4-acetyl-2-methylbenzoic 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:

Experimental start: 5 December 2017, experimental completion: 22 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-acetyl-2-methylbenzoic acid

EC Number:

856-079-4

Cas Number:

55860-35-0

Molecular formula:

C10H10O3

IUPAC Name:

4-acetyl-2-methylbenzoic acid

Test material form:

solid: particulate/powder

Method

Target gene:

Histidine locus (Salmonella typhimurium), genes for tryptophan biosynthesis (Escherichia coli)

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

A mammalian metabolic system was used (S9-mix). Phenobarbital/ß-naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Wistar rats (RjHan:WI; weight approx. 220 – 320 g, Janvier Labs, 53941 Saint-Berthevin Cedex, France) induced by peroral administration of 80 mg/kg b.w. phenobarbital (Sigma-Aldrich Chemie GmbH, 82024 Taufkirchen, Germany) and by peroral administrations of ß-naphthoflavone (Acros Organics, 2440 Geel, Belgium) each, on three consecutive days. The livers were prepared 24 hours after the last treatment. The S9 fractions were produced by dilution of the liver homogenate with a KCl solution (1+3 parts) followed by centrifugation at 9000 g. Aliquots of the supernatant were frozen and stored in ampoules at –80 °C. Small numbers of the ampoules can be kept at –20 °C for up to one week. Each batch of S9 mix was routinely tested with 2-aminoanthracene as well as benzo[a]pyrene.
The protein concentration in the S9 preparation was 32.6 mg/mL (lot no. 010617E) in the pre-experiment / Experiment I and 30.4 mg/mL (lot no. 261017K) in Experiment II.

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate
Since no relevant cytotoxic effects were observed 5000 µg/plate was chosen as the maximal concentration in experiment II.

Vehicle / solvent:

Dimethylsulfoxide (DMSO), Fisher Leics LE11 5RG, batch 1684307, purity 99.98%

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other:

Remarks:

NaN3: TA1535, TA100 (without S9-mix), 4-NOPD: TA1537, TA98 (without S9-mix), MMS: WP2 uvrA (pKM101), WP2 (pKM101) (without S9-mix), 2-AA: all strains (with S9-mix)

Details on test system and experimental conditions:

For each strain and dose level including the controls, three plates were used.
The following materials were mixed in a test tube and poured onto the selective agar plates:
- 100 µL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control),
- 500 µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation, 7 parts of the 100 mM sodium-ortho-phosphate-buffer pH 7.4 with 3 parts of KCl solution 0.15 M),
- 100 µL bacteria suspension (cf. test system, pre-culture of the strains),
- 2000 µL overlay agar

In the pre-incubation assay 100 µL test solution, 500 µL S9 mix / S9 mix substitution buffer and 100 µL bacterial suspensions were mixed in a test tube and shaken at 37° C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45° C) was added to each tube. The mixture was poured on selective agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37° C in the dark .
The test substance was assessed for its potential to induce gene mutations in the plate incorporation test (Experiment I) and the pre-incubation test (Experiment II) using S. typhimurium strains TA1535, TA1537, TA98, and TA100, and the E. coli strains WP2 uvrA pKM101 and WP2 pKM101.
In the pre-experiment the concentration range tested was 3 - 5000 µg/plate. The pre-experiment is reported as Experiment I since the acceptability criteria of the assay were met. Since no relevant cytotoxic effects were observed 5000 µg/plate was chosen as the maximal concentration in experiment II.
The assay was performed with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The concentration range included two logarithmic decades.

Rationale for test conditions:

The test conditions were selected in accordance with the test guideline.

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 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:

Not applicable

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No precipitation of the test item occurred up to the highest investigated dose. The plates incubated with the test item showed reduced background growth at 5000 µg/plate in experiment I. No cytotoxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation. No substantial increase in revertant colony numbers of any of the six tester strains was observed following treatment with the substance at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
In Experiment I, the data in the negative and solvent control of strain TA 100 without metabolic activation were slightly above the current historical control range however were considered acceptable for inclusion to the historical control data. Two out of three values each were weakly increased in these control groups. Besides, no relevant effect on the numbers of revertant colonies was observed in this experiment. Thus, this observation has no detrimental impact on the outcome and validity of this study.

Any other information on results incl. tables

Table 1: Summary of results of experiment I

			Revertant colony counts (mean ± SD)
S9-mix	Test group	Concentration (μg/plate)	TA1535	±	TA1537	±	TA98	±	TA100	±	WP2 pKM101	±	WP2 uvrA pKM101	±
-	DMSO		13	1	11	1	31	5	214	23	197	11	334	21
-	Untreated		9	2	7	1	37	9	235	37	240	19	376	21
-	Substance	3	10	1	10	2	34	12	197	13	225	25	364	6
-		10	13	3	11	3	36	10	199	6	211	19	351	22
-		33	12	4	11	2	23	4	202	29	201	21	304	5
-		100	10	1	8	2	26	5	201	26	215	24	316	18
-		333	14	2	8	1	27	5	173	23	193	18	349	10
-		1000	10	2	10	2	28	3	184	28	193	27	309	11
-		2500	9	2	8	3	29	4	176	12	161	2	276	12
-		5000	11	3	6 R	1	39 R	3	165	37	110 R	20	250 R	24
-	NaN3	10	1251	32					1944	199
-	4-NOPD	10					329	36
-	4-NOPD	50			141	6
-	MMS	2.0									3474	223	3610	285
+	DMSO		13	4	15	1	44	4	168	2	221	9	369	32
+	Untreated		9	1	15	1	43	5	196	3	259	8	404	36
+	Substance	3	13	2	13	3	47	1	154	11	237	23	421	18
+		10	10	1	19	3	38	2	183	6	221	17	409	3
+		33	14	4	15	1	33	10	168	18	208	23	364	24
+		100	12	5	13	3	35	2	179	5	172	34	344	35
+		333	15	3	15	4	39	9	163	8	214	8	411	23
+		1000	14	6	15	3	37	11	179	22	200	39	414	30
+		2500	9	2	15	2	39	2	159	30	189	33	342	32
+		5000	11 R	1	12 R	3	32 R	4	168 R	10	121 R	15	251 R	22
+	2-AA	2.5	498	18	113	7	3406	251	3834	106
+	2-AA	10.0									1218	75	2282	344

NaN3 = sodium azide, 2-AA = 2-aminoanthracene, 4-NOPD = 4-nitro-o-phenylene-diamine, MMS = methylmethanesulfonate, R = reduced background growth

Table 2: Summary of results of experiment II

			Revertant colony counts (mean ± SD)
S9-mix	Test group	Concentration (μg/plate)	TA1535	±	TA1537	±	TA98	±	TA100	±	WP2 pKM101	±	WP2 uvrA pKM101	±
-	DMSO		9	2	9	3	23	4	142	7	172	9	312	34
-	Untreated		12	3	6	1	19	4	192	9	235	13	342	19
-	Substance	33	9	33	11	1	19	4	141	9	197	15	313	18
-		100	10	1	10	2	16	2	149	16	183	7	298	20
-		333	11	2	11	1	25	5	146	11	163	20	279	8
-		1000	7	2	8	2	22	1	137	5	175	19	314	43
-		2500	10	4	8	2	19	4	150	26	147	6	294	10
-		5000	8	3	8	3	20	5	157	14	127	26	248	15
-	NaN3	10	1156	51					2031	205
-	4-NOPD	10					370	15
-	4-NOPD	50			148	8
-	MMS	2.0									3272	471	2767	689
+	DMSO		8	2	10	3	29	4	118	11	211	15	357	20
+	Untreated		12	3	8	1	31	5	198	8	252	38	380	13
+	Substance	33	8	1	8	3	33	4	128	8	213	13	388	32
+		100	9	3	7	1	29	3	126	12	225	23	331	10
+		333	10	3	10	4	26	3	134	3	178	38	340	13
+		1000	10	2	11	4	29	3	138	21	229	19	362	25
+		2500	8	2	8	1	31	1	125	19	185	8	311	43
+		5000	7	2	11	4	27	8	151	10	123	31	278	53
+	2-AA	2.5	374	44	117	7	2770	79	4570	732
+	2-AA	10.0									1182	27	1809	135

NaN3 = sodium azide, 2-AA = 2-aminoanthracene, 4-NOPD = 4-nitro-o-phenylene-diamine, MMS = methylmethanesulfonate

Applicant's summary and conclusion

Conclusions:

The substance was considered to be non-mutagenic in the bacterial reverse mutation assay with S. typhimurium and E. coli.

Executive summary:

A study on the mutagenicity of the substance to bacteria was performed under GLP to OECD TG 471. The substance was tested in the plate incorporation test (Experiment I) and the pre-incubation test (Experiment II) using the Salmonella typhimurium (S. typhimurium) strains TA1535, TA1537, TA98, and TA100, and the Escherichia coli (E. coli) strains WP2 uvrA pKM101 and WP2 pKM101. In Experiment I the plates incubated with the substance showed reduced background growth in all strains at the highest concentration analysed (5000 μg/plate) in the presence and absence of metabolic activation (S9 mix) with the exception of TA 1535 and TA 100 in the absence of metabolic activation. In Experiment II normal background growth was observed in all strains with and without metabolic activation. No cytotoxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains with and without metabolic activation. No relevant increase in revertant colony numbers of any of the six tester strains was observed following treatment with the substance at any concentration level, neither in the presence or absence of metabolic activation. There was also no observed tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls, which showed a distinct increase of induced revertant colonies. The substance was considered to be non-mutagenic in the bacterial reverse mutation test.