p-anisic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial gene mutation (OECD 471): negative

Cytogenicity/chromosome aberration in mammalian cells (OECD 487): negative

Gene mutation in mammalian cells (OECD 476): negative

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:

14 Sep - 02 Oct 2017

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:

adopted in 1997

Deviations:

yes

Remarks:

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

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

Target gene:

his operon and trp operon

Species / strain / cell type:

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

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.

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.

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

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.

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Sep - 21 Nov 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

adopted in 2016

Deviations:

yes

Remarks:

In-house validation studies were performed with proficiency chemicals. To achieve appropriate responses the test design, specifically for the treatment, the recovery phase and harvest time, was slightly modified compared to the guideline proposals.

GLP compliance:

yes (incl. QA statement)

Remarks:

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

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

not applicable

Species / strain / cell type:

lymphocytes:

Remarks:

cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Suitability of cells: lymphocytes of the respective donors have been shown to respond well to stimulation of proliferation with PHA and to positive control substances; donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes
- Sex, age and number of blood donors if applicable: healthy non-smoking donors; Experiment I: male, 27 years old; Experiment II; female, 35 years old
- Methods for maintenance in cell culture if applicable: blood cultures were established by preparing an 11 % mixture of whole blood in medium within 30 h after blood collection; culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 μg/mL), the mitogen PHA (3 μg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL).
All incubations were done at 37 °C with 5.5 % CO2 in humidified air.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1); already supplemented with 200 mM GlutaMAX™; additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 μg/mL), the mitogen PHA (3 μg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL); all incubations were done at 37 °C with 5.5 % CO2 in humidified air

Additional strain / cell type characteristics:

not applicable

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 (4 h exposure, with and without S9): 9.9, 17.3, 30.3, 53.0, 92.7, 162, 284, 497, 870 and 1522 µg/mL

Experiment II (20 h exposure, without S9): 162, 284, 497, 870 and 1522 µg/mL

In all experiments only the three highest concentrations were evaluated.
The highest concentration tested corresponds to approximately 10 mM the maximum required concentration according to OECD 487.

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: DMSO was chosen due to its solubility properties and its relative non-toxicity to the cell cultures

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

culture medium with 0.5 dimethyl sufoxide (DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: demecolcine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- The first experiment was based on pulse exposure; the second was based on continuous exposure

DURATION
- Stimulation period: 48 h
- Exposure duration: 4 and 20 h
- Expression time (cells in growth medium): 16 h
- Selection time (if incubation with a selection agent): 20 h
- Fixation time (start of exposure up to fixation or harvest of cells): 40 h

SPINDLE INHIBITOR (cytogenetic assays): cytochalasin B (4 μg/mL)

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide; the cells were stained with Giemsa

NUMBER OF CELLS EVALUATED: 2000 binucleated cells per concentration

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: micronuclei have to be stained in the same way as the main nucleus; the area of the micronucleus should not extend the third part of the area of the main nucleus; at least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides

DETERMINATION OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index (CBPI)
- Any supplementary information relevant to cytotoxicity: CBPI was determined in 500 cells per culture

Evaluation criteria:

A test item is considered to be clearly positive if:
− At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− The increase is concentration-related in at least one experimental condition
− The results are outside the range of the laboratory historical solvent control data (95% control limit realized as 95% confidence interval)

A test item is considered to be clearly negative if:
− None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− There is no concentration-related increase
− The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data (95% control limit realized as 95% confidence interval)

Statistics:

Statistical significance was confirmed by the Chi square test (α < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.

Key result

Species / strain:

lymphocytes: cultured peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in experiment II in the absence of S9 mix after continuous treatment, moderate cytotoxicity of 43.4 % cytostasis was observed at 1522 µg/mL (the highest concentration tested)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none observed
- Effects of osmolality: none observed
- Precipitation: none observed

RANGE-FINDING/SCREENING STUDIES: A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Cytotoxicity is characterized by the percentages of reduction in the CBPI in comparison with the controls (% cytostasis) by counting 500 cells per culture. The experimental conditions in this pre-experimental phase were identical to those required and described below for the mutagenicity assay.

The pre-test was performed with 10 concentrations of the test item separated by no more than a factor of √10 and a solvent and positive control. All cell cultures were set up in duplicate. Exposure time was 4 h (with and without S9 mix). The preparation interval was 40 h after start of the exposure. This preliminary test was designated Experiment I, since the cultures fulfilled the acceptability criteria and appropriate concentrations could be selected for cytogenetic evaluation.

CYTOKINESIS BLOCK
- Distribution of mono-, bi- and multi-nucleated cells: not specified

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: 2000
- Indication whether binucleate or mononucleate where appropriate: binucleate

HISTORICAL CONTROL DATA
- Positive historical control data: please see Tables 1 and 2
- Negative (solvent/vehicle) historical control data: please see Tables 3 and 4

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI

Table 5. Summary of results

Exp.	Preparation	Test item	Proliferation	Cytostasis	Micronucleated
	interval	concentration	index	in %*	cells
		in µg/mL	CBPI		in %**
Exposure period 4 hrs without S9 mix
I	40 hrs	Solvent control1	2.00		0.70
		Positive control2	1.64	36.0	12.50S
		497	1.95	4.8	0.60
		870	1.89	10.8	0.60
		1522	1.94	5.8	0.90
Exposure period 20 hrs without S9 mix
II	40 hrs	Solvent control1	1.98		0.85
		Positive control3	1.70	28.6	4.65S
		497	1.78	20.6	0.40
		870	1.78	20.1	1.00
		1522	1.55	43.4	0.45
Exposure period 4 hrs with S9 mix
I	40 hrs	Solvent control1	2.05		0.85
		Positive control4	1.57	46.3	5.70S
		497	1.95	9.5	0.30
		870	1.90	14.4	0.55
		1522	1.94	11.1	0.65

*      For the positive control groups and the test item treatment groups the values are related to the solvent controls

**    The number of micronucleated cells was determined in a sample of 2000 binucleated cells

S           The number of micronucleated cells is statistically significantly higher than corresponding control values

n.c.   Not calculated as the CBPI is equal or higher than the solvent control value

1           DMSO  0.5 % (v/v)

2           Mitomycin C 0.8 µg/mL

3           Demecolcine 50 ng/mL

4           Cyclophosphamide 17.5 µg/mL

Conclusions:

The test item was tested for its potential to induce clastogenicity in cultured peripheral human lymphocytes according to OECD 487. Experiments were performed with and without metabolic activation at concentrations of up to 1522 µg/mL. Based on the results of the conducted study the test item did not induce micronuclei in human lymphocytes.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 Dec 2018 - 08 Feb 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adopted in 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

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:

other: in vitro mammalian cell gene mutation test using the Hprt and xprt genes (migrated information)

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Source of cells: Laboratory for Mutagenicity Testing; Technical University, Darmstadt, Germany
- Cell cycle length: doubling time 12 - 16 h in stock cultures
- Methods for maintenance in cell culture: in liquid nitrogen in the cell bank of Envigo CRS GmbH
- Modal number of chromosomes: 22

- Type and identity of media: MEM (minimal essential medium) containing Hank’s salts supplemented with 10% foetal bovine serum (FBS), neomycin (5 μg/mL) and amphotericin B (1%)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

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:

First experiment: 23.8, 47.6, 95.1, 190.3, 380.5, 761.0 and 1522.0 µg/mL with and without metabolic activation

The maximum test item concentration of the pre-experiment and the main experiment was 1522 µg/mL, corresponding to 10 mM, and was selected based on the recommendations of the OECD guideline 476.

Only the five highest concentrations were used for mutation rate analysis.

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: 0.7 to 1.2 x 10E7

DURATION
- Preincubation period: 24 h
- Exposure duration: 4 h with and without metabolic activation
- Expression time (cells in growth medium): 6 days
- Selection time: 11 days
- Fixation time (start of exposure up to fixation or harvest of cells): 17 days

SELECTION AGENT: 6-thioguanine (6-TG)

NUMBER OF REPLICATIONS: 2

STAINING TECHNIQUE USED: 10 % methylene blue in 0.01 % KOH solution

NUMBER OF CELLS EVALUATED: 10E6

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
- Any supplementary information relevant to cytotoxicity: The colonies used to determine the cloning efficiency I (test item induced cytotoxicity) were fixed and stained 6 to 8 days after treatment. The colonies for cloning efficiency II (cell viability) were stained after 8 days following expression time.

Evaluation criteria:

- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
- the increase is dose-related when evaluated with an appropriate trend test
- any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits)

Statistics:

A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. Both, biological and statistical significance were considered together.
A t-Test was not performed since all mean mutant frequencies were well within the 95% confidence interval of the laboratory’s historical negative control data.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Precipitation: no

RANGE-FINDING/SCREENING STUDIES: A pre-test was performed in order to determine the toxicity of the test item. In addition the pH and osmolarity were measured. The general culturing and experimental conditions in this pre-test were the same as described below for the mutagenicity experiment.

In this pre-test approximately 1.5 million cells were seeded in 25 cm² flasks 24 hours prior to treatment. After approximately 24 hours the test item was added and the treatment proceeded for 4 hours (duplicate cultures per concentration level). Immediately after treatment the test item was removed by rinsing with PBS. Subsequently, the cells were trypsinized and suspended in complete culture medium. After an appropriate dilution the cell density was determined with a cell counter. Toxicity of the test item is evident as a reduction of the cell density compared to a corresponding solvent control. A cell density of approximately 1.5 million cells in 25 cm² flasks is about the same as approximately 10 million cells seeded in 175 cm² bottles 24 hours prior to treatment with the main experiment.

HISTORICAL CONTROL DATA : see Table 1

Table 2. Summary of results

				relative	relative	rel. adjusted	mutant	95%
	conc.	P/	S9	cloning	cell	cloning	colonies/	confidence
	µg/mL	PS	mix	efficiency I	density	efficiency I	10E6 cells	interval
				%	%	%
Main Experiment / 4 h treatment				mean values of culture I and II
Solvent control with DMSO		-	-	100.0	100.0	100.0	11.1	1.7 - 30.2
Positive control (EMS)	300.0	-	-	82.3	83.8	71.0	269.3	1.7 - 30.2
Test item	23.8	-	-	87.1	96.3	85.6	#	#
Test item	47.6	-	-	98.5	93.3	91.7	#	#
Test item	95.1	-	-	98.6	94.2	93.3	20.0	1.7 - 30.2
Test item	190.3	-	-	95.3	91.0	87.4	16.6	1.7 - 30.2
Test item	380.5	-	-	91.4	79.5	72.8	15.7	1.7 - 30.2
Test item	761.0	-	-	95.1	86.0	82.3	19.4	1.7 - 30.2
Test item	1522.0	-	-	95.8	81.7	78.6	14.7	1.7 - 30.2
Solvent control with DMSO		-	+	100.0	100.0	100.0	15.0	2.0 - 29.4
Positive control (DMBA)	2.3	-	+	87.3	95.6	83.8	168.5	2.0 - 29.4
Test item	23.8	-	+	100.3	96.8	97.0	#	#
Test item	47.6	-	+	97.3	96.2	93.3	#	#
Test item	95.1	-	+	101.9	85.2	87.2	14.8	2.0 - 29.4
Test item	190.3	-	+	99.7	95.4	95.2	16.8	2.0 - 29.4
Test item	380.5	-	+	99.9	80.4	80.3	9.4	2.0 - 29.4
Test item	761.0	-	+	94.8	86.3	81.1	14.7	2.0 - 29.4
Test item	1522.0	-	+	93.4	71.4	65.9	16.4	2.0 - 29.4

P/PS = precipatation/phase separation

#   culture was not continued as a minimum of only four analyzable concentrations arerequired

Conclusions:

The test item was tested for its potential to induce reverse mutations at the HPRT locus in V79 Chinese hamster lung fibroblasts according to OECD 476. Cells were treated with the test material with and without meatbolic activation at concentrations of up to 1522 µg/mL. Based on the results of the conducted study the test item did not exhibit mutagenic properties in mammalian cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

In the absence of any evidence for species specific effects or modes of action (beside changes in the forestomach) the effects observed in animals and the absence of effects are regarded as relevant for humans.

Additional information

Reliable in vitro genotoxicity studies available for p-anisic acid.

- Gene mutation in bacteria:

Key study: A GLP guideline study according to OECD guideline 471 is available for p-anisic acid (Schulz, 2017). Salmonella typhimurium strains TA 1535, TA 1537, TA98, TA100 as well as the Escherichia coli strain WP2 uvrA were treated with the test material for 48 h using the Ames plate incorporation and the preincubation method at concentrations ranging from 10 to 5000 µg/plate, in triplicate, both with and without metabolic activation. Phenobarbital/β-naphthoflavone induced rat liver S9 was used as the metabolic activation system (10% liver S9 in standard co-factors). The test material was dissolved in the solvent dimethylsulfoxide (DMSO). The negative control, in both the plate incorporation and the pre-incubation studies, was a solvent control. The positive control agents used in this assay were sodium azide (TA 1532 and TA 100), methylmethanesulfonate (WP2 uvrA), 4-nitro-1,2-phenylene diamine (TA1537 and TA98) for the incubations without S9, and 2-aminoanthracene for the incubations with S9 for all strains and all incubation methods. Colonies were counted with an automatic counter after gross evaluation of the background growth. 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. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in strain WP2 uvrA with and without metabolic activation in both experiments from 2500 to 5000 μg/plate. In the Salmonella typhimurium strains no cytotoxic toxic effects were observed in both experiments neither with nor without metabolic activation. The background revertant levels reported are in line with both historical data from the laboratory. The incidence of positive control induced revertants is also consistent with historical data. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. Neither the plate incorporation nor the preincubation trials produced any indication of mutagenicity in any bacterial strain, either with or without metabolic activation.

Accordingly, the test material was evaluated as not mutagenic under the conditions of this test.

A supporting study performed equivalent or similar to OECD guideline 471 is available with p-anisic acid (Marquardt, 1994), due to insufficient documentation the reliability of the study is not assignable (RL 4). In this study the substance was tested for mutagenicity with Salmonella typhimurium strains TA 98, TA100, TA 1535, TA 1537 and TA 1538. However, no strain to detect cross-linking mutagens (TA 102 / E.coli) was tested. Testing was conducted in the absence and in the presence of a metabolising system derived from rat liver homogenate and positive and vehicle controls confirmed the validity of the assay. In a cytotoxicity test doses ranging from 10 to 2000 µg/plate were used. The test substance proved to be non-toxic to the bacterial strains but precipitation was observed at 2000 µg/plate. Accordingly 1000 µg/plate was chosen as top dose level for the mutagenicity study. Neither in the absence nor in the presence of metabolic activation the test compound showed a dose dependent increase in the number of revertants in any of the bacterial strains. Summarizing, it can be stated also in this test that p-anisic acid was not mutagenic in bacteria either with or without exogenous metabolic activation.

 

- Cytogenicity/ micronucleus test in mammalian cells

 A GLP guideline study performed according to OECD guideline 487 is available for p-anisic acid (Naumann, 2018). The study was performed to investigate the potential of the test material to induce micronucleated cells in cultured peripheral human lymphocytes. Cells were treated with the test material both with and without the addition of a metabolising system derived from rat liver homogenate. The S9 fraction used for the metabolic activation was prepared from livers of male rats induced with phenobarbital/β-naphthoflavone. The test material was dissolved in the solvent dimethylsulfoxide (DMSO). In a pre-test, concentrations of the test material ranged from 9.9 to 1522 µg/mL with and without S9 mix. At the end of the 4-hour incubation period, the medium with the test item was replaced with fresh medium and cells were further incubated for 36 h before cytochalasin B (4 μg/mL) was added to each flask 20 hours prior to the end of the incubation period. Then cells were harvested after a total time of 40 hours. There was no effect on osmolality or pH of the culture medium and no precipitation was noted. In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied and evaluated concentration.Therefore, this preliminary test was designated Experiment I, since the cultures fulfilled the acceptability criteria and appropriate concentrations could be selected for cytogenetic evaluation.

The concentrations in Experiment II ranged from 162 to 1522 µg/mL (20-hour exposure without S9). After exposure to the test item for 20 hours the cells were incubated with cytochalasin B (4 μg/mL) for further 20 hours. The positive controls used were mitomycin C and demecolcine in the absence and cyclophosphamide in the presence of metabolic activation. Duplicate cultures were tested for every concentration of test substance and for the negative and positive controls. From each culture, the cytogenetic damage was determined by counting 1000 binucleated cells per culture and determining the percentage of cells with micronucei. To determine a cytotoxic effect the cytokinesis-block proliferation index (CBPI) was determined in 500 cells per culture and cytotoxicity was described as % cytostasis. In Experiment II in the absence of S9 mix after continuous treatment, moderate cytotoxicity of 43.4% cytostasis was observed at the highest applied and evaluated concentration. In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying micronuclei was observed. The positive controls produced distinct increases in cells with micronuclei.

The test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. 

- Gene mutation in mammalian cells:

A GLP guideline study performed according to OECD guideline 476 is available for p-anisic acid (Sokolowski, 2018). The study was performed to investigate the ability of the test material to induce reverse mutations at the HPRT locus in V79 Chinese hamster lung fibroblasts. Cells were treated with the test material both with and without the addition of a rat liver homogenate metabolising system (S9 in standard co-factors). The S9 used for metabolic activation in this study was prepared from the livers of male rats induced by administration of phenobarbital/β-naphthoflavone. The test material was suspended in the solvent dimethylsulfoxide. In a preliminary cytotoxicity test treatment concentrations of the test material of up to 1522 µg/mL were applied followed by a 4 h-incubation. After treatment the test item was removed by rinsing with PBS. Subsequently, the cells were trypsinized and suspended in complete culture medium. After an appropriate dilution the cell density was determined with a cell counter. Osmolality and pH in the test medium did not change at any concentration and no precipitation was noted. Accordingly, 1522 µg/mL (10 mM) was selected as the maximum concentration for the main test.  Cells were treated with the test substance for 4 h, and incubated for 6 days for cytotoxicity determination. For mutagenicity cells were additionally incubated with selective medium containing 6-thioguanidine for 6 to 8 days before colonies were counted. The positive control agents were ethylmethanesulfonate (EMS) at a final concentration of 300 µg/mL in the absence of metabolic activation and dimethylbenzanthracene (DMBA) in DMSO at a final concentration of 2.3 µg/mL in the presence of metabolic activation.

The positive control substances produced clear biologically and statistically significant increases in mutation frequency. For the test substance, there was no increase in mutation frequency related to dose, and all findings were within the historical control frequency, either with or without metabolic activation. Accordingly, the test substance was evaluated as not mutagenic in mammalian cells under the conditions of this test.

Justification for classification or non-classification

The available data on genetic toxicity do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.