Plum, ext.

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:

16 February 2019 - 15 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Plum, ext.

EC Number:

290-179-3

EC Name:

Plum, ext.

Cas Number:

90082-87-4

Molecular formula:

not applicable

IUPAC Name:

Extract obtained from fruits of Prunus domestica L. Rosaceae

Test material form:

liquid

Details on test material:

- Physical appearance: blackish brown to dark brown liquid
- Storage conditions: at room temperature

Specific details on test material used for the study:

- pH: 3.6
- Stability at higher temperatures: yes, maximum temperature: 40°C, maximum duration: 120 minutes

Method

Target gene:

Salmonella typhimurium: histidine gene
Escherichia coli: tryptophan gene

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:

Rat liver microsomal enzymes (S9), prepared from male Sprague Dawley rats injected with Aroclor 1254 (500 mg/kg body weight).

Test concentrations with justification for top dose:

Dose range finding test (TA100 and WP2uvrA, with and without S9): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate (reported as part of the first experiment)
First experiment (TA1535, TA 1537 and TA98, with and without S9): 52, 164, 512, 1600 and 5000 μg/plate
Second experiment (all strains, with and without S9): 52, 164, 512, 1600 and 5000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulfoxide
- Justification for choice of solvent/vehicle: The test item was dissolved in DMSO. The vehicle was according to OECD guideline 471.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191; 2-aminoanthracene

Remarks:

For more details see 'any other information on materials and methods'

Details on test system and experimental conditions:

Two individual experiments were performed: the first experiment was a direct plate assay and the second experiment was a pre-incubation assay. A dose-range finding study was performed and reported as part of the first experiment.

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 +/- 4 hours (for both experiments)
- Pre-incubation period: 30 +/- 2 minutes (for the second experiment only)

NUMBER OF REPLICATIONS: 3

PLATE PREPARATION: Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (10^9 cells/mL) of one of the tester strains, 0.1 ml of a dilution of the test item in DMSO and either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of nonactivation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0°C for 48 ± 4 h.

NUMBER OF CELLS EVALUATED: 10^9 cells/mL

CYTOTOXICITY:
- Cytotoxicity was examined by the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies

COLONY COUNTING:
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three (3) times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Acceptability 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 this laboratory.
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 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Only in experiment 2, in the presence of S9-mix at a dose level of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

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:

not applicable

Positive controls validity:

valid

Test results 3

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:

not applicable

Positive controls validity:

valid

Test results 4

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:

not applicable

Positive controls validity:

valid

Test results 5

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:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: in both experiments, no precipitation was observed at the start or at the end of the incubation period in any of the strains and at any of the doses tested.

CYTOTOXICITY:
- Experiment 1: No cytotoxicity observed in any of the tester strains.
- Experiment 2: Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was only observed in tester strain TA1537 at the concentration of 5000 μg/plate in the presence of S9-mix.

MUTAGENICITY: In both experiments, in all strains and at all doses tested, no biologically relevant increase in the number of revertants was observed upon treatment with the test item, with and without S9.

HISTORICAL CONTROL DATA: 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 2 Positive control historical data

	TA1535		TA1537		TA98		TA100		WP2uvrA
S9-mix	-	+	-	+	-	+	-	+	-	+
Range	3 – 29	3 - 27	3 – 20	3 – 23	8 - 41	8 - 55	63 – 176	54 - 160	10 – 59	9 - 69
Mean	10	11	6	7	16	23	108	107	25	32
SD	3	4	2	3	5	7	19	20	7	8
n	2356	2336	2264	2235	2319	2360	2341	2336	2075	2078

Table 3 Negative control historical data

	TA1535		TA1537		TA98
S9-mix	-	+	-	+	-	+
Range	125 – 1248	73 – 1206	55 – 1353	54 – 1051	365 – 1995	250 – 1977
Mean	846	219	787	353	1406	887
SD	146	119	345	162	258	349
n	2348	2229	2003	2234	2200	2276

	TA100		WP2uvrA
S9-mix	-	+	-	+
Range	439 – 1848	408 - 2651	93 – 1951	111 - 1359
Mean	901	1232	1094	437
SD	168	343	477	149
n	2335	2327	2021	2085

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between Oct 2015 and Oct 2017. 

Applicant's summary and conclusion

Conclusions:

The results of an Ames test, performed according to OECD guideline 471 and GLP principles, showed that Plum concentrate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.