Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptably documented collaborative study according to scientifically reasonable procedures (Ames test) which included also the registered substance.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1996
Report date:
1996

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: as described by Maron and Ames: Maron, D.M. and B.M. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215.
Deviations:
no
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Pyridine-2-carbaldehyde oxime
EC Number:
212-849-6
EC Name:
Pyridine-2-carbaldehyde oxime
Cas Number:
873-69-8
Molecular formula:
C6H6N2O
IUPAC Name:
pyridine-2-carbaldehyde oxime
Constituent 2
Reference substance name:
Pikolinealdoxime
IUPAC Name:
Pikolinealdoxime
Test material form:
not specified
Details on test material:
- Name of test material (as cited in study report): Pikolinealdoxime
- Substance type: pure substance
- Other: Source: Wako Pure Chemical Industries, Ltd., Osaka (Japan)

Method

Target gene:
his (S. typhimurium) / trp (E. coli)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 102
Species / strain / cell type:
S. typhimurium, other: TA2638
Species / strain / cell type:
E. coli, other: WP2/pKM101
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and 5,6-benzoflavone induced Sprague-Dawley rat liver S9
Test concentrations with justification for top dose:
0, 62.5, 78, 125, 156, 200, 250, 313, 400, 500, 600, 625, 800, 1000, 1250 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
yes
Remarks:
vehicle control / water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
other: 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide; 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48h

SELECTION AGENT (mutation assays): his / trp minimal agar

NUMBER OF REPLICATIONS: The chemicals were tested in at least two independent experiments using five dose levels and three plates per dose, and the tests were performed in two laboratories per chemical to assess reproducibility.

DETERMINATION OF CYTOTOXICITY
- Method: toxicity to the background lawn and/or a reduction in the number of revertant colonies
Statistics:
The results were analyzed for statistical significance using a linear regression test. This statistical analysis recommended by UKEMS was conducted based on the dose-response relationship and carried out at the 1% significance level. Doses with observed cytotoxicity, which was judged by a toxicity to the background lawn and/or a reduction in the number of revertant colonies, were excluded from the statistical analysis.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
positive in 2/2 laboratories according to the present publication
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not specified
Species / strain:
S. typhimurium, other: TA2638
Metabolic activation:
with
Genotoxicity:
negative
Remarks:
negative in 2/2 laboratories
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not specified
Species / strain:
E. coli, other: WP2/pKM101
Metabolic activation:
with
Genotoxicity:
negative
Remarks:
negative in 2/2 laboratories
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not valid
Untreated negative controls validity:
valid
Positive controls validity:
not specified
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
positive in 2/2 laboratories according to the present publication
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not specified

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
ambiguous with metabolic activation

Within an acceptably documented collaborative (inter-laboratory) study according to scientifically reasonable procedures (Ames test) which included also the registered substance, but was initially aiming to identify a suitable fifth strain to complement the existing four standard strains in the Ames test. Hence, two of the employed strains in this test (S. typhimurium TA2638 and E. coli WP2/pKM101) do not represent the currently recommended ones, but are nevertheless suitable to detect mutagens as shown in this study. Also, S. typhimurium TA102 and E. coli WP2 uvrA/pKM101 are currently recommended. Further, testing of Pikolinealdoxime was only performed in the presence of a metabolic activation system, but this deviation from the currently recommended procedures is considered minor as it mimics the situation in humans (mammals) more precisely than testing without. Hence, the performance of the study is considered scientifically reasonable and the results are sufficiently reliable to asses the potential of Pikolinealdoxime to induce mutations in bacteria.
However, care should be taken with the interpretation of the results. In the present publication the criteria for a positive result are not mentioned. Nevertheless, it is denoted that Pikolinealdoxime was tested positive in strains S. typhimurium TA102 and E. coli WP2 uvrA/pKM101. As no criteria are mentioned and these strains are part of the recent OECD 471 testing guideline, it seems most reasonable to consult those criteria and commonly accepted cut-off values to re-evaluate the conclusions drawn in the present publication. According to OECD TG 471, “there are several criteria for determining a positive result, such as a concentration-related increase over the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system. Biological relevance of the results should be considered first“.
Commonly, a test item is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor  2) in at least one strain can be observed.
As obvious from the presented individual data of mutagenic activity of Pikolinealdoxime, neither a dose-related increase nor an increase 2-fold over background could be observed in two out of four strains, i.e. S. typhimurium TA2638 and E. coli WP2/pKM101. In strain TA102 a very slight dose-related increase in revertants could be observed by both laboratories from the control up to cytotoxic levels, i.e. from 408 to 515 resp. 505 to max. 633. This increase of approx. the 1.25-fold value of the control does not qualify as an 2-fold increase and can hardly be considered biologically significant as such an slight, although dose-dependent, increase can be considered as covered within the biological variance of repair enzymes and should so not be regarded as biologically relevant. Similar effects were seen in laboratory 2 on tester strain E. coli WP2 uvrA/pKM101. Only laboratory 1 detected an increase in this strain from 104 (control) to max. 222 (625 µg/plate). This increase is dose-dependent and two-fold over background, which can be considered as biologically relevant. However, taking into account the fact that this increase was only observed in one strain in one out of two laboratories close to the cytotoxic range, that the other observed increases were only very slight and so hardly biologically relevant, and two out of four strains were tested negative in both laboratories, the assessment of the potential of Pikolinealdoxime to induce gene mutations in bacteria must be considered as ambiguous, based on these results.
Within this publication, not only the genotoxic potential of pyridine-2-carbaldehyde oxime was evaluated, but also the one of Isonicotinaldoxime, i.e. of pyridine-4-carbaldehyde oxime. This substance may be consulted as read-across substance as the only difference between these compounds is the localization of the aldoxim group, in position 2 resp. 4 to the nitrogen of the pyridine ring. Generally, ortho- and para-position are toxicologically and with regard to their reactivity more comparable than ortho- or para-position compared to meta-position. Regarding the effects induced by Isonicotinaldoxime may so additional information on the biological relevance of the effects obtained from Pikolinealdoxime. Isonicotinaldoxime did neither induce a dose-dependent increase in revertants nor a 2-fold one over background in three strains, i.e. S. typhimurium TA102, TA2638 and E. coli WP2/pKM101. However, in E. coli WP2 uvrA/pKM101 an approx. 2-fold, dose-dependent increase was observed by both testing laboratories. So it is obvious that both related substance only induced potentially considerable effects in this strain. In this publication, in total 29 different substances were evaluated. In E. coli WP2 uvrA/pKM101, 17 substances gave a positive result as interpreted by the researchers, whereas only 10 / 7 / 9 gave positive results in TA102 / TA2638 / WP2/pKM101. WP2 uvrA/pKM101 is the only employed strain which is excision repair deficient. Being excision repair deficient enhances the sensitivity of this particular strain, and may lead to (for human risk assessment) irrelevant results as humans are excision repair proficient. Or it may lead to potentially false positive results, the latter potentially based on confounding factors which are less relevant in the other strains.
Consequently, a positive result in this strain should be seen less critical, and the results of the present study should be regarded in a precautionary approach as max. ambiguous, a classification as positive is scientifically not reasonable.
According to REACH Annex VII column 2, “further mutagenicity studies shall be considered in case of a positive result“. As this is not the case here, additional testing is not required, no data gaps were identified and the tonnage-driven data requirements under REACH are fully met.
Executive summary:

In a reverse gene mutation assay in bacteria (Ames test), strains S. typhimurium TA102, TA2638, E. coli WP2/pKM101 and E. coli WP2 uvrA/pKM101 were exposed to Pikolinealdoxime in DMSO at concentrations of 0, 62.5, 78, 125, 156, 200, 250, 313, 400, 500, 600, 625, 800, 1000, 1250 µg/plate in the absence of mammalian metabolic activation (induced rat S9, co-incubation)

 

Pikolinealdoxime was tested up to cytotoxic concentrations. In S. typhimurium TA2638 and E. coli WP2/pKM101, no increase of revertants was seen compared to control. In strains S. typhimurium TA102 and E. coli WP2 uvrA/pKM101 only a slight dose-dependent increase was seen, which only resulted in one out of two tests in a 2-fold increase in mutant colonies in E. coli WP2 uvrA/pKM101 over background. Hence, the results should be considered precautionarily as ambiguous.

 

This study is classified as acceptable. This study satisfies the requirements for a scientifically reasonable bacterial reverse gene mutation data.