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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:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
6th February 2012 to 21st May 2012.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted according to relevant testing guidelines. There was a minor protocol deviation, but this was not considered to have affected the validity of the study.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Minor protocol deviation: In Experiment 1, mean number of revertants in the vehicle control for strain TA1537 without S-9 was below the 99% confidence interval and 1 of the individual vehicle counts was outside the 99% reference range.
Principles of method if other than guideline:
Not applicable.
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Fatty acids, C18-unsatd., dimers, oligomeric reaction products with tall-oil fatty acids and triethylenetetramine
EC Number:
500-191-5
EC Name:
Fatty acids, C18-unsatd., dimers, oligomeric reaction products with tall-oil fatty acids and triethylenetetramine
Cas Number:
68082-29-1
IUPAC Name:
Fatty acids, C18-unsatd, dimers, polymers with tall-oil fatty acids and triethylenetetramine
Constituent 2
Reference substance name:
TOFA_DimerFA_TETA_PAA
IUPAC Name:
TOFA_DimerFA_TETA_PAA
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): TOFA_DimerFA_TETA_PAA
- Physical state: Brown liquid.
- Analytical purity: 100% (UVCB)
- Lot/batch No.: BB 001030 V1
- Storage condition of test material: Stored at 15-30°C protected from light in a tightly closed container

Method

Target gene:
Histidine
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
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).
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix.
Test concentrations with justification for top dose:
Experiment 1: 5, 15.81, 50, 158.1, 500, 1581 and 5000 μg/plate
Experiment 2: 2.62, 6.55, 16.38, 40.96, 102.4, 256, 640 and 1600 μg/plate
Vehicle / solvent:
- Vehicle used: acetone
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Without metabolic activation (S9 mix)

Migrated to IUCLID6: TA98; 5 μg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without metabolic activation (S9 mix)

Migrated to IUCLID6: TA100 and TA1535; 2 μg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Without metabolic activation (S9 mix)

Migrated to IUCLID6: TA1537; 50 μg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Without metabolic activation (S9 mix)

Migrated to IUCLID6: TA102; 0.2 μg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With metabolic activation (S9 mix)

Migrated to IUCLID6: TA98; 10 μg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
TA100, TA1535 and TA 1537; 5μg/plate
Positive control substance:
other: 2-aminoanthracene
Remarks:
With metabolic activation (S9 mix)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
TA102; 20μg/plate
Positive control substance:
other: 2-aminoanthracene
Remarks:
With metabolic activation (S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) and pre-incubation.

In the first experiment, the platings were achieved by the addition of 0.1 mL bacterial culture, 0.05 mL test article solution or vehicle control or 0.1 mL positive control and 0.5 mL 10% S-9 mix or buffer solution to 2.5 mL molten agar at 46±1°C. This was then mixed rapidly and poured on to Vogel-Bonner E agar plates. When set, the plates were inverted and incubated at 37±1°C in the dark for 2 or 3 days. Following incubation, these plates were examined for evidence of toxicity to the background lawn, and where possible revertant colonies were counted.

In Experiment 2, a pre-incubation step was included prior to plate incorporation. Quantities of test article or vehicle control solution (reduced to 0.025 mL) or positive control solution (reduced to 0.05 mL), bacteria and S9 mix, were mixed together and incubated for 20 minutes at 37±1°C, before the addition of 2.5 mL molten agar at 46±1°C. Plating of these treatments then proceeded as for the normal plate-incorporation procedure.

NUMBER OF REPLICATIONS: Triplicate plates were used at each concentration level. Negative (vehicle) controls were included in quintuplicate and positive controls were included in triplicate in both assays with and without S9 mix.

Colonies were counted electronically using a Sorcerer Colony Counter or manually where confounding factors such as bubbles or splits in the agar 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. When assessed using Dunnett's test, an increase in revertant numbers gave a significant response (p≤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 within and between concentrations and between experiments.
Statistics:
Dunnett's test was used to compare the counts at each concentration with the control. The presence or otherwise of a concentration response was checked by non-statistical analysis, up to limiting levels.

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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In Experiment 1, there was evidence of toxicity which ranged from a slight thinning of the background bacterial lawn, with or without a concurrent reduction in revertant numbers, to a complete killing of the test bacteria which was observed at 158.1 μg/plate and above in strain TA1537 in the absence of S9; 500 μg/plate and above in strains TA98 and TA100 in the absence of S9, strain TA1537 in the presence of S9 and strains TA1535 and TA102 in the absence and presence of S9; and 1581 μg/plate and above in strains TA98 and TA100 in the presence of S9. Precipitation was observed on the test plates at 5000 μg/plate in the absence and presence of S9.

In Experiment 1, the mean number of revertants in the vehicle control for strain TA1537 in the absence of S9 was below the 99% confidence interval and one of the individual vehicle counts was outside the 99% reference range. Since the value was only marginally below the 99% confidence interval and four of the five remaining individual vehicle counts were within the reference range, these data were considered to be characteristic of the strain. Therefore the correct strain and assay functioning was confirmed and the data were accepted as valid.

In Experiment 2, evidence of toxicity ranging from a slight thinning of the background bacterial lawn, with or without a reduction in revertant numbers, to a complete killing of test bacteria was observed at 256 μg/plate and above in strains TA1535 and TA1537 in the absence of S9 and 640 μg/plate and above in strains TA98, TA100 and TA102 in the absence and presence of S9 and strains TA1535 and TA1537 in the presence of S9. Precipitation was observed at 1600 μg/plate in all strains in the presence of S9 only.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

No additional information.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation.

Under the conditions of this study, it was determined that TOFA_DimerFA_TETA_PAA did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested in the presence and absence of metabolic activation.
Executive summary:

The mutagenic potential of TOFA_DimerFA_TETA_PAA was evaluated in five histidine-requiring strains of Salmonella typhimurium (TA98, TA100, TA102, TA1535 and TA1537) according to OECD Test Guideline 471. The bacterial strains were tested in the presence and absence of metabolic activation (S9 mix) in two separate experiments involving the plate incorporation method and pre-incubation. In Experiment 1, the test material was tested at concentrations of 5, 15.81, 50, 158.1, 500, 1581 and 5000 μg/plate with negative and positive controls. Evidence of toxicity was observed at 158.1 μg/plate and above strain TA1537 in the absence of S9; 500 μg/plate and above in strains TA98 and TA100 in the absence of S9, strain TA1537 in the presence of S9 and strains TA1535 and TA102 in the absence and presence of S9; and 1581 μg/plate and above in strains TA98 and TA100 in the presence of S9.

In Experiment 2, a pre-incubation step was included prior to plate incorporation. At the concentrations used in experiment 2 (for strain TA102 in the presence and absence of S9 mix: 2.62, 6.55, 16.38, 40.96, 102.4, 256 and 640 μg/plate and for remaining bacterial strains in the presence and absence of S9 mix: 2.62, 6.55, 16.38, 40.96, 102.4, 256, 640 and 1600 μg/plate), evidence of toxicity was observed at 256 μg/plate and above in strains TA1535 and TA1537 in the absence of S9 and 640 μg/plate and above in strains TA98, TA100 and TA102 in the absence and presence of S9 and strains TA1535 and TA1537 in the presence of S9.

Under the conditions employed in this study, the test material, TOFA_DimerFA_TETA_PAA did not induce mutation in five histidine-

requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, in the presence and absence of S9 mix.