Sodium anisate

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

Read-across from p-anisic acid (CAS 100-09-4)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Refer to analogue justification provided in IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

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 read across approach is justified in the analogue justification. The target and source substances are considered unlikely to differ in their bacterial mutagenicity potential. A bacterial reverse mutation assays has been performed with the source substance p-anisic acid (CAS 100-09-4) 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, in the presence and absence of metabolic activation. All results obtained were negative, i.e. no gene mutation in bacteria was observed. Therefore, no mutagenic potential in bacteria is expected for the target substance sodium anisate.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Refer to analogue justification provided in IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

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 read across approach is justified in the analogue justification. The target and source substances are considered unlikely to differ in their genotoxic potential. In an in vitro mammalian cell gene mutation test using the HPRT locus performed according to OECD guideline 476 with the source substance p-anisic acid (CAS 100-09-4) no mutagenicity was found in V79 Chinese hamster lung fibroblasts. Therefore, no mutagenic potential in mammalian cells is expected for the target substance sodium anisate.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Refer to analogue justification provided in IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

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 applicable

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 read across approach is justified in the analogue justification. The target and source substances are considered unlikely to differ in their genotoxic potential. In an in vitro mammalian cell cytogenicity/micronucleus test according to OECD guideline 487 with the source substance p-anisic acid (CAS 100-09-4) no clastogenic potential was identified in cultured human peripheral lymphocytes. Therefore, no clastogenic potential in mammalian cells is expected for the target substance sodium anisate.

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

Justification for read-across

There are no experimental data available regarding the genotoxic potential of sodium anisate (CAS 536-45-8). Thus, read-across from an appropriate surrogate substance (p-anisic acid, CAS 100-09-4) is conducted in accordance with Regulation (EC) No 1907/2006, Annex XI, 1.5 in order to fulfil the standard information requirements defined in Regulation (EC) No 1907/2006, Annex VII and VIII, 8.1. Common functional groups and structural similarities of the source and target substance are the basis of read-across. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).

- 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.

- 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 these 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 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 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

Based on read-across the data on genetic toxicity as available from a source source substance do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.