Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 May 2013 to 04 April 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Conducted according to current test guidelines and GLP compliant

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
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Name: Oxypyrion-Acetate
CAS Number: 1742-79-6
Batch number: 11085913
Purity: Not stated
Date of arrival: 20 November 2012
Storage conditions: Refrigerated (2 to 8°C)

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Mammalian liver post-mitochondrial fraction (S-9)
Test concentrations with justification for top dose:
Mutation Experiment 1:
5,000, 15,81, 50,00, 158,1, 500,0, 1581 and 5000 µg/plate (with and without S-9)
Mutation Experiment 2:
15,63 (strains TA98, TA1535 and TA102 only), 31,25, 62,50, 125,0, 250,0, 500,0, 1000 and 2000 (strains TA100 and TA1537 only) µg/plate (without S-9)
31,25, 62,50, 125,0, 250,0, 500,0, 1000 and 2000 µg/plate (with S-9)

Positive controls:
2-nitrofluorene (2NF): 5 µg/plate (TA98, - S-9)
Sodium azide (NaN3): 2 µg/plate (TA100, TA1535, - S-9)
9-aminoacridine (AAC): 50 µg/plate (TA1537, - S-9)
Mitomycin C (MMC): 0,2 µg/plate (TA102, - S-9)
Benzo[a]pyrene (B[a]P): 10 µg/plate (TA98, + S-9)
2-aminoanthracene (AAN): 5 µg/plate (TA100, TA1535, TA1537, + S-9) and 20 µg/plate (TA102, + S-9)
Vehicle / solvent:
Anhydrous analytical grade dimethyl sulphoxide (DMSO)
Controls
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
mitomycin C
other: 2-aminoanthracene
Details on test system and experimental conditions:
Five strains of Salmonella typhimurium bacteria (TA98, TA100, TA1535, TA1537 and TA102) were used in this study. 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. For all assays, bacteria were cultured at 37±1°C for 10 hours in nutrient broth, containing ampicillin (TA98, TA100) or ampicillin and tetracycline (TA102) as appropriate, to provide bacterial cultures in the range of approximately 108 to 109 cells/ml, based on cell count data from testing of each strain batch. Incubation was carried out with shaking in an anhydric incubator, set to turn on using a timer switch. All treatments were completed within 6 hours of the end of the incubation period.
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 (Maron and Ames, 1983) and De Serres and Shelby (De Serres and Shelby, 1979).


Evaluation criteria:
For valid data, the test article was considered to be mutagenic if:
1. When assessed using Dunnett's test, an increase in revertant numbers gave a significant response (p equal or less than 0,01) which was concentration related.
2. The positive trend/effects described above were reproducible.
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none 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:
Not applicable

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Toxicity, solubility and concentration selection:
Experiment 1 treatments of all the tester strains were performed in the absence and in the presence of S 9, using final concentrations of Oxypyrionacetate at 5, 15,81, 50, 158,1, 500, 1581 and 5000 µg/plate, plus negative (vehicle) and positive controls. Following these treatments, 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 500 and/or 1581 µg/plate and above in all strains in the absence and in the presence of S-9.
Experiment 2 treatments of all the tester strains were performed in the absence and in the presence of S-9. For all strains, the maximum test concentrations were reduced based on strain specific toxicity observed in Experiment 1. Narrowed concentration intervals were employed covering the ranges of 15,63 – 1000 µg/plate in strains TA98, TA1535 and TA102 in the absence of S-9 or 31,25 – 2000 µg/plate in strains TA100 and TA1537 in the absence and presence of S-9 and strains TA98, TA1535 and TA102 in the presence of S-9. These narrowed concentration intervals were employed in order to examine more closely those concentrations of Oxypyrionacetate approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre-incubation step. Following Experiment 2 treatments, 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 at 500 µg/plate in strains TA98 and TA102 in the absence of S-9 and at 1000 and/or 2000 µg/plate in all strains in the absence and in the presence of S-9.
The test article was completely soluble in the aqueous assay system at all concentrations treated, in each of the experiments performed.

Mutation:
Following treatments of all the test strains in the absence and presence of S-9, only Experiment 2 treatments of strain TA1537 in the absence of S-9 at 125 µg/plate resulted in an increase in revertant numbers that was statistically significant when the data were analysed at the 1% level using Dunnett’s test. The increase occurred at a single low concentration with no indication of a concentration relationship. Accordingly this increase was considered to have been due to normal biological variability and not evidence of mutagenic activity.
No other increases in revertant numbers were observed that were statistically significant when the data were analysed at the 1% level using Dunnett’s test. This study was considered therefore to have provided no evidence of any Oxypyrionacetate mutagenic activity in this assay system.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
It was concluded that Oxypyrionacetate did not induce mutation in five histidine requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments up to toxic concentrations, in the absence and in the presence of a rat liver metabolic activation system (S-9).
Executive summary:

Oxypyrionacetate was assayed for mutation in five histidine‑requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation by an Aroclor 1254‑induced rat liver post‑mitochondrial fraction (S‑9), in two separate experiments.

All Oxypyrionacetate treatments in this study were performed using formulations prepared inanhydrousanalytical grade dimethyl sulphoxide (DMSO).

Experiment 1 treatments of all the tester strains were performed in the absence and in the presence of S‑9, using final concentrations of Oxypyrionacetate at 5, 15,81, 50, 158,1, 500, 1581 and 5000 µg/plate, plus negative (vehicle) and positive controls. Following these treatments,evidence of toxicity was observed at 500 and/or 1581 µg/plate and above in all strains in the absence and in the presence of S-9.

Experiment 2 treatments of all the tester strains were performed in the absence and in the presence of S-9. For all strains, the maximum test concentrations were reduced based on strain specific toxicity observed in Experiment 1. Narrowed concentration intervals were employed covering the ranges of 15,63 – 1000 mg/plate in strains TA98, TA1535 and TA102 in the absence of S-9 or 31,25 – 2000 µg/plate in strains TA100 and TA1537 in the absence and presence of S-9 and strains TA98, TA1535 and TA102 in the presence of S-9. These narrowed concentration intervals were employed in order to examine more closely those concentrations of Oxypyrionacetate approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. In addition, all treatmentsin the presence of S-9 were further modified by the inclusion of a pre-incubation step. Following Experiment 2 treatments, evidence of toxicity was observed at 500 µg/plate in strains TA98 and TA102 in the absence of S-9 and at 1000 and/or 2000 µg/plate in all strains in the absence and in the presence of S-9.

The test article was completely soluble in the aqueous assay system at all concentrations tested, in each of the experiments performed.

Negative (vehicle) and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies all fell within acceptable ranges for negative control treatments, and were significantly elevated by positive control treatments.

Results of formulation analyses demonstrated achieved concentrationswithin 100±10% of the nominal test article concentrations and were therefore considered acceptable.

No concentration related increases in revertant numbers were observed that were statistically significant when the data were analysed at the 1% level using Dunnett’s test. This study was considered therefore to have provided no evidence of any Oxypyrionacetate mutagenic activity in this assay system.

It was concluded that Oxypyrionacetate did not induce mutation in five histidine‑requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments up to toxic concentrations, in the absence and in the presence of a rat liver metabolic activation system (S‑9).