3-ethoxy-4-hydroxybenzaldehyde

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Two studies with reliability 1 have been selected as key study:

- Ames test (OECD 471 guideline study, Kr.1): negative in Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and in Escherichia coli (E. coli: WP2uvrA) in the presence or absence of an exogenous mammalian metabolic activation system (S9)

- In vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells (OECD 490 guideline study, Kr.1): positive in the presence or absence of an exogenous mammalian metabolic activation system (S9) at the highest tested doses which are also cytotoxic.

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:

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

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:

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).

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.

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

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

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

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

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.

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

 

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 19 January 2017 to 22 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

Thymidine kinase gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: American Type Culture Collection, (ATCC, Manassas, USA) (2001).
Stock cultures of the cells were stored in liquid nitrogen (-196°C). The cultures were checked for mycoplasma contamination. Cell density was kept below 1 x 106 cells/ml.

MEDIA USED
- Type and identity of media including CO2 concentration:
Horse serum:
Horse serum (Life Technologies) was inactivated by incubation at 56°C for at least 30 minutes.
Basic medium:
RPMI 1640 Hepes buffered medium (Dutch modification) (Life Technologies) containing
penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively) (Life Technologies), 1 mM sodium pyruvate (Sigma, Zwijndrecht, The Netherlands) and 2 mM L-glutamin (Life Technologies).
Growth medium:
Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).
Exposure medium:
Cells were exposed to the test item in basic medium supplemented with 5% (v/v) heatinactivated horse serum (R5-medium).
Selective medium:
Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium) and 5 μg/ml trifluorothymidine (TFT) (Sigma).
Non-selective medium:
Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium).
Environmental conditions:
All incubations were carried out in a humid atmosphere (80 - 100%, actual range 35 – 94%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 33.7 – 37.4 °C).
- Properly maintained: not specified
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: not specified
- Periodically 'cleansed' against high spontaneous background:
Prior to dose range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in R10-medium containing 10-4 M hypoxanthine (Sigma), 2 x 10-7 M aminopterine (Fluka Chemie AG, Buchs, Switzerland) and 1.6 x 10-5 M thymidine (Sigma) (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10-medium containing hypoxanthine and thymidine only. After this period cells were returned to R10-medium for at least 1 day before starting the experiment.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate)

Test concentrations with justification for top dose:

Test concentrations:
Based on the results of the dose range finding test, the following dose range was selected for the mutagenicity test in the absence and presence of S9-mix: 100, 400, 600, 800, 1000, 1100, 1200, 1300, 1400, 1500 and 1600 μg/ml exposure medium.
The test item was tested in the absence and presence of S9-mix with a 3 hour treatment period.

Justification for top dose:
The highest doses that were tested gave a cell survival of approximately 10-20% and the survival in the lowest doses was approximately the same as the cell survival in the solvent control. Also some intermediate doses were tested.
In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test item concentration range of 63 to 1662 μg/ml in the absence and presence of S9-mix with a 3 hour treatment period.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: The solvent for the test item was DMSO (dimethyl sulfoxide) (Merck Darmstadt, Germany). The final concentration of the solvent in the exposure medium was 1% (v/v).
- Justification for choice of solvent/vehicle: solvent in which the test item was found soluble.

Untreated negative controls:

yes

Remarks:

= Solvent control

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: Per culture 8 x 106 cells (106 cells/ml for 3 hours treatment) or 6 x 106 cells (1.25 x 105 cells/ml for 24 hours treatment) were used.

DURATION
- Preincubation period: non
- Exposure duration: 3 hours or 24 hours (not done because positive results after 3 h of incubation)
- Expression time (cells in growth medium): For expression of the mutant phenotype, the remaining cells were cultured for 2 days after the treatment period.
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays): Selective medium: Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20) and 5 μg/ml trifluorothymidine (TFT) (Sigma).

SPINDLE INHIBITOR (cytogenetic assays): N/A

STAIN (for cytogenetic assays): N/A

NUMBER OF REPLICATIONS: N/A

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: N/A

NUMBER OF CELLS EVALUATED: For determination of the CEday2 the cell suspensions were diluted and seeded in wells of a 96-well dish. One cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective medium.
For determination of the mutation frequency (MF) a total number of 9.6 x 105 cells/concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 105 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection). The microtiter plates for CEday2 and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 2 hours, by adding 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma) to each well. The plates for the CE day2 and MF were scored with the naked eye or with the microscope.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): N/A

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: N/A

DETERMINATION OF CYTOTOXICITY
- Method: Parameters: Cloning efficiency; rRelative Total Growth; relative survival; Suspension Growth; Relative Suspension Growth.

OTHER EXAMINATIONS:
- Determination of polyploidy: N/A
- Determination of endoreplication: N/A
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): N/A

- OTHER:

Rationale for test conditions:

L5178Y mouse lymphoma cells are used because they are sensitive indicators of mutagenic activity of a broad range of chemical classes. The TK mutational system is able to detect base pair alterations, frame shift mutations and small deletions and clastogenic effect.
Cells deficient in thymidine kinase (TK), due to the forward mutation (TK+/- to TK-/-) are resistant to the cytotoxic effects of the pyrimidine analogue trifluorothymidine (TFT).
TK deficient cells cannot incorporate the analogue into its phosphorylated derivative (nucleotide); the nucleotides needed for cellular metabolism are obtained solely from de novo synthesis. In the presence of TK, TFT is converted into nucleotides, which is lethal to the cells. Thus, cells that are able to proliferate in culture medium containing TFT are mutated, either spontaneously or by the action of the test item, to a TK deficient phenotype. Furthermore, by applying the TFT-selection procedure it is possible to discriminate between two different classes of TFT-resistant mutants (small and large colonies). The large colonies are believed to be the result of mutants with single gene mutations (substitutions, deletions of base-pairs) affecting the TK gene. The small colonies are believed to be the result of chromosomal damage to the TK and adjacent genes.
A test article, which induces a positive response in this assay, is presumed to be a potential mammalian cell mutagen.

Evaluation criteria:

In addition to the criteria stated below, any increase of the mutation frequency should be evaluated for its biological relevance including comparison of the results with the historical control data range.
The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
- A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
- A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.

Statistics:

Not performed.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

Negative at non-cytotoxic doses (see more details in the fields below).

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at the highest dose.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

Negative at non-cytotoxic doses (see more details in the fields below)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at the highest doses.

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: The pH and osmolarity at a concentration of 1662 μg/ml were 7.071 and 0.448 Osm/kg respectively (compared to 7.282 and 0.459 Osm/kg in the solvent control) in the solubility test.
- Effects of osmolality: The pH and osmolarity at a concentration of 1662 μg/ml were 7.071 and 0.448 Osm/kg respectively (compared to 7.282 and 0.459 Osm/kg in the solvent control) in the solubility test.
- Evaporation from medium: Not assessed
- Water solubility: Not assessed
- Precipitation: Ethylvanillin did not precipitate in the exposure medium up to and including the concentration of 1662 μg/ml (= 10 mM). Since testing up to 0.01 M is recommended in the guidelines, this concentration was used as the highest test item concentration in the dose range finding test.
- Definition of acceptable cells for analysis: Not assessed
- Other confounding effects: N/A

RANGE-FINDING/SCREENING STUDIES:
In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test item concentration range of 63 to 1662 μg/ml in the absence and presence of S9-mix with a 3 hour treatment period.
In the absence of S9-mix, the relative suspension growth was 39% at the test item concentration of 1000 μg/ml compared to the relative suspension growth of the solvent control. No cell survival was observed at the test item concentration of 1662 μg/ml.
In the presence of S9-mix, the relative suspension growth was 34% at the test item concentration of 1000 μg/ml compared to the relative suspension growth of the solvent control. No cell survival was observed at the test item concentration of 1662 μg/ml.


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
- Negative (solvent/vehicle) historical control data: The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: cloning efficiency, relative survival, Relative Total Growth, Suspension Growth, Relative Suspension Growth.
- Other observations when applicable: The growth rate, as an indicator of optimally growing cultures, was calculated for the solvent control cultures.

ADDITIONAL INFORMATION ON MUTAGENECITY:
- In absence of S9, Ethylvanillin induced mutations only at the highest tested dose (1200 µg/L) that was also cytotoxic (RTG = 16%)
- In presence of S9, Ethylvanillin induced mutations only at the two highest doses (1000 and 1100 µg/L) that were also cytotoxic (RTG = 16 and 11% respectively).

  Table : Mutation experiment : Cytotoxic and mutagenic response of Ethylvanillin in the mouse lymphoma L5178Y test system

Dose (µg/ml)	RSG (%)	CE day 2 (%)	RCE (%)	RTG (%)	Mutation frequency per 10^6survivors Total               (small            large)
without metabolic activation 3 hours treatment
SC1 SC2 100 400 600 800 1000 1100 1200 1300^1) MMS	100   88 85 78 73 53 57 22 4 42	108 93 93 94 118 111 102 91 70 72 60	100   92 94 117 111 102 91 70 76 60	100   81 80 91 81 54 52 16 3 25	62 71 61 75 63 75 81 122 210 394 479	(45 (59 (44 (60 (37 (60 (71 (94 (166 (306 (265	15) 10) 15) 13) 24) 13) 8) 23) 35) 55) 180)
With metabolic activation 3 hours treatment
SC1 SC2 100 400 600 800 1000 1100 CP	100   97 92 85 73 23 14 55	91 90 83 97 70 116 63 74 34	100   91 107 78 128 70 81 37	100   89 98 66 94 16 11 20	77 61 68 60 93 87 241 315 1225	(55 (51 (54 (41 (80 (71 (193 (286 (1035	20) 9) 13) 18) 12) 14) 37) 19) 133)

 

Note: all calculations were made without rounding off

RSG = Relative Suspension Growth; CE = Cloning Efficiency; RCE = Relative Cloning Efficiency; RTG = Relative Total Growth; SC = Solvent control = DMSO; MMS = Methylmethanesulfonate; CP: Cyclophosphamide

1)     RTG below 10%, not used for mutagenicity determination

 

 

Conclusions:

Ethylvanillin is mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report but only at the highest tested doses which were also cytotoxic.

Executive summary:

The objective of this study was to evaluate the mutagenic activity of Ethylvanillin in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells.

This report describes the effects of Ethylvanillin on the induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells. The test was performed with a 3 hour treatment period in the absence and presence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone). The study procedures described in this report were based on the most recent OECD guideline (no 490, adopted 29 July 2016). The test item was dissolved in dimethyl sulfoxide.

In the mutation experiment, the test item was tested up to concentrations of 1200 and 1100 μg/ml in the absence and presence of S9-mix, respectively. The treatment period was 3 hours. Relative total growth (RTG) was 16 and 11% in the absence and presence of S9-mix, respectively. Above the dose level of 1200 μg/ml the RTG was below the acceptable limit of 10% in the absence of S9-mix. The test item did not precipitate in the culture medium up to the concentration of 1200 μg/ml.

The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database.

Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, Ethylvanillin induced an up to 3.2-fold dose related increase in the mutation frequency at the highest tested concentrations (1200 μg/ml) which is also cytotoxic. The increase was above the 95% control limits of the distribution of the historical negative control database and also above the GEF + MF(controls) (193 per 106 survivors).

In the presence of S9-mix, Ethylvanillin induced an up to 4.6-fold dose related increase in the mutation frequency at the two highest tested concentrations (1000 and 1100 μg/ml) which are also cytotoxic. The increase was above the 95% control limits of the distribution of the historical negative control database and also above the GEF + MF(controls) (195 per 106 survivors).

Although the increases in the mutation frequency at the TK locus were only observed at toxic dose levels, RTG between 10- 20%, the mutation frequencies at these concentrations were above the GEF and the results are considered to be biological relevance and the test item is mutagenic at toxic dose levels. In addition, a statistical significant dose related trend (p<0.001) was observed.

It is concluded that Ethylvanillin is mutagenic in the mouse lymphoma L5178Y test system at the hihest tested concentrations that are also cytotoxic and under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Three in vivo studies (mammalian erythrocyte micronuleus) performed on mice with reliability 2 are available (NTP, 1994, Wild 1983, Drebs laboratory, 1979). These three studies showed that Ethylvanillin do not induce chromosome aberration.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Study well described, and meets generally the strandard guidelines.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Study done according to NTP standard protocols

GLP compliance:

not specified

Type of assay:

micronucleus assay

Species:

mouse

Strain:

B6C3F1

Sex:

male

Details on test animals or test system and environmental conditions:

No more information on animals. No data on environmental conditions.

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: no data
- Concentration of test material in vehicle: no data

Duration of treatment / exposure:

72 hours

Frequency of treatment:

Daily during 3 days

Post exposure period:

24 hours after last dosing.

Remarks:

Doses / Concentrations:
0; 47; 94; 187; 375; 750 mg/kg in corn oil
Basis:
nominal conc.

No. of animals per sex per dose:

5 males per dose

Control animals:

yes, concurrent no treatment

Positive control(s):

none

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Doses extend up to the maximum tolerated dose.
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Based on the cell cycle and maturation times of the erythrocytes, harvesting of the bone marrow usually occurs 24 hours after the last dosing; this interval is indicated in the data tables as "sample collection time."

DETAILS OF SLIDE PREPARATION: The bone marrow is flushed from the femurs and spread onto slides. The slides are air-dried, fixed, and stained with a fluorescent DNA-specific stain that easily illuminates any micronuclei that may be present.

METHOD OF ANALYSIS: no data

Evaluation criteria:

Typically, 2000 polychromatic erythrocytes (PCEs, reticulocytes; immature erythrocytes) are scored per animal for frequency of micronucleated cells in each of 5 animals per dose group. In addition, the percentage of PCEs among the total erythrocyte population in the bone marrow is scored for each dose group as an indicator of chemical-induced toxicity. In non-treated healthy mice and rats, the %PCE in bone marrow is usually around 50-60%. If a chemical interferes with the production of erythrocytes in the bone marrow, then the %PCE in the bone marrow may decline from the typical normal level. Conversely, if erythrocyte production is stimulated by chemical exposure, then a higher percentage of immature erythrocytes may be observed.

Statistics:

no data

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Conclusions:

negative

Executive summary:

In the NTP report (1984), male mice (5 per dose) were exposed by i.p. route to ethylvanillin at doses 0; 47; 94; 187; 375; 750 mg/kg in corn oil for 72 hours. 2000 polychromatic erythrocytes were scored per animal for frequency of micronucleated cells.

Negative result was reported.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Study well described, but no information about purity of substance.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Ivanovas GmbH
- Age at study initiation: 10 to 14 week old
- Weight at study initiation: no data
- Assigned to test groups randomly: no data
- Fasting period before study: no data
- Housing: no data
- Diet (e.g. ad libitum): no data
- Water (e.g. ad libitum): no data
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS: nodata

IN-LIFE DATES: no data

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: gum arabic
- Justification for choice of solvent/vehicle: no data
- Concentration of test material in vehicle: 3 %
- Amount of vehicle (if gavage or dermal): no data

Duration of treatment / exposure:

24 hours

Frequency of treatment:

once

Post exposure period:

30 hours

Remarks:

Doses / Concentrations:
0; 666; 1332; 2000 mg/kg bw in gum arabic
Basis:
nominal conc.

No. of animals per sex per dose:

4

Control animals:

yes, concurrent no treatment

Positive control(s):

no data

Tissues and cell types examined:

Micronucleated PE/ 1000 PE (PE: polychromatic erythrocytes)
No more information.

Evaluation criteria:

If the value differs significantly (P<0.05) from the concurrent control.

Statistics:

no data

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Conclusions:

Negative

Executive summary:

In a NMRI mouse bone marrow micronucleus assay (Wild, 1983), 4 animals per sex per dose were treated by i.p. route with ethylvanillin at doses of 0 to 1000 mg/kg bw. Bone marrow cells were harvested at 30 hours post-treatment. The vehicle was gum arabic.

There was no signs of toxicity during the study with ethylvanillin.

There was not a significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time.

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

The study is sufficiently documented and the test conditions are similar to current test guidelines (the deviations are not judged to affect the validity of the study).

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

(no positive control, only females were treated, sampling 6 hrs after the last exposure)

GLP compliance:

no

Type of assay:

micronucleus assay

Species:

mouse

Strain:

other: OF1

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: IFFA CREDO
- Age at study initiation: no data
- Weight at study initiation: ca. 20 g
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: no data
- Diet (e.g. ad libitum): no data
- Water (e.g. ad libitum): no data
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS: no data

IN-LIFE DATES: no data

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: arachis oil
- Justification for choice of solvent/vehicle: no data
- Amount of vehicle: 0.2 mL / 20 g

Duration of treatment / exposure:

2 exposures

Frequency of treatment:

30 and 6 hours before sampling

Post exposure period:

6 hours

Remarks:

Doses / Concentrations:
0.5 and 1 g/kg
Basis:
no data

No. of animals per sex per dose:

10 females

Control animals:

yes, concurrent vehicle

Positive control(s):

none

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: a preliminary study showed no deaths at 2 g/kg, 1 and 0.5 g/kg.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): treatment 30 and 6 hours before sampling

DETAILS OF SLIDE PREPARATION: cell suspensions were centrifugated for 5 minutes at 1000 tr/min, the supernatent discarded, cells were coloured with Giemsa

METHOD OF ANALYSIS: microscope (immersion) X 100

OTHER: 1000 polychromatic cells were observed

Evaluation criteria:

no data

Statistics:

no data

Sex:

female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Conclusions:

Negative

Executive summary:

In an OF1 mouse bone marrow micronucleus assay (Marzin, 1979), 10 females per dose were treated with ethylvanillin at doses of 0.5 and 1 g/kg bw. Bone marrow cells were harvested at 30 and 6 hours post-treatment. The vehicle was arachis oil.

There were no signs of toxicity during the study.

There was not a significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Ethylvanillin was assessed for its genotoxic potential in in vitro studies (Ames tests, in vitro gene mutation tests in mammalian cells, in vitro mammalian chromosome aberation tests and in vitro DNA damage and/or repair studies ):

 

Ames test:

One study of reliability 1 according to Klimisch cotation critera is available (2017) and was selected as a key study. In this reverse gene mutation assay in bacteria, performed according to the OECD No.471 guideline, Ethylvanillin dissolved in dimethyl sulfoxide (DMSO) was tested in S. typhimurium TA1535, TA1537, TA100, TA98 strains and E. coli WP2 uvrA strain in the presence or the absence of mammalian metabolic activation (S9 mix) . 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.

In the literature, several studies regarding the Ames tests are also available. Two of them were chosen as supporting studies with reliability 2 (Ishidate, 1984 and NTP, 1982). In these two studies different strains of Salmonella Typhimurium were tested, TA98, TA100, TA1535, TA1537 and TA1538 with and without metabolic activation system. These tests showed negative results. These results are consistent with the result obtained in the new Ames tests performed in 2017 (E. coli WP2 uvrA strain has been added).

Therefore ethylvanillin does not induce mutation in bacteria cells.

 

In vitro gene mutation test in mammalian cells:

One study of reliability 1 according to Klimisch cotation critera is available (2017) and was selected as a key study. The objective of this study was to evaluate the mutagenic activity of Ethylvanillin in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells. The test was performed with a 3 hour treatment period in the absence and presence of S9-mix. The study procedures described in this report were based on the most recent OECD guideline (no 490, adopted 29 July 2016). In the absence of S9-mix, Ethylvanillin induced an up to 3.2-fold dose related increase in the mutation frequency at the highest tested concentrations (1200 μg/ml). The increase was above the 95% control limits of the distribution of the historical negative control database and also above the GEF + MF(controls) (193 per 106 survivors). In the presence of S9-mix, Ethylvanillin induced an up to 4.6-fold dose related increase in the mutation frequency at the two highest tested concentrations (1000 and 1100 μg/ml). The increase was above the 95% control limits of the distribution of the historical negative control database and also above the GEF + MF(controls) (195 per 106 survivors). Although the increases in the mutation frequency at the TK locus were only observed at toxic dose levels, RTG between 10- 20%, the mutation frequencies at these concentrations were above the GEF and the results are considered to be biological relevance and the test item is mutagenic at toxic dose levels. In addition, a statistical significant dose related trend (p<0.001) was observed. It is concluded that Ethylvanillin is mutagenic in the mouse lymphoma L5178Y test system at the highest tested concentrations that were also cytotoxic and under the experimental conditions described in this report.

Two other gene mutation tests in mammalian cells are available, one with reliability 3 and one with reliability 4. In the publication (Heck et al., 1989 - reliability 4) positive results have been observed in mouse lymphoma L 5178Y cells with and without metabolic activation. However no information on the cytotoxicity is available and according to the authors additional studies are needed to clarify the basis for the positive mouse lymphoma Assay. In the RIFM report, 1983 (reliability 3), performed also on mouse lymphoma L 5178Y cells, ambiguous results were noted in presence of metabolic activation and negative results were observed in absence of metabolic activation.

Based on the positive results observed at the highest tested concentrations that were also cytotoxic in the new in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells performed on 2017, ethylvanillin is considered to induce mutations in mammalian cells.

 

In vitro chromosome aberration in mammalian cells :

Only one in vitro study is available with reliability 3 (Ishida et al., 1984). This in vitro mammalian chromosome aberration test performed on Chinese Hamster Lung fibroblasts (V79) showed positive results without metabolic activation (only the condition without metabolic activation has been conducted). However three reliable in vivo micronucleus tests showed negative results (see below, in vivo data). Therefore ethylvanillin is not considered to induce chromosome aberration.

 

Other in vitro studies with reliability 3 and 4, dealing with SCE tests showed negative results and other studies demonstrated the anti-mutagenic potential of Ethylvanillin.

 

Ethylvanillin was also assessed for its genotoxic potential in in vivo studies. Three in vivo studies (Mammalian erythrocyte micronucleus) performed on mice with reliability 2 are available (NTP, 1994, Wild 1983, Drebs laboratory, 1979). These three studies showed that Ethylvanillin do not induce chromosome aberration. Therefore ethylvanillin is not considered to induce chromosome aberration.

 

The only positive results were noted in in vitro gene mutation in mammalian cells. The in vitro tests performed on Mouse Lymphoma L5178Y cells in 2017 showed positive results at the highest doses which were also cytotoxic. This finding probably reflect the known activity of alcohols or aldehydes in biological systems. In Becker et al., 1996 publication para-Methoxybenzaldehyde alone did not induce strand breaks in supercoiled DNA from the phage PM2, although positive results were reported with the substance in the presence of CuCl2. The finding that the effect depended on the concentration of copper suggests that DNA-damaging species are produced during redox reactions of aromatic (and aliphatic) aldehydes with CuCl2. Due to the structure of ethylvanillin and its antioxidant properties it is not surprising to see mutations in mammalian cells in a in vitro test.

In the EFSA document 3 July 2007, Flavouring group evaluation consideration of hydroxy- and alkoxy- substituted benzyl derivatives, ethylvanillin is not considered as genotoxic. The EFSA Committee concluded that the hydroxy- and alkoxy-substituted benzyl derivatives do not have genotoxic potential in vivo.

Short description of key information:
The different in vitro and in vivo studies showed that Ethylvanillin has no genotoxic potential.

Justification for classification or non-classification

Based on a weight of evidence approach and taken into account the in vitro and in vivo data available on ethylvanillin, the substance is not considered to be genotoxic in vivo. Positive effect noted in in vitro mammalian cells mutation tests is probably an artefact due to the activity of ethylvanillin in biological systems (antioxidant properties, redox reaction induces by alcohols or aldehydes). Therefore ethylvanillin is considered to be not genotoxic and no classification is required according to the CLP 1272/2008/EC criteria.