Ethyl 4-dimethylaminobenzoate

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:

12 January 2006 to 02 February 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

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:

other: Commission Directive 2000/32/EC L1362000 Annex 4D

Version / remarks:

2000

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:

Ethyl 4-dimethylaminobenzoate

EC Number:

233-634-3

EC Name:

Ethyl 4-dimethylaminobenzoate

Cas Number:

10287-53-3

Molecular formula:

C11H15NO2

IUPAC Name:

ethyl 4-(dimethylamino)benzoate

Test material form:

solid

Method

Target gene:

- S. typhimurium: Histidine locus
- E. coli: Tryptophan locus

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Obtained from Trinova Biochem GmbH (35394 Gießen, Germany)
- Method of culture: The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO in liquid nitrogen.

MEDIA USED
- Type and identity of media for pre-cultures: From the thawed ampoules of the strains, 0.5 mL suspension was transferred into 250 mL Erlenmeyer flasks containing 20 mL nutrient medium. A solution of 20 ML ampicillin (25 µg/mL) was added to the strains TA 98 and TA 100. This nutrient medium contains per litre: 8 g Merck Nutrient Broth and 5 g NaCl. The bacterial cultures were incubated in a shaking water bath for 4 hours at 37 °C.
- Properly maintained: Yes. Regular checking of the properties of the strains regarding the membrane permeability and ampicillin resistance as well as spontaneous mutation rates is performed.

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Obtained from Trinova Biochem GmbH (35394 Gießen, Germany)
- Method of culture: The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO in liquid nitrogen.

MEDIA USED
- Type and identity of media for pre-cultures: From the thawed ampoules of the strains, 0.5 mL suspension was transferred into 250 mL Erlenmeyer flasks containing 20 mL nutrient medium. This nutrient medium contains per litre: 8 g Merck Nutrient Broth and 5 g NaCl. The bacterial cultures were incubated in a shaking water bath for 4 hours at 37 °C.
- Properly maintained: Yes. Regular checking of the properties of the strains regarding the membrane permeability as well as spontaneous mutation rates is performed.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

- Pre-Experiment /Experiment I: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
- Experiment II: 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

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

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

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

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) (experiment I) and pre-incubation (experiment II)
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 or positive control), 500 µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation), 100 µL bacteria suspension and 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 suspension 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.

NUMBER OF REPLICATIONS: The study was performed in triplicate

DETERMINATION OF CYTOTOXICITY
- Method: Assessment of the background lawn
The colonies were counted using the Petri Viewer Mk2 (Perceptive Instruments Ltd, Suffolk CB 7BN, UK) with the software program Ames Study Manager. The counter was connected to an IBM AT compatible PC with printer to print out the individual values and the means from the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates. Due to precipitation of the test material and reduced background growth, the revertant colonies were partly counted manually.

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- Regular background growth in the negative and solvent control
- The spontaneous reversion rates in the negative and solvent control are in the range of historical data
- The positive control substances should produce a significant increase in mutant colony frequencies

EVALUATION OF RESULTS
A test material 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:

A statistical analysis of the data was not carried out

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:

cytotoxicity

Remarks:

5000 µg/plate +S9 mix

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:

cytotoxicity

Remarks:

2500 and 5000 µg/plate -S9mix; 1000 to 5000 µg/plate +S9mix

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:

cytotoxicity

Remarks:

5000 µg/plate -S9mix; 2500 and 5000 µg/plate +S9mix

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:

cytotoxicity

Remarks:

5000 µg/plate -S9mix; 1000 to 5000 µg/plate +S9mix

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 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

1000 to 5000 µg/plate +S9mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Remarks:

The historical control range was slightly exceeded in the untreated WP2 uvrA with/without activation in experiment I. This deviation is based on biologically irrelevant fluctuations in the number of colonies and has no impact on the outcome of the study.

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No visible reduction of the background growth was observed with or without metabolic activation in experiment I. In experiment II, reduced background growth was observed at higher concentrations with and without metabolic activation in all strains used.
Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5) were observed at the following concentrations (µg/plate):
TA 1535: 5000 µg/plate with S9 mix (Experiment II)
TA 1537: 5000 µg/plate with and without S9 mix (Experiment I); 2500 and 5000 µg/plate without S9 mix, 1000 - 5000 µg/plate with S9 mix (Experiment II)
TA 98: 5000 µg/plate with and without S9 mix (Experiment I); 2500, 5000 µg/plate with S9 mix (Experiment II)
TA 100: 5000 µg/plate without S9 mix, 1000 - 5000 µg/plate with S9 mix (Experiment II)
WP2 uvrA: 5000 µg/plate with S9 mix (Experiment I); 1000 - 5000 µg/plate with S9 mix (Experiment II).
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test material at any dose 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.
Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.
The laboratory´s historical control range was slightly exceeded in the untreated control of strain WP2 uvrA with and without metabolic activation in experiment I. This deviation is judged to be based on biologically irrelevant fluctuations in the number of colonies and has no impact on the outcome of the study.
Under the experimental conditions reported, the test material did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Any other information on results incl. tables

Table 1: Summary of Pre-Experiment and Experiment I

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
-	DMSO Untreated 3 10 33 100 333 1000 2500 5000	114 121 110 123 112 113 95 112 101P 85P	21 16 19 19 23 19 16 22 23P 20P	47 50 56 51 55 61 45 41 39P 30P	27 28 27 25 30 26 28 29 28P 11P	13 14 8 12 11 13 10 6 7P 6P
+	DMSO Untreated 3 10 33 100 333 1000 2500 5000	128 123 115 125 131 130 111 110 98P 74P	26 21 25 18 25 26 17 24 20P 16P	63 68 69 64 64 69 61 45 38P 13P	30 29 28 31 29 28 25 28 26P 8P	15 16 19 15 12 13 19 20 18P 5P
Positive Controls
-	Name	NaN3	NaN3	MMS	4-NOPD	4-NOPD
	Concentration (µg/plate)	10	10	4.0 µL	10	50
	Mean no. colonies/plate	2013	1236	1435	357	109
+	Name	2AA	2AA	2AA	2AA	2AA
	Concentration (µg/plate)	2.5	2.5	10	2.5	2.5
	Mean no. colonies/plate	1749	286	294	1364	134

P = Precipitate

NaN3 = sodium azide

4-NOPD = 4-nitro-o-phenylene-diamine

MMS = methyl methane sulfonate

2AA = 2-aminoanthracene

Table 2: Summary of Experiment II

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
-	DMSO Untreated 10 33 100 333 1000 2500 5000	115 164 139 147 129 104 117 61P 44P	16 17 19 18 19 15 18 10P 9P	57 66 50 53 53 49 47 31P 28P	24 24 28 26 27 18 16 16P 11P	9 13 8 7 8 5 4 2P 2P
+	DMSO Untreated 10 33 100 333 1000 2500 5000	163 198 172 158 155 129 62 50P 53P	21 28 30 26 29 15 12 10P 9P	68 72 77 69 82 64 28 18P 10P	38 36 29 29 33 34 26 12P 12P	12 8 12 9 14 16 3 3P 2P
Positive Controls
-	Name	NaN3	NaN3	MMS	4-NOPD	4-NOPD
	Concentration (µg/plate)	10	10	4.0 µL	10	50
	Mean no. colonies/plate	1865	1347	416	390	95
+	Name	2AA	2AA	2AA	2AA	2AA
	Concentration (µg/plate)	2.5	2.5	10	2.5	2.5
	Mean no. colonies/plate	3476	521	272	4953	143

P = Precipitate

NaN3 = sodium azide

4-NOPD = 4-nitro-o-phenylene-diamine

MMS = methyl methane sulfonate

2AA = 2-aminoanthracene

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the test material was determined to be non-mutagenic in both the presence and absence of metabolic activation.

Executive summary:

The genetic toxicity of the test material was investigated in accordance with the standardised guidelines OECD 471 and Commission Directive 2000/32/EC L1362000 Annex 4D under GLP conditions using the bacterial reverse mutation assay.

The study was performed to investigate the potential of the test material to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test material was tested at the following concentrations: Pre-Experiment /Experiment I: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate; Experiment II: 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate.

No visible reduction of the background growth was observed with and without metabolic activation in experiment I. In experiment II, reduced background growth was observed at higher concentrations with and without metabolic activation in all strains used. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5) were observed at 5000 µg/plate in strains TA 1537 and TA 98 with and without metabolic activation and in strain WP2 uvrA with metabolic activation in experiment I. In experiment II, toxic effects were observed at higher concentrations with metabolic activation in all strains and without metabolic activation in strains TA 1537 and TA 100.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test material at any dose 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.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. Appropriate untreated and solvent controls were also used and the study was considered to be valid.

During the described mutagenicity test and under the experimental conditions reported, the test material did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Under the conditions of this study, the test material is considered to be non-mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay.