monoesters of C16 and C18 (branched and linear) fatty acids with decan-1-ol

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 date: 12 April 2011 Experimental end date 23 May 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

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:

monoesters of C16 and C18 (branched and linear) fatty acids with decan-1-ol

EC Number:

953-178-5

Molecular formula:

not applicable, substance is UVCB

IUPAC Name:

monoesters of C16 and C18 (branched and linear) fatty acids with decan-1-ol

Method

Target gene:

histidine locus

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:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Preliminary Toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500, and 5000 μg/plate
Experiment 1: 50, 150, 500, 1500 and 5000 μg/plate
Experiment 2: 50, 150, 500, 1500 and 5000 μg/plate
All with and without metabolic activation
toxicity wasn't sufficiently severe enough to prevent the test item being tested up to the maximum
recommended dose level of 5000 pg/plate

Vehicle / solvent:

The test item was immiscible in sterile distilled water and dimethyl sulphoxide at
50 mg/ml but was fully miscible in acetone at the same concentration in solubility checks
performed in-house. Acetone was therefore selected as the vehicle.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

2 μg/plate for WP2uvrA, 3 μg/plate for TA100, 5 μg/plate for TA1535

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

80 μg/plate for TA1537

Positive control substance:

9-aminoacridine

Remarks:

without metabolic activation

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

0.2 μg/plate for TA98

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without metabolic activation

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

1 μg/plate for TA100, 2 μg/plate for TA1535 and TA1537, 10 μg/plate for WP2uvrA

Positive control substance:

other: 2-Aminoanthracene

Remarks:

with metabolic activation

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

5 μg/plate for TA98

Positive control substance:

benzo(a)pyrene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

Preliminary Toxicity Test
In order to select appropriate dose levels for use in the main test, a preliminary assay
was carried out to determine the toxicity of the test item. The concentrations tested were
0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 pg/plate. The assay was performed
by mixing 0.1 ml of bacterial culture (TA100 or WP2uvrA), 0.1 ml of test item formulation,
0.5 ml of S9-mix or phosphate buffer and 2 ml of molten, trace histidine or tryptophan
supplemented, top agar and overlaying onto sterile plates of Vogel-Bonner Minimal agar
(30 ml/plate). Ten concentrations of the test item and a vehicle control (acetone) were
tested. In addition, 0.1 ml of the maximum concentration of the test item and 2 ml of
molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile
nutrient agar plate in order to assess the sterility of the test item. After approximately 48
hours incubation at 37°C the plates were assessed for numbers of revertant colonies
using a Domino colony counter and examined for effects on the growth of the bacterial
background lawn.

Mutation Test — Experiment 1 (Range-finding Test)
Five concentrations of the test item (50, 150, 500, 1500 and 5000 pg/plate) were
assayed in triplicate against each tester strain, using the direct plate incorporation
method.
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of
test tubes followed by 2 ml of molten, trace histidine or tryptophan supplemented, top
agar, 0.1 ml of the test item formulation, vehicle or positive control and either 0.5 ml of
S9-mix or phosphate buffer. The contents of each test tube were mixed and equally
distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate).
This procedure was repeated, in triplicate, for each bacterial strain and for each
concentration of test item both with and without S9-mix.
All of the plates were incubated at 37°C for approximately 48 hours and the frequency of
revertant colonies assessed using a Domino colony counter.

Mutation Test — Experiment 2 (Main Test)
The second experiment was performed using fresh bacterial cultures, test item and
control solutions. The test item dose range was the same as the range-finding test (50
to 5000 pg/plate).
As it is good scientific practice to alter one condition in the replicate assay, the exposure
condition was changed from plate incorporation to pre-incubation. The test item
formulations and vehicle control were therefore dosed as follows:
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of
test tubes followed by 0.5 ml of S9-mix or phosphate buffer and 0.05 ml of the vehicle or
test item formulation and incubated for 20 minutes at 37°C with shaking at approximately
130 rpm prior to the addition of 2 ml of molten, trace histidine or tryptophan
supplemented, top agar. The contents of the tube were then mixed and equally
distributed on the surface of Vogel-Bonner Minimal agar plates (one tube per plate).
This procedure was repeated, in triplicate, for each bacterial strain and for each
concentration of test item both with and without S9-mix. The positive and untreated
controls were dosed using the standard plate incorporation method.
All of the plates were incubated at 37°C for approximately 48 hours and the frequency of
revertant colonies assessed using a Domino colony counter.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the
following can be used to determine the overall result of the study:
1 A dose-related increase in mutant frequency over the dose range tested (De
Serres and Shelby (1979)).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al (1989)).
5. Fold increase greater than two times the concurrent solvent control for any tester
strain (especially if accompanied by an out-of-historical range response).
A test item will be considered non-mutagenic (negative) in the test system if the above
criteria are not met.
Although most experiments will give clear positive or negative results, in some instances
the data generated will prohibit making a definite judgement about test item activity.
Results of this type will be reported as equivocal.

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:

no cytotoxicity

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:

no cytotoxicity

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:

Preliminary Toxicity Test
The test item was non-toxic to the strains of bacteria used (TA100 and WP2uvrA). The
test item formulation and S9-mix used in this experiment were both shown to be sterile.

Mutation Test
Prior to use, the master strains were checked for characteristics, viability and
spontaneous reversion rate (all were found to be satisfactory). These data are not given
in the report. The amino acid supplemented top agar and the S9-mix used in both
experiments was shown to be sterile.

In the range-finding test (plate incorporation method) the test item caused no visible
reduction in the growth of the bacterial background lawns of any of the tester strains in
either the presence or absence of S9-mix. In the second experiment (pre-incubation
method) the test item induced toxicity to the bacterial background lawns of all of the
Salmonella strains dosed in the absence of S9-mix at 5000 μg/plate. No toxicity was
noted to the Salmonella strains dosed in the presence of S9-mix or to Escherichia coli
strain WP2uvrA in either experiment. These results were not indicative of toxicity
sufficiently severe enough to prevent the test item being tested up to the maximum
recommended dose level of 5000 μg/plate. An oily test item precipitate was observed at
and above 1500 μg/plate, this observation did not prevent the scoring of revertant
colonies.
No toxicologically significant increases in the frequency of revertant colonies were
recorded for any of the bacterial strains, with any dose of the test item, either with or
without metabolic activation or exposure method. Small but statistically significant
increases in WP2uvrA revertant colony frequency were observed in the range-finding
test in the absence of S9-mix at 150 and 5000 μg/plate. These increases were
considered to be of no biological relevance because there was no evidence of a
dose-response relationship or reproducibility. Furthermore, the individual revertant
counts at 150 and 5000 μg/plate were within the in-house historical untreated/vehicle
control range for the tester strain and the fold increase was only up to 1.5 times the
concurrent vehicle control.
All of the positive control chemicals used in the test induced marked increases in the
frequency of revertant colonies thus confirming the activity of the S9-mix and the
sensitivity of the bacterial strains

Any other information on results incl. tables

Test Period		From: 20 May 2011 To: 23 May 2011
With or without S9-Mix	Test substance concentration (pg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type TA100                TA1535           WP2uvrA			Frameshift type TA98                   TA1537
+	0	125	25	32	23	7
		133   (120)	36 (30)	32  (30)	20 (2.1)	7  (9)
		101   16.7#	29  5.6	25   4.0	20  1.7	12  2.9
+	50	123	30	19	24	11
		101  (111)	21  (24)	29  (23)	18  (20)	5   (9)
		110  11.1	20   5.5	21   5.3	18   3.5	12   3.8
+	150	121	30	30	21	10
		100  (110)	16  (24)	25   (26)	22  (19)	8   (11)
		109  10.5	27   7.4	24   3.2	13   4.9	14  3.1
+	500	115	29	37	21	4
		108   (105)	29   (29)	26  (31)	10   (16)	10   (9)
		93    11.2	29    0.0	30   5.6	18  5.7	13  4.6
+	1500	81 P	29 P	27 P	16 P	4.0 P
		108 P  (106)	31 P  (30)	22 P (29)	21 P (17)	4 P  (4)
		129 P   24.1	CP    1.4	38 P  8.3	14 P  3.6	5 P  0.6
+	5000	60 *P	0 *P	38 P	10 *P	0 *P
		56 *P    (62)	0 *P  (0)	43 P (36)	8 *P  (9)	0 *P  (0)
		69 *P    6.7	0 *P  0.0	26 P  8.7	8 *P  1.2	0 *P  0.0
Positice Control S9-mix +	Name Concentration (p9/plate) No. colonies per plate	ENNG	ENNG	ENNG	ENQO	9AA
		3	5	2	0.2	80
		299	348	597	119	773
		315   (312)	272   (313)	673 (644)	154  (132)	927   (856)
		321   11.4	318  38.3	661 40.9	123  19.2	868   77.7

 

Applicant's summary and conclusion

Conclusions:

The test item,Monoesters of C16 and C18 (branched and linear) fatty acids with decan-1-ol, was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Introduction. The test method was designed to be compatible with the guidelines for

bacterial mutagenicity testing published by the major Japanese Regulatory Authorities

including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No.

471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC)

number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised

guidelines.

Methods. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and

Escherichia coli strain WP2uvrA were treated with the test item, decyl isostearate, using

both the Ames plate incorporation and pre-incubation methods at five dose levels, in

triplicate, both with and without the addition of a rat liver homogenate metabolising

system (10% liver S9 in standard co-factors). The dose range for the range-finding test

was determined in a preliminary toxicity assay and was 50 to 5000 pg/plate. The

experiment was repeated on a separate day (pre-incubation method) using the same

dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test

item formulations.

Results. The vehicle (acetone) control plates gave counts of revertant colonies within

the normal range. All of the positive control chemicals used in the test induced marked

increases in the frequency of revertant colonies, both with or without metabolic

activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were

validated.

In the range-finding test (plate incorporation method) the test item caused no visible

reduction in the growth of the bacterial background lawns of any of the tester strains in

either the presence or absence of S9-mix. In the second experiment (pre-incubation

method) the test item induced toxicity to the bacterial background lawns of all of the

Salmonella strains dosed in the absence of S9-mix at 5000 μg/plate. No toxicity was

noted to the Salmonella strains dosed in the presence of S9-mix or to Escherichia coli

strain WP2uvrA in either experiment. These results were not indicative of toxicity

sufficiently severe enough to prevent the test item being tested up to the maximum

recommended dose level of 5000 μg/plate. An oily test item precipitate was observed at

and above 1500 μg/plate, this observation did not prevent the scoring of revertant

colonies.

No toxicologically significant increases in the frequency of revertant colonies were

recorded for any of the bacterial strains, with any dose of the test item, either with or

without metabolic activation or exposure method. Small but statistically significant

increases in WP2uvrA revertant colony frequency were observed in the range-finding

test in the absence of S9-mix at 150 and 5000 μg/plate. These increases were

considered to be of no biological relevance because there was no evidence of a

dose-response relationship or reproducibility. Furthermore, the individual revertant

counts at 150 and 5000 μg/plate were within the in-house historical untreated/vehicle

control range for the tester strain and the fold increase was only up to 1.5 times the

concurrent vehicle control.

Conclusion. The test item, Monoesters of C16 and C18 (branched and linear) fatty acids with decan-1-ol, was considered to be non-mutagenic

under the conditions of this test.

Categories Display