p-anisic acid

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:

14 Sep - 02 Oct 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in 1997

Deviations:

yes

Remarks:

2-AA is used as the sole indicator of the efficacy of the S9-mix

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2008

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:

p-anisic acid

EC Number:

202-818-5

EC Name:

p-anisic acid

Cas Number:

100-09-4

Molecular formula:

C8H8O3

IUPAC Name:

4-methoxybenzoic acid

Method

Target gene:

his operon and trp operon

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:

Experiment I:
All strains: 3, 10, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation

Experiment II:
Strain WP2 uvrA: 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate
Samonella strains: 33, 100, 333, 1000, 2500 and 5000 μg/plate

The test item precipitated in the overlay agar in the test tubes from 2500 to 5000 μg/plate in experiment I and at 5000 μg/plate in experiment II. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment I at 5000 μg/plate. In experiment II no precipitation of the test item occurred up to the highest investigated dose. The undissolved particles had no influence on the data recording. The concentration of 5000 µg/plate was selected as highest dose as recommended by the OECD guideline.

Vehicle / solvent:

- Vehicle 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

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine and 2-aminoanthracene

Details on test system and experimental conditions:

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

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

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

Evaluation criteria:

A test item was 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 threefold (strains TA 1535 and TA 1537) of 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

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

both experiments: 2500 and 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: The test item precipitated in the overlay agar in the test tubes from 2500 to 5000 μg/plate in experiment I and at 5000 μg/plate in experiment II. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment I at 5000 μg/plate. In experiment II no precipitation of the test item occurred up to the highest investigated dose. The undissolved particles had no influence on the data recording.

Any other information on results incl. tables

Table 2. Summary of Experiment 1

EXPERIMENT 1 (Plate Incorporation Test)
S9-Mix	Without
Test item (µg/plate)	TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
NC (DMSO)	10 ± 4	8 ± 2	29 ± 6	123 ± 22	35 ± 5
Untreated	8 ± 2	11 ± 2	24 ± 6	151 ± 18	33 ± 6
3	11 ± 2	10 ± 2	27 ± 10	122 ± 12	37 ± 9
10	10 ± 2	9 ± 2	25 ± 8	141 ± 10	33 ± 3
33	9 ± 2	8 ± 3	26 ± 4	137 ± 10	33 ± 2
100	12 ± 4	8 ± 3	25 ± 1	121 ± 11	29 ± 9
333	9 ± 2	9 ± 5	30 ± 8	138 ± 18	31 ± 1
1000	8 ± 3	13 ± 2	29 ± 9	140 ± 1	19 ± 5
2500	8 ± 1	7 ± 3	24 ± 4	132 ± 13	11 ± 1
5000	8 ± 3 P	5 ± 1 P	18 ± 4 P	114 ± 5 P	5 ± 1 P
NaN3 10 µg	1063 ± 138			2216 ± 61
4-NOPD 10			778 ± 63
4-NOPD 50		69 ± 5
MMS 2 µL					962 ± 7
S9-Mix	With
Test item (µg/plate)	TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
NC (DMSO)	14 ± 4	13 ± 3	35 ± 7	92 ± 12	57 ± 11
Untreated	12 ± 2	12 ± 1	40 ± 7	108 ± 12	47 ± 2
3	12 ± 4	13 ± 3	37 ± 9	101 ± 8	38 ± 6
10	14 ± 2	14 ± 3	42 ± 10	111 ± 8	46 ± 4
33	10 ± 3	15 ± 1	41 ± 11	114 ± 18	46 ± 5
100	13 ± 4	17 ± 1	37 ± 14	113 ± 9	49 ± 4
333	11 ± 1	19 ± 3	42 ± 6	89 ± 4	40 ± 5
1000	8 ± 2	18 ± 1	29 ± 7	105 ± 11	34 ± 4
2500	12 ± 3	20 ± 4	34 ± 2	112 ± 8	14 ± 5
5000	7 ± 2 P	18 ± 4 P	32 ± 2 P	112 ± 3 P	5 ± 1 P
2-AA 2.5	369 ± 32	117 ± 22	3863 ± 401	1419 ± 235
2-AA 10					466 ± 27
NC = Negative/Vehicle Control P = Precipitate

 

Table 3: Summary of Experiment 2

EXPERIMENT 2 (Pre-incubation Test)
S9-Mix	Without
Test item (µg/plate)	TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
NC (DMSO)	9 ± 1	10 ± 5	14 ± 2 B M	170 ± 13	29 ± 5
Untreated	9 ± 5	9 ± 3	19 ± 3 B M	199 ± 11	42 ± 7
10					38 ± 12
33	10 ± 2	7 ± 3	15 ± 2 B M	160 ± 15	26 ± 3
100	10 ± 4	9 ± 1	16 ± 1 B M	148 ± 18	33 ± 5
333	13 ± 3	7 ± 4	12 ± 2 B M	165 ± 10	35 ± 1
1000	10 ± 5	8 ± 2	11 ± 3 B M	165 ± 8	27 ± 7
2500	11 ± 3	9 ± 4	13 ± 1 B M	164 ± 23	12 ± 3
5000	7 ± 3	6 ± 2	10 ± 2 B M	161 ± 7	6 ± 2
NaN3 10	1212 ± 118			2181 ± 31
4-NOPD 10			328 ± 11 B M
4-NOPD 50		82 ± 12
MMS 2 µL					836 ± 14
S9-Mix	With
Test item (µg/plate)	TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
NC (DMSO)	13 ± 2	13 ± 1	16 ± 1 B M	159 ± 22	48 ± 8
Untreated	12 ± 3	13 ± 3	13 ± 2 B M	207 ± 11	48 ± 5
10					46 ± 1
33	9 ± 2	14 ± 4	16 ± 2 B M	159 ± 17	43 ± 2
100	12 ± 3	11 ± 1	16 ± 2 B M	159 ± 10	42 ± 5
333	12 ± 5	14 ± 5	12 ± 3 B M	158 ± 5	40 ± 2
1000	13 ± 3	14 ± 5	14 ± 1 B M	154 ± 14	33 ± 6
2500	12 ± 3	15 ± 1	13 ± 4 B M	145 ± 4	13 ± 3
5000	11 ± 5	11 ± 4	8 ± 2 B M	136 ± 9	4 ± 2
2-AA 2.5	429 ± 32	116 ± 12	3939 ± 222 B M	3982 ± 399
2-AA 10					501 ± 5
NC = Negative/Vehicle control B = Extensive bacterial growth M = Manual count

Applicant's summary and conclusion

Conclusions:

The test item was tested for bacterial mutagenicity according to OECD 471, with S. typhimurium strains TA 1535, 1537, 98 and 100 as well as E. coli WP2 uvr A at concentration levels up to 5000 µg/plate with and without metabolic activation. Based on the results of the conducted study the test item did not exhibit mutagenic properties in bacterial cells.