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Toxicological information

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:
experimental study
Adequacy of study:
key study
Study period:
6th June to 14th July 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Comparable to OECD guideline and conducted to GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Phenol, (tetrapropenyl) derivatives (CAS No. 74499-35-7)

Test material purity not provided.

Method

Target gene:
Histidine synthesis
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
histidine-deficient strains of Salmonella typhimurium
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
0.1, 0.33, 1.0, 3.33 and 10.0 mg/plate with and without activation
Vehicle / solvent:
The test material was diluted with 25% Pluronic F127 (w/w in ethanol)
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: With S9: 2-aminoanthracene(2 µg); without S9: TA98 = 2-nitrofluorene (10 µg), TA100/1535 = Sodium azide (1 µg), TA1537 = ICR-191 (2 µg)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium.
Histidine-deficient strains of Salmonella typhimurium were grown on medium that contained only minimal amounts of histidine and biotin. Only bacteria that reverted and were able to synthesize histidine were expected to grow into colonies after 2-3 days incubation at 37°C. The number of colonies per plate was an estimate of the mutation rate.

DURATION
The bacterial tester strain culture was grown by inoculating nutrient broth with a tester strain and incubating with shaking at 37 ± 1°C to a density of 1-2 x 10ˆ9 cells/ml. Without metabolic activation, 100 µl of tester strain and 100 µl of solvent or test material were added to 2.5 ml of molten selective top agar at 45 ± 1°C. With metabolic activation, 100 µl of tester strain, 100 µl of solvent or test material, and 0.5 ml of S-9 mix were added to 2.0 ml of molten selective top agar at 45 ± 1°C. After vortexing, the mixture was poured onto the surface of 25 ml of minimal bottom agar. After the overlay had solidified, the plates were inverted and incubated for 48 hours at 37 ± 1°C. The number of revertant colonies was counted and the condition of the bacterial lawn was noted. Solvent and positive controls were run concurrently.
All dose levels were plated in triplicate. Results were confirmed in an independent experiment.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
Criteria for a Valid Test: The following criteria were met before the results of the mutagenicity assay were judged as positive or negative:
(1) the high dose showed some toxicity, or was 10 mg/plate or the limit of solubility;
(2) the spontaneous controls were within the laboratory's acceptable range;
(3) the positive controls produced at least a three-fold increase in the number of revertants over the values for the respective negative controls.

Repsonse criteria:
NEGATIVE: Does not meet criteria for equivocal {+/-) or positive (+,++,+++).
+/- EQUIVOCAL: Two consecutive dose levels produced less than 2-fold (2.5-fold for TA1535, TA1537, aged TA1538) but statistically significant responses that were dose responsive .
+ WEAKLY POSITIVE: Three consecutive dose levels produced less than 2-fold (2.5-fold for TA1535, TA1537, aged TA1538) but statistically significant responses that were dose responsive or meets criteria for "positive" response but lacks a dose response.
++ POSITIVE: Two consecutive dose levels (or the highest non-toxic dose level) produced responses at least twice (2.5-fold for TA1535, TA1537, TA1538) that of the negative/solvent control and these consecutive doses showed a dose-response relationship.
+++ STRONGLY POSITIVE: Two consecutive dose levels (or the highest non-toxic dose level) produced responses at least five times that of the negative/solvent control and these consecutive doses showed a dose response relationship.

Significant response (p≤0.05): treated mean minus one maximal standard deviation > control mean plus one maximal standard deviation. Maximal standard deviation - observed standard deviation or counting (Poisson) error, whichever is greater.
Statistics:
No data

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
The test material appeared to form a stable emulsion with the Pluronic F127 and the dilutions appeared to be well dispersed in the top agar; however, after incubation, test material was observed on the surface of the agar at > 1.0 mg/plate.

RANGE-FINDING/SCREENING STUDIES:
The test material was checked for toxicity on strain TA100 with and without S-9 mix at dose levels of 0.003 to 10 mg/plate at half-log intervals. Toxicity was observed at 10 mg/plate with S9 and ≥3.3 mg/plate without S9.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Testing at 3.3 mg/plate with 25-400 µl S-9/plate on strains TA98 and TA100 indicated that increasing the concentration of S-9 did not significantly increase the induction of revertants; therefore, 25 µl S-9/plate was used for the mutagenicity assay.
As a result of the toxicity test, dose levels of 0.1 to 10 mg/plate were selected for the mutagenicity assays.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

No statistically significant increases in mutant frequency were observed in the test material. Under the conditions tested, at dose levels of 0.1 to 10 mg/plate, the test material was not mutagenic to TA98, TA100, TA1535, or TA1537 with or without metabolic activation.

The tester strains responded to the positive controls, known mutagens, as expected.

The test material was cytotoxic at 10 mg/plate to TA100 with and without S-9 and at ≥3.3 mg/plate to TA1353 with S9.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the conditions of this study, the test material was not mutagenic.
Executive summary:

The test material was diluted with 25% Pluronic F127 (w/w in ethanol) and tested in the histidine-deficient strains of Salmonella typhimurium TA98, TA100, TA1535, and TA1537 at dose levels of 0.1 to 10 mg/plate with and without metabolic activation provided by Aroclor-induced rat liver S-9. The test material appeared to form a stable emulsion with Pluronic F127 and the dilutions were well dispersed in the top agar; however, after incubation, test material was observed on the surface of the top agar at 10 mg/plate. The test material was cytotoxic at 10 mg/plate to TA100 with and without S-9 and at > 3.3 mg/plate to TA1535 with S-9.

No statistically significant increases in mutant frequency were observed in any strain. Under the conditions tested, the test material was not mutagenic to strain TA98, TA100, TA1535, or TA1537 with or without metabolic activation.