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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
14 February to 31 March 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Well conducted and well described study in accordance with GLP and OECD Guideline 471 without any deviation.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Test material form:
liquid
Details on test material:
Batch No.: 141767
Purity: 98.1%
Name of test material (as cited in study report): NOPOL
Physical state: colourless to very pale yellow liquid
Storage conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry date: 01 April 2015

Method

Target gene:
None
Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell lines (if applicable):
Not applicable
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
10 % S9 mix; S9 fraction prepared from liver homogenates of male Sprague Dawley rats induced with Aroclor 1254
Test concentrations with justification for top dose:
Experiment 1 (plate-incorporation method):
- TA1535, TA1537, TA98, TA100 and TA102: 5, 16, 50, 160, 500, 1600 and 5000 μg/plate, with and without S9-mix

Experiment 2 (plate-incorporation method without S9 mix; preincubation method with S9 mix):
- TA1535, TA1537, TA98, TA100 and TA102: 25, 50, 100, 200, 400, 800 and 1600 μg/plate, with and without S9-mix

Experiment 3 (preincubation method):
- TA1535, TA1537, TA98, TA100 and TA102: 6.25, 12.5, 25, 50, 100, 200 and 400 μg/plate, with S9-mix
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that Nopol was soluble in anhydrous analytical grade dimethyl sulphoxide (DMSO) at concentrations up to at least 100 mg/mL. Therefore, DMSO was selected as vehicle.
- Test substance preparation: Test substance stock solutions were prepared by formulating Nopol under subdued lighting in DMSO with the aid of vortex mixing as required, to give the maximum required treatment concentration. Subsequent dilutions were made using DMSO. The test article solutions were protected from light and used within approximately 4 h of initial formulation.
Controlsopen allclose all
Negative controls:
no
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
Remarks:
without metabolic activation
Negative controls:
no
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-aminoanthracene
Remarks:
with metabolic activation
Details on test system and conditions:
SOURCE OF TEST SYSTEM:
Strains TA98, TA1535 and TA1537 were originally obtained from the UK NCTC. Strains TA100 and TA102 were derived from cultures originally obtained from Covance Laboratories Inc., USA.

METHOD OF APPLICATION: In agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 minutes at 37 ± 1 °C, with shaking
- Incubation period: Plates were inverted and incubated at 37 ± 1 °C in the dark for 3 days.

NUMBER OF REPLICATIONS:
- Vehicle and positive controls were included in quintuplicate and triplicate plates, respectively.
- Treatment (test item) groups were included in triplicate plates

DETERMINATION OF CYTOTOXICITY
- Method: The background lawns of the plates were examined for signs of toxicity. Other evidence of toxicity may have included a marked reduction in revertants compared to the concurrent vehicle controls and/or a reduction in mutagenic response.

OTHER:
- Strain characteristics: The inocula were taken from master plates or vials of frozen cultures, which had been checked for strain characteristics (histidine dependence, rfa character, uvrB character and resistance to ampicillin or ampicillin plus tetracycline). Checks were carried out according to Maron and Ames, 1983 and De Serres and Shelby, 1979.
- Colony counting: Colonies were counted electronically using a Sorcerer Colony Counter (Perceptive Instruments) or manually where confounding factors such as bubbles, splits in the agar or contamination affected the accuracy of the automated counter. The background lawn was inspected for signs of toxicity.
Evaluation criteria:
For valid data, the test article was considered to be mutagenic if:
1. A concentration related increase in revertant numbers was ≥1.5-fold (in strain TA102), 2-fold (in strains TA98 or TA100) or 3-fold (in strains TA1535 or TA1537) the concurrent vehicle control values.
2. Any observed response was reproducible under the same treatment conditions.
The test article was considered positive in this assay if both of the above criteria were met.
The test article was considered negative in this assay if either of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Biological relevance was taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments.
Statistics:
The presence or otherwise of a concentration response was checked by non-statistical analysis, up to limiting levels (for example toxicity, precipitation or 5000 μg/plate). However, adequate interpretation of biological relevance was of critical importance (OECD, 1997; ICH S2(R1), 2011).

Results and discussion

Test results
Key result
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed on the test plates during scoring, in any of the experiments performed.
- Other confounding effects: None

COMPARISON WITH HISTORICAL CONTROL DATA: Mean vehicle control counts fell within the laboratory’s historical ranges.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Experiment 1: Following the treatment, evidence of toxicity in the form of a slight thinning of the background bacterial lawn, was observed at 500 μg/plate in all strains in the absence and presence of S-9. A complete killing of the test bacteria was observed at 1600 μg/plate and above in all strains in the absence and presence of S-9. In addition, a reduction in revertant numbers was observed at 160 μg/plate in strain TA1537 only in the absence and presence of S-9.
- Experiment 2: Following the treatment, evidence of toxicity ranging from a diminution of the background bacterial lawn, with or without a concurrent marked reduction in revertant numbers, to a complete killing of the test bacteria was observed in all strains from 800 and from 400 μg/plate in the absence and presence of S-9, respectively. In addition, a reduction in revertant numbers was observed at 400 μg/plate in strain TA1537 in the absence of S-9 only. Since mutation data were only available for four concentrations in the presence of S-9 for all tester strains due to toxicity, a further Experiment (Experiment 3) was performed.
- Experiment 3: Following the treatment, evidence of toxicity ranging from a slight thinning of the background bacterial lawn, with or without a concurrent marked reduction in revertant numbers, to a complete killing of the test bacteria was observed at 200 μg/plate and above in all strains.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
The test item is not considered as mutagenic in S. typhimurium (TA1535, TA1537, TA98, TA100 and TA102) strains, with and without metabolic activation.
Executive summary:

In a reverse gene mutation assay in bacteria, performed according to Guideline OECD 471 and in compliance with GLP, strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100 and TA102) were exposed to the test item, Nopol, at the concentrations below. 

Experiment 1 (plate-incorporation method):

- TA1535, TA1537, TA98, TA100 and TA102: 5, 16, 50, 160, 500, 1600 and 5000 μg/plate, with and without S9-mix

 

Experiment 2 (plate-incorporation method without S9 mix; preincubation method with S9 mix):

- TA1535, TA1537, TA98, TA100 and TA102: 25, 50, 100, 200, 400, 800 and 1600 μg/plate, with and without S9-mix

 

Experiment 3 (preincubation method):

- TA1535, TA1537, TA98, TA100 and TA102: 6.25, 12.5, 25, 50, 100, 200 and 400 μg/plate, with S9-mix

 

Metabolic activation system was S9 fraction prepared from liver homogenates of male Sprague Dawley rats induced with Aroclor 1254. Vehicle and positive controls were also included.

 

In Experiment 1, evidence of toxicity was observed at 160 and/or 500 μg/plate and above in all strains in the absence and presence of S-9. In Experiment 2, evidence of toxicity was observed at 800 μg/plate and above in all strains in the absence of S-9 and at 400 μg/plate and above in the presence of S-9 in all strains and in strain TA1537 in the absence of S-9 only. Since mutation data were only available for four concentrations in the presence of S-9 for all tester strains due to toxicity, a further Experiment (Experiment 3) was performed. In Experiment 3, evidence of toxicity was observed at 200 μg/plate and above in all strains.

 

The mean numbers of revertant colonies fell within acceptable ranges for vehicle control treatments, and were elevated by positive control treatments. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation. 

 

Therefore, the test item is not considered as mutagenic in this bacterial system.