Registration Dossier

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:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
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Constituent
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Constituent
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Constituent
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Constituent
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Constituent
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Constituent
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Constituent
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Constituent
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Constituent
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
Identity: BACCARTOL CRUDE
Batch no.: VE00364716
Expiry date: 02 April 2017
Storage conditions: Room temperature, protected from light
RTC number: 14844

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Test concentrations with justification for top dose:
Toxicity Test
The test item BACCARTOL CRUDE was assayed in the toxicity test at a maximum concentration of 5000 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 µg/plate. No precipitation of the test item was observed at the end of the incubation period at any concentration.

Main Assays
Three Main Assays were performed. The maximum concentration of the test item to be used in the main experiments was selected as the concentration which elicits a moderate toxicity. The number of lower dose levels included in each treatment series was selected in order to have a sufficient number of analysable concentrations (at least three concentrations without toxicity).
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-Aminoanthracene
Details on test system and experimental conditions:
One batch of S9 tissue fraction, provided by Trinova Biochem GmbH, was used in this study and had the following characteristics:
Species: Rat
Strain: Sprague Dawley
Tissue: Liver
Inducing Agents: Phenobarbital – 5,6-Benzoflavone
Producer: MOLTOX,Molecular Toxicology, Inc.
Batch Number: 3488
Evaluation criteria:
Acceptance criteria
The assay was considered valid if the following criteria were met:
1. Mean plate counts for untreated and positive control plates should fall within 2 standard deviations of the current historical mean values.
2. The estimated numbers of viable bacteria/plate should fall in the range of 100 – 500 millions for each strain.
3. No more than 5% of the plates should be lost through contamination or other unforeseen event.

Criteria for outcome of the assays
For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.

Evaluation
Results show that mean plate counts for untreated and positive control plates fell within the normal range based on historical control data. The estimated numbers of viable bacteria/plate (titre) fell in the range of 100 - 500 million for each strain. No plates were lost through contamination or cracking. The study was accepted as valid.
The test item did not induce two-fold increases in the number of revertant colonies, at any dose level, in any tester strain, in the absence or presence of S9 metabolism.

Results and discussion

Test results
Species / strain:
other: TA1535, TA1537, TA98, TA100 and a strain of Escherichia coli (WP2 uvrA)
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:
Toxicity test
No precipitation of the test item was observed at the end of the incubation period at any concentration. Toxicity, as indicated by reduction in revertant colonies, thinning of the background lawn and/or microcolony formation was noted at higher concentrations with all tester strains, in the absence and presence of S9 metabolism. A more pronounced toxic effect was noted with TA100 tester strain.

Main Assays

In Main Assay I, using the plate incorporation method, the test item was assayed at the following dose levels:
Tester strain S9 Dose level (µg/plate)
TA1535, TA1537 ± 2500, 1250, 625, 313 and 156
TA98 ± 5000, 2500, 1250, 625, 313 and 156
WP2 uvrA − 5000, 2500, 1250, 625, 313 and 156
WP2 uvrA + 5000, 2500, 1250, 625 and 313
TA100 − 2500, 1250, 625, 313, 156, 78.1 and 39.1
TA100 + 2500, 1250, 625, 313, 156 and 78.1

Toxicity, as indicated by thinning of the background lawn and/or reduction in revertant number, was observed at dose levels of 2500 and 1250 µg/plate with TA1535, TA 1537 and TA100 tester strains, both in the absence and presence of S9 metabolic activation. A more pronounced toxic effect was observed with TA100 tester strain in the absence of S9 metabolism, where slight thinning of the background lawn was noted also at 625 µg/plate. No toxicity was observed with WP2 uvrA, while slight toxicity was noted with TA98 tester strain at 5000 µg/plate, both in the absence and presence of S9 metabolism. As no relevant increase in revertant numbers was observed at any concentration tested, a pre-incubation step was included for all treatments of Main Assay II. The dose-range was slightly modified to take into account the toxicity results of Main Assay I.

Main Assay II:

Tester strain S9 Dose level (µg/plate)
TA1535, TA1537 ± 2000, 1000, 500, 250, 125 and 62.5
TA100 ± 2000, 1000, 500, 250, 125, 62.5 and 31.3
WP2 uvrA ± 5000, 2500, 1250, 625 and 313
TA98 ± 5000, 2500, 1250, 625, 313 and 156

Toxicity, as indicated by thinning of the background lawn, microcolony formation and/or reduction in revertant number, was observed at all or almost all concentrations tested, in the absence and presence of S9 metabolism, with all tester strains with the exception of TA100, where a sufficient number of analysable concentrations was obtained. In order to analyse the test item at not cytotoxic concentration, a Main Assay III was performed with TA1535, TA1537, TA98 and WP2 uvrA tester strains, in the absence and presence of S9 metabolism, using the pre-incubation method.

Main Assay III:
Tester strain S9 Dose level (µg/plate)
TA1535, TA1537 − 250, 125, 62.5, 31.3 and 15.6
TA1535, TA1537 + 62.5, 31.3, 15.6, 7.81, 3.91 and 1.95
WP2 uvrA, TA98 + 250, 125, 62.5, 31.3, 15.6 and 7.81
WP2 uvrA − 500, 250, 125, 62.5, 31.3 and 15.6
TA98 − 500, 250, 125, 62.5 and 31.3

Toxicity, as indicated by thinning of the background lawn, was observed at the highest or two highest dose levels with all tester strains, in the absence and presence of S9 metabolism. No precipitation of the test item was observed at the end of the incubation period at any
concentration, in any experiment. No relevant increase in the number of revertant colonies was observed in any experiment, using the plate incorporation or pre-incubation method, at any dose level, with any tester strain, in the absence or presence of S9 metabolism.

The sterility of the S9 mix and of the test item solutions was confirmed by the absence of colonies on additional agar plates spread separately with these solutions. Marked increases in revertant numbers were obtained in these tests following treatment with the positive control items, indicating that the assay system was functioning correctly.
Remarks on result:
other: results provided in 'Additional information on results'

Applicant's summary and conclusion

Conclusions:
It is concluded that the test item BACCARTOL CRUDE does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.