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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:
1995-04-24 to 1995-07-11
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline (OECD 471) study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(1983)
Deviations:
yes
Remarks:
see below
Principles of method if other than guideline:
Ames et al. (1975), Mutation Research 31, 347-364; latest OECD guideline 471 requires additional bacterial strain: E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA 102.
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: flakes

Method

Target gene:
The amino acid histidine locus is the target gene in the selected Salmonella typhimurium strains (TA 1537, TA 1535, TA 100, TA 98), where back mutations will transform the histidine auxotrophy (his-) to histidine prototrophy (his+). The strains were specifically constructed to differentiate between mutations of base pairs (TA 1535, TA 100) and frameshifts (TA 1537, TA 98).
Species / strain
Species / strain / cell type:
other: S. typhimurium TA 98, TA 100, TA 1535, TA 1537
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9
Test concentrations with justification for top dose:
0, 50, 160, 500, 1600, or 5000 µg/plate (both: experiment I and II)
Vehicle / solvent:
Solvent used: bidest water
Vehicles for respective pos. controls:
-S9-mix: DMSO for TA 98 TA and 1537, aqua bidest for TA 100 and TA 1535;
+S9-mix: DMSO for all four Salmonella strains.
Controls
Untreated negative controls:
yes
Remarks:
aqua bidest
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: (-S9-mix): 2-Nitrofluorene for TA 98, sodium azide for TA 100 and TA 1535, 9-aminoacaridine with TA 1537; (+S9-mix): 2-aminoanthracene for all four strains (TA 98, TA 100, TA 1535 and TA 1537).
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation twice); no pre-incubation method

DURATION
- Incubation duration: 72 hrs at 37°C

NUMBER OF REPLICATIONS: each concentration in triplicate, full repeat in Exp. II

DETERMINATION OF CYTOTOXICITY
- Method: reduction of revertant colony no.'s or clearing of background lawn
Evaluation criteria:
- Solvent control with characteristic numbers of spontaneous revertants for each tester strain
- Overnight titres must be in excess of 10 to the 8 bacteria/mL
- Mean of positive controls must exhibit at least 3-fold increase compared to respective negative control
- at least four non-toxic dose levels necessary for evaluation
Statistics:
No statistics

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test substance induced a mutagenic effect in S. typhimurium strains TA 100 and TA 1537 with metabolic activation (S9 mix), which was confirmed in two independent experiments. Therefore, under the conditions of this S. typhimurium reverse mutation assay, 4-OH-TEMPO is considered to be a weak bacterial mutagen, including base-pair and frameshift mutations after metabolic activation by rat liver S9 mix.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not detected
- Precipitation: no precipitate observed
- Other confounding effects: none reported
Remarks on result:
other: other: S. typhimurium TA 98, TA 100, TA 1535, TA 1537
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive with metabolic activation

The genetic toxicity in vitro of 4-Hydroxy TEMPO was tested in a GLP compliant OECD 471 guideline study (Ames test). Classification: weakly mutagenic with metabolic activation.