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Genetic toxicity in vitro

Description of key information

- Evaluation of The Mutagenic Activity Of Nectaryl In The Salmonella Typhimurium Reverse Mutation Assay And The Escherichia Coli Reverse Mutation Assayp performed in 2015, 2006 and 1989 according to the OECD Guideline No. 471 shows that NECTARYL is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

- Evaluation of the potential of NECTARYL to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5l78Y performed in 2014 according the OECD Guideline No. 476 shows that NECTARYL did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5 l 78Y  in the absence and presence of metabolic activation.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 December 2014 to 08 January 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
There were no deviations from standard operating procedures that affected the integrity of the study.
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Name as stated in the report: NECTARYL
Batch No.: VE00318744
Appearance: Clear colourless liquid (determined at WIL Research Europe B.V.)
Expiration date: 14 March 2015
Target gene:
Four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA).
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:
rat liver S9-mix induced by induced by Aroclor 1254
Test concentrations with justification for top dose:
Experiment I : 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate were tested in triplicate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the absence and presence of 5% (v/v) S9-mix.
Experiment II : based on the results of the first mutation assay, six doses (increasing with approximately half-log steps) of the test substance were selected and tested in triplicate in each strain in the second experiment in the absence and presence of 10% (v/v) S9-mix.. The highest concentration of NECTARYL used in the second mutation assay was 5 mg/plate.
Vehicle / solvent:
NECTARYL was dissolved in dimethyl sulfoxide. Test substance concentrations were used within 3 hours after preparation.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
The vehicle of the test article, being DMSO.
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191 at 2.5 μg/plate (DMSO) and 2-aminoanthracene at 2.5 μg in DMSO
Details on test system and experimental conditions:
The test substance was tested both in the absence and presence of S9-mix in each strain, in two independent experiments.
The vehicle control and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix. To avoid indistinctness about interference of precipitation of the test substance at the colony counting, the solubility of the test substance was assessed at the beginning and end of the treatment period. In parallel selection plates with the highest dose level were used during the incubation period in the second mutation experiment.
Top agar in top agar tubes was melted by heating to 45°C. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (109 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test substance 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 non-activation 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 h. After this period revertant colonies (histidine independent for Salmonella typhimurium bacteria and tryptophan independent for Escherichia coli) were counted.
Rationale for test conditions:
according to guideline
Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the
concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537,
TA98 or WP2uvrA is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one independently repeated experiment.

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

A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The negative control data (number of spontaneous revertants per plate) should be within the laboratory historical range for each tester strain.
b) The positive control chemicals should produce responses in all tester strains, which are within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be at least three times the concurrent vehicle control group mean.
c) 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.
d) No more than 5% of the plates are lost through contamination or some unforeseen event.
Key result
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A
Remarks:
first mutation assay (5% (v/v) S9-mix)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Toxicity was observed in all tester strains in the absence and presence of S9-mix, except in tester strain WP2uvrA, and test item precipitated (oily droplets) at 5000µg/plate in all experiments.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A
Remarks:
Second mutation assay (10% (v/v) S9-mix)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Toxicity was observed in all tester strains+/- S9-mix, except in tester strain WP2uvrA +/- S9-mix and TA1535 +S9-mix, and test item precipitated (oily droplets) at 5000µg/plate in all experiments.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The negative and strain-specific positive control values were within the laboratory background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

In the first mutation assay, NECTARYL was tested up to concentrations of 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix. NECTARYL precipitated on the plates as oily droplets at the dose level of 5000 μg/plate. Toxicity was observed in all tester strains in the absence and presence of S9-mix, except in tester strain WP2uvrA.

In the second mutation assay, NECTARYL was tested up to concentrations of 5000 μg/plate in the absence and presence of 10% (v/v) S9-mix. NECTARYL precipitated (observed as droplets) on the plates at the dose level of 5000 μg/plate. Toxicity was observed in all tester strains in the absence and presence of S9-mix, except in tester strain WP2uvrA in the absence and presence of S9-mix and TA1535 in the presence of S9-mix. NECTARYL 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 an independently repeated experiment.

Conclusions:
Based on the results of this study it is concluded that NECTARYL is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

Evaluation of the mutagenic activity of NECTARYL in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay.

NECTARYL was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by induced by Aroclor 1254).

The study procedures described in this report were based on the most recent OECD, EC and Japanese guidelines.

Batch VE00318744 of NECTARYL was a clear colourless liquid with a purity of 97.1%. The test substance was dissolved in dimethyl sulfoxide.

In the first mutation assay, NECTARYL was tested up to concentrations of 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix. NECTARYL precipitated on the plates as oily droplets at the dose level of 5000 μg/plate. Toxicity was observed in all tester strains in the absence and presence of S9-mix, except in tester strain WP2uvrA.

In the second mutation assay, NECTARYL was tested up to concentrations of 5000 μg/plate in the absence and presence of 10% (v/v) S9-mix. NECTARYL precipitated (observed as droplets) on the plates at the dose level of 5000 μg/plate. Toxicity was observed in all tester strains in the absence and presence of S9-mix, except in tester strain WP2uvrA in the absence and presence of S9-mix and TA1535 in the presence of S9-mix.

NECTARYL 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 an independently repeated experiment.

In this study, 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.

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

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 March 2014 to 28 April 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Specific details on test material used for the study:
Name as stated in the report: NECTARYL
Expiration date: 28 .January 2015
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
L5178Y/TK+/- 3.7.2C mouse lymphoma cells.
Metabolic activation:
with and without
Test concentrations with justification for top dose:
Experiment I (with and without S9 mix): 2.2, 4.4, 8.8, 17.5, 35.0, 52.5 and 70.0 µg/mL (4 hours exposure)
Experiment II (with S9 mix): 4.4, 8.8, 17.5, 35.0, 52.5 , 70.0 and 105.0 µg/mL (24 hours exposure)
Experiment II (without S9 mix): 8.8, 17.5, 35.0, 70.0, 105, 140 and 245.0 µg/mL (4 hours exposure)
The dose range of the first experiment was adjusted to toxicity data generated in the pre­ experiment In the second experiment, toxicity data of the pre-experiment and the first main experiment were considered. In both main experiments the individual concentrations were generally spaced by a factor of 2.0 in the lower range. A narrower spacing was used at the highest concentrations to cover the cytotoxic range more closely. To overcome problems with possible deviations in toxicity the main experiments were started with more than fom concentrations.
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Remarks:
solvent control (DMSO)
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
- Source: the L5178Y/TK mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit in Brighton, UK. The cel ls were originally obtained from Dr. D. Clive of Bw-roughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.
- Storage: large stocks of the cleansed L5178Y cell line are stored in liquid nitrogen in the cell bank of Harlan CCR allowing the repeated use of the same cell culture batch in experiments. Each batch is screened for mycoplasm contamination and checked for karyotype stability and spontaneous mutant frequency . Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells.
Rationale for test conditions:
Rationale: Recommended test system in international guidelines (e.g. OECD).
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See "additional information on results" field
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Relevant cytotoxic effect indicated by a relative total growth of less than 50% of survival in both parallel cultures was observed in experiment I at 35.0 µg/mL without metabolic activation. In experiment II cytotoxicity as described above was noted at 35.0 µg/ml and above in the absence of metabolic activation and at 70.0 µg/mL in the presence of metabolic activation.
Conclusions:
ln conclusion it can be stated that under the experimental conditions reported the test item NECTARYL did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5 l 78Y in the absence and presence of metabolic activation.
Therefore, NECTARYL is considered to be non-mutagenic in this mouse lymphoma assay.
Executive summary:

The study was performed according to the OECD Guideline 476 investigate the potential of NECTARYL to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5l78Y.

The assay was perfomed in two independent experiments, using two parallel cultures each. The first experiment was performed with and without liver microsomal activation and a treatment period of 4h.The second experiment was performed with a treatment period of 4 hours with and 24 hours without metabolic activation.

The highest concentration of 2260 µg/mL applied in the pre-experiment was equal to approximately 10mM. The concentration range of the main experiments was limited by cytotoxic effects.

No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item.

Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid. 

ln conclusion it can be stated that under the experimental conditions reported the test item NECTARYL did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5 l 78Y  in the absence and presence of metabolic activation.

Therefore, NECTARYL is considered to be non-mutagenic in this mouse lymphoma assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The mutagenic potential of the test substance NECTARYL was tested in the mouse using the micronucleus test OECD Guideline No. 474 performed in 1989.

Under the experimental conditions employed,the test substance NECTARYL did not induce clastogenic or aneugenic effects in the mouse.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 March 1989 to 12 October 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test
Specific details on test material used for the study:
Name (as stated in the report) : NECTARYL - LRG 1371
Identification of the test item for the study: 07683 D9 001
Aspect: colourless slightly viscous liquid
Batch Number: AS1241001
Species:
mouse
Strain:
other: OFl mouse (IOPS Caw).
Details on species / strain selection:
The mouse is currently used in this type of test and is recommended by the different legislative authorities.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Iffa-Credo (69210 L'Arbresle - France).
- Age at study initiation: young adults, aged from 6 to 8 weeks
- Weight at study initiation: values between 28.3 g and 36.9 g for the males, and between 21.2 g and 28.8 g for the females.
- Cages : housed in groups of 5 (or 2 in the preliminary studies), by sex in translucent polypropylene VS cages (internal dimensions 205 x 115 x 129 mm).
- Air changes: at least 10 per hour
- Temperature: 22, 3° C
- Humidity: 30 to 70% R.H
- Lighting: artificial, 12 hours out of 24 (photoperiod = 07,30 - 19,30).
- Hygiene: cages changed for each study.
Route of administration:
other: Oral route (test substance and negative control) and intraperitoneal route (positive control).
Vehicle:
The test substance was diluted in sterile Codex liquid paraffin (Aguettant, Batch 6170 B, March 1993)
Details on exposure:
Preliminary study: 4 groups made up of 2 males and 2 females each treated respectively at the dose levels of 9600*, 4800, 960 and 192 mg/kg. A complementary study was performed at the dose levels of 4800 and 960 mg/kg to confirm the clinical observations.
Main study: The dose level of 4800 mg/kg was administered in the main study
Duration of treatment / exposure:
Oral administration: 10 ml/kg of live body weight
Frequency of treatment:
The test substance was administered once only
Post exposure period:
Observations were carried out within the first hour and then 15 minutes, 24, 48 and 72 hours after administration.
Dose / conc.:
4 800 mg/kg bw/day (nominal)
Remarks:
Dose conc of the main study was determined from the complementary study where no mortality occured at the dose level of 4800 mg/kg bw
No. of animals per sex per dose:
5 males and 5 females per group / 1 dose concentration per group
Control animals:
yes
yes, concurrent vehicle
Positive control(s):
A solution of cyclophosphamide (Sigma, Batch 114 F 0393) in sterile water for injection (Aguettant, Batch 85456, July 1991) was prepared. Used concentration: 10 mg/ml
Details of tissue and slide preparation:
For each animal both femurs were removed. The femoral bone marrow was extracted with the aid of syringe, and suspended in foetal calf serum. After centrifugation and resuspension the smears were prepared (2 slides per animal). The slides were stained with May-Grunwald- Giemsa. Microscopic examination of the slides was performed. For each animal 1000 erythrocytes were examined (500 cells per slide, 2 slides). A count of the number of polychromatophile erythrocytes bearing micronuclei was performed. At the same time the ratio of normochromatophile erythrocytes to polychromatophile erythrocytes was established by the observation of 400 cells per animal (200 cells per slide, 2 slides).
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
see "additional information on results" field below
Vehicle controls validity:
not examined
Remarks:
test item was administered pure
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The normochromatic / polychromatic erythrocytes ratio, is enhanced by a factor of 2 to 3 in the animals treated by the test substance. This observation shows the slightly toxic effect of the test substance on the bone marrow cells.
Conclusions:
No significant increase of the number of polychromatophile erythrocytes bearing micronuclei was observed in the animals treated with the test substance, at the dose level of 4800 mg/kg and killed 24, 48 or 72 hours after administration.
In contrast, significant results were observed in the animals treated with the positive control. The normochromatic / polychromatic erythrocytes ratio, is enhanced by a factor of 2 to 3 in the animals treated by the test substance. This observation shows the slightly toxic effect of the test substance on the bone marrow cells.
From these experimental results, it may be concluded that the test substance does not induce any mutagenic effect in the mouse.
Executive summary:

The mutagenic potential of the test substance was tested in the mouse using the micronucleus test OECD Guideline No. 474. The test substance was administered by the oral route at the dose level of 4800mg/kg as described in the preliminary study.

A negative control (administration of vehicle) and a positive control (administration of a standard mutagenic substance) were included in this study.

The animals (5 males and 5 females per group) were killed 24, 48 or 72 hours after the administration of the test substance. For each animal an examination of 1000 polychromatophile erythrocytes obtained from the femoral bone marrow was performed. The frequency of micronuclei was calculated for each animal and for each group.

Under the experimental conditions employed,the test substance NECTARYL did not induce mutagenic effects in the mouse.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

Based on the data available and key results described in this summary, the substance has shown no genotoxic potential and should therefore not be classified for mutagenicity according to the (EC) No 1272/2008 Regulation (CLP).