfonate

M: Manual count

P: Precipitate

R: Reduced background growth

 

Table 3. Reduction in the number of spontaneous revertants (below the induction factor of 0.5) in Experiment I and II at different concentrations (µg/plate).

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	1000 - 5000/	1000 - 5000	1000 - 2500	2500 - 5000
TA 1537	1000 - 5000/	2500 - 5000	/	2500 - 5000
TA 98	1000 - 5000/	2500 - 5000	1000 - 2500	2500 - 5000
TA 100	1000 - 5000	2500 - 5000	1000 - 2500	2500 - 5000
WP2 uvrA	/	/	/	/

/ = no toxic effects

Applicant's summary and conclusion

Conclusions:

Under the conditions of the Ames test the substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 98, TA 100 and WP2 uvrA) tested with and without metabolic activation.