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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2012-2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: Ajinomoto Co., Inc.
- Lot/batch No.of test material: 110825
- Expiration date of the lot/batch: 25.08.2014
- Purity: 100%

Method

Species / strainopen allclose all
Species / strain / cell type:
other: TA 98, TA 100, TA 1535, TA 1537, TA 1538
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver
Test concentrations with justification for top dose:
3.16 - 1000 µg/plate
Vehicle / solvent:
water for injection
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 2-amino-anthracene
Details on test system and experimental conditions:
The test item was completely dissolved in water for injection and the vehicle served as the negative control.
Prior to the main test two preliminary cytotoxicity tests (plate incorporation test, without and with metabolic activation) were carried out in test strain TA 100.
In the main study 6 different concentrations of the test item were tested, with half-log intervals between plates (i.e. 3.16, 10.0, 31.6, 100, 316 and 1000 µg per plate).
Evaluation criteria:
In this study, the test item was considered to show a positive response if:
the number of revertants is significantly increased compared to the solvent control to at least 2-fold of the solvent control for TA 98, TA 100 and Escherichia coli and 3-fold of the solvent control for TA 1535, TA 1537 and TA 1538 in both independent experiments
a significant concentration (log value)-related effect is observed
positive results have to be reproducible and the histidine or tryptophan independence of the revertants has to be confirmed by streaking random samples on histidine- or tryptophan-free agar plates.
Cytotoxicity is defined as a reduction in the number of colonies by more than 50% compared with the solvent control and/or a scarce background lawn.
Statistics:
yes

Results and discussion

Test results
Key result
Species / strain:
other: TA 98, TA 100, TA 1535, TA 1537, TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1000 µg/plate
Vehicle controls validity:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
The test item showed no mutagenic effect neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation when tested up to a cytotoxic concentration of 1000 µg/plate in the Salmonella typhimurium strains TA 98, TA 100, TA 1535 TA 1537 and TA 1538 and the Escherichia coli strain WP2 uvr A.
Executive summary:

In a study performed according to OECD Guideline 471 with GLP comliance, the test item was examined in the 5 Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 and in the Escherichia coli strain WP2 uvr A in two independent experiments, each carried out without and with metabolic activation. The first experiment was carried out as a plate incorporation test and the second as a preincubation test.

The test item was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA 100. Ten concentrations ranging from 0.316 to 5000 µg/plate were tested. Pronounced cytotoxicity was noted starting at a concentration of 1000 µg/plate. Hence, 1000 µg/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test, respectively. Six concentrations ranging from 3.16 to 1000 µg/plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation.

In the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation cytotoxicity was noted at the top concentration of 1000 µg/plate, in all Salmonella typhimurium strains and in the Escherichia coli strain WP2 uvr A.

No mutagenic effect was observed tested up to a cytotoxic concentration of 1000 µg/plate, in the Salmonella typhimurium and in the Escherichia coli test strains in two independent experiments without and with metabolic activation, respectively.