3-ethoxy-4-hydroxybenzaldehyde

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:

from 2017-02-02 to 2017-04-05

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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:

3-ethoxy-4-hydroxybenzaldehyde

EC Number:

204-464-7

EC Name:

3-ethoxy-4-hydroxybenzaldehyde

Cas Number:

121-32-4

Molecular formula:

C9H10O3

IUPAC Name:

3-ethoxy-4-hydroxybenzaldehyde

Test material form:

solid: particulate/powder

Details on test material:

see confidential details

Method

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:

S9-mix

Test concentrations with justification for top dose:

Dose range finding test: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate with and without S9-mix.
Experiment 1 and 2: 52, 164, 512, 1600 and 5000 µg/plate with and without S9-mix.

Vehicle / solvent:

The vehicle of the test item was dimethyl sulfoxide (DMSO, Merck, Darmstadt, Germany).

Controls

Untreated negative controls:

yes

Remarks:

dimethyl sulfoxide (DMSO, Merck, Darmstadt, Germany)

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulfoxide (DMSO, Merck, Darmstadt, Germany).

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191 and 2-aminoanthracene (2AA)

Details on test system and experimental conditions:

Test system: Salmonella typhimurium bacteria and Escherichia coli bacteria

Source: Trinova Biochem GmbH, Germany [Master culture from Dr. Bruce N. Ames (TA1535: 2006, TA1537: 2016, TA98: 2015, TA100: 2015; and Master culture from The National Collections of Industrial and Marine Bacteria, Aberdeen, UK (WP2uvrA:
2008)]

Strain Histidine mutation Mutation type
TA1537 hisC3076 Frameshift
TA98 hisD3052/R-factor* Frameshift
TA1535 hisG46 Base-pair substitutions
TA100 hisG46/R-factor* Base-pair substitutions
*: R-factor = plasmid pKM101 (increases error-prone DNA repair)

Each tester strain contained the following additional mutations:
rfa : deep rough (defective lipopolysaccharide cellcoat)
gal : mutation in the galactose metabolism
chl : mutation in nitrate reductase
bio : defective biotin synthesis
uvrB : loss of the excision repair system (deletion of the ultraviolet-repair B gene)

The Salmonella typhimurium strains are regularly checked to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.

The Escherichia coli WP2uvrA strain detects base-pair substitutions. The strain lacks an excision repair system and is sensitive to agents such as UV. The sensitivity of the strain to a wide variety of mutagens has been enhanced by permeabilization of the strain using Tris-EDTA treatment. The strain is regularly checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants.

Stock cultures of the five strains were stored in liquid nitrogen (-196°C).

Cell culture
Preparation of bacterial cultures
Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37 ± 1°C, 150 rpm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (109 cells/ml). Freshly grown cultures of each strain were used for a test.

Agar plates
Agar plates (ø 9 cm) contained 25 ml glucose agar medium. Glucose agar medium contained per liter: 18 g purified agar (Oxoid LTD) in Vogel-Bonner Medium E, 20 g glucose (Fresenius Kabi, Bad Homburg, Germany). The agar plates for the test with the Salmonella typhimurium strains also contained 12.5 μg/plate biotin (Merck) and 15 μg/plate histidine (Sigma) and the agar plates for the test with the Escherichia coli strain contained 15 μg/plate tryptophan (Sigma).

Top agar
Milli-Q water containing 0.6% (w/v) bacteriological agar (Oxoid LTD) and 0.5% (w/v) sodium chloride (Merck) was heated to dissolve the agar. Samples of 3 ml top agar were transferred into 10 ml glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 ± 3°C.

Environmental conditions
All incubations were carried out in a controlled environment at a temperature of 37.0 ± 1.0°C (actual range 35.0 - 39.4°C). The temperature was continuously monitored throughout the experiment. Due to addition of plates (which were at room temperature) to the incubator or due to opening and closing the incubator door, temporary deviations from the temperature may occur. Based on laboratory historical data these deviations are considered not to affect the study integrity.
All the test were evaluated in triplicate.

Evaluation criteria:

Acceptability of the assay

A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Charles River Den Bosch.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

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

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:

In presence of S9 mix at the highest tested concentration (5000 μg/plate).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

In the absence of S9 mix at the highest tested concentration (5000 μg/plate).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

- Water solubility: the substance was soluble at all concentration tested
- Precipitation: no precipitation observed

RANGE-FINDING: yes
In the dose range finding study, the test item was initially tested up to concentrations of 5000 μg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The test item did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.


COMPARISON WITH HISTORICAL CONTROL DATA: yes
The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Any other information on results incl. tables

Table 1   
Dose-Range Finding Test: Mutagenic Response of Ethylvanillin in the Salmonella Typhimurium Reverse Mutation Assay and in the Escherichia Coli Reverse Mutation Assay

Direct plate assay

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with one Salmonella typhimurium and one Escherichia coli strain.
	TA100	WP2uvrA

 Without S9-mix

Positive control	933	±	57		1642	±	126
Solvent control	115	±	9		19	±	4
1.7	101	±	31		12	±	8
5.4	109	±	23		24	±	9
17	102	±	16		22	±	3
52	99	±	14		18	±	6
164	91	±	3		19	±	1
512	92	±	6		17	±	5
1600	80	±	10		17	±	8
5000	73	±	4	^n NP	10	±	6	^n NP

 

With S9-mix 

Positive control	1320	±	239		311	±	8
Solvent control	106	±	7		21	±	2
1.7	113	±	20		24	±	2
5.4	82	±	17		24	±	13
17	109	±	37		28	±	10
52	106	±	7		24	±	5
164	94	±	17		20	±	2
512	86	±	12		22	±	9
1600	79	±	5		20	±	3
5000	72	±	24	^n NP	18	±	6	^n NP

 

NP	No precipitate
n	Normal bacterial background lawn

 

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, it is concluded that Ethylvanillin is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

The objective of this study was to determine the potential of Ethylvanillin and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9). The study was performed according to international guidelines (OECD guideline No. 471 adopted 21 July 1997 and test method No. B13/14 of EC regulation No. 440/2008) and in compliance with the principles of Good Laboratory Practice.

The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay.

The test item was dissolved in dimethyl sulfoxide (DMSO). In the dose range finding study, the test item was initially tested up to concentrations of 5000 μg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The test item did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In the first mutation experiment, the test item was tested up to concentrations of 5000 μg/plate in the strains TA1535, TA1537 and TA98. The test item did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In the second mutation experiment, the test item was tested up to concentrations of 5000 μg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay. The test item did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in the tester strains TA98 and TA1537 in the absence and presence of S9-mix, respectively, at the highest tested concentration.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

In conclusion, based on the results of this study it is concluded that Ethylvanillin is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay in the presence or in the absence of S9-metabolic activation.