Ethyl nicotinate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

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:

yes

Remarks:

The test item was not stored protected from light as per the study plan.The test item is not normally stored under conditions to prevent exposure to light and is not known to be overly sensitive to light, thus this deviation had no impact on the study.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl nicotinate

EC Number:

210-370-7

EC Name:

Ethyl nicotinate

Cas Number:

614-18-6

Molecular formula:

C8H9NO2

IUPAC Name:

ethyl nicotinate

Test material form:

liquid

Method

Target gene:

Histidine (Salmonella spp) and Tryptophan (E.coli)

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: rfa uvrB

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction (Aroclor 1254 induced male Sprague Dawley rat liver)

Test concentrations with justification for top dose:

as µg/plate: 1.29, 3.86, 11.6, 34.7, 104, 313, 625, 1250, 2500, 5000. Top dose as per limit dose in the guideline

Vehicle / solvent:

sterile water

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene

Details on test system and experimental conditions:

3.1 Test System
The bacteria were originally supplied by Moltox, NC, USA. Each batch of frozen bacteria was
tested for appropriate phenotype characteristics and spontaneous reversion rates; response to
diagnostic mutagens was also routinely assessed. The following bacterial strains were employed:
S. typhimurium TA1535 hisG46 rfa ∆uvrB
S. typhimurium TA1537 hisC3076 rfa ∆uvrB
S. typhimurium TA98 hisD3052 rfa ∆uvrB pKM101
S. typhimurium TA100 hisG46 rfa ∆uvrB pKM101
E. coli WP2 trp uvrA
Fresh bacterial cultures were prepared and were in the late-log phase of growth at the time of
use. The density of the cultures was confirmed to be ≥ 1000 x 10^6 bacteria/mL using a bacterial
counting chamber before the cultures were used in the study.

3.2 S9 Mix
The S9 mixture, used as a model of intact mammalian metabolism, was prepared on the day of
use and contained 10% v/v S9 fraction (Aroclor 1254 induced male Sprague Dawley rat liver
fraction supplied by Moltox) and the following sterile cofactors: 8 mM MgCl2, 33 mM KCl,
100 mM sodium phosphate buffer pH 7.4, 5 mM glucose-6-phosphate and 4 mM NADP. The
S9 mixture was stored in a refrigerator set to maintain 4°C or on ice until required. A copy of the
manufacturer’s quality control certificate for the S9 fraction was retained as raw data.

3.3 Sterility and Spontaneous Mutation Rates
The sterility of the test item formulation (high dose) was confirmed on the day of the study using
appropriate preparations without bacteria.
The spontaneous mutation rates of the bacterial strains were assessed using concurrent control
samples in which the bacteria were exposed to the vehicle control.

3.4 Preparation of Dose Formulations
All formulations of the test item were freshly prepared on the day of dosing, just prior to use. An
appropriate quantity of the test item was dissolved in water to create the high concentration stock
(50 mg/mL) on the day of dosing. All lower level formulations were made by serial dilution.
Positive control solutions were prepared in advance, stored frozen, and thawed for use on the day
of the study.

3.5 Plate Incorporation Method
A 0.5 mL aliquot of S9 mix (+S9) or phosphate buffer 0.2 M pH 7.4 (0S9) was combined with
0.1 mL bacterial culture in a sterile container. A 0.1 mL aliquot of the test item, vehicle control
or positive control was added, and approximately 2 mL of molten top agar supplemented with
0.05 mM biotin and minimal (0.05 mM) histidine and minimal (0.05 mM) tryptophan was added
immediately afterward. The solution was mixed and overlaid onto a minimal glucose plate (1.5%
agar, Vogel-Bonner medium E, 2% glucose). After the overlay solidified, the plates were
inverted and placed in an incubator set to maintain 37°C for 65 hours and 23 minutes in the
initial test and for 64 hours and 4 minutes in the repeat test.

Due to invalid vehicle control results obtained in the initial test with strain WP2 uvrA in the
absence of metabolic activation, this portion of the test was repeated.

Evaluation criteria:

4 EVALUATION AND INTERPRETATION OF RESULTS
Means and standard deviations for appropriate dose levels were calculated using Microsoft®
Excel 2007/2013. The mean number of revertant colonies for all treatment groups was compared
with those obtained for the concurrent vehicle control group. The mutagenic activity of the test
item was assessed by applying the following criteria:
• Positive: If treatment with the test item produced a dose-related increase in revertant colony
numbers at least twice the concurrent vehicle control levels with bacterial strains TA98,
TA100 and WP2 uvrA, (3-fold for TA1535 and TA1537) either in the presence or absence
of S9 mix, the test item was considered to show evidence of mutagenic activity in the test
system (provided mean value(s) lay outside the historical control range).
• Negative: If treatment with the test item did not produce a dose-related increase in revertant
colony numbers at least twice the concurrent vehicle control levels with strains TA98,
TA100, and WP2 uvrA (3-fold for strains TA1535 and TA1537) the test item was
considered to show no evidence of mutagenic activity in the test system.
• Equivocal: If the results obtained failed to satisfy the criteria for a clear “positive”
or “negative” response, the results were considered equivocal. It was acceptable to conclude
an equivocal response if no clear conclusion could be made. Note that the reproducibility of
any apparent effect was taken into account in making any clear conclusion.

Statistics:

For the study to be considered valid, the mean revertant colony counts of the vehicle control for
strains TA98, TA100, TA1535 and WP2 uvrA should not be below 2 standard deviations of the
respective historical vehicle control mean of the laboratory. For strain TA1537, the mean
revertant count of the vehicle control should not be below 5. In addition, the mean revertant
colony counts of the vehicle control for all strains should not exceed the respective defined 98%
upper tolerance limit of the historical vehicle control values. All positive controls (with S9
where required) had to produce increases in revertant colony numbers indicative of a positive
response.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The results of the study are presented in Table 1 to Table 5. Means and standard deviations have
been rounded to the nearest whole number.
Ten concentrations of Ethyl Nicotinate (ranging from 1.29 to 5000 µg/plate) were tested in
triplicate cultures of each strain in the initial test. The mean revertant colony counts of
the vehicle control for all strains met the Assay Acceptance Criteria (see also
Historical Control Results), with the exception of strain WP2 uvra in the absence of metabolic
activation where the mean revertant colony count was slightly above the 98% tolerance limit
making this portion of the test invalid. A repeat test was conducted using six test item
concentrations (ranging from 104 to 5000 µg/plate) with strain WP2 uvra in the absence of
metabolic activation. All results in the repeat test were valid. All positive control cultures
exhibited revertant count increases indicative of a mutagenic response, confirming the sensitivity
of the test system and the activity of the S9 mix for both the initial and repeat tests (Table 3 and
Table 5; see also Historical Control Results). The absence of colonies on sterility check plate
confirmed absence of microbial contamination (result not shown).
No meaningful increases in the revertant colony numbers were obtained with any of the tester
strains following exposure to Ethyl Nicotinate at any test concentration, in either the presence or
absence of the metabolic activation system.

Applicant's summary and conclusion

Conclusions:

In conclusion, Ethyl Nicotinate was not mutagenic in the Salmonella typhimurium and
Escherichia coli strains when tested in accordance with regulatory guidelines.