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Toxicological information

Genetic toxicity: in vitro

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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:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1981

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Phenolphthalein
EC Number:
201-004-7
EC Name:
Phenolphthalein
Cas Number:
77-09-8
Molecular formula:
C20H14O4
IUPAC Name:
phenolphthalein

Method

Target gene:
HIS operon (S. thyphimurium)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
S. typhimurium TA 1537
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
A supernatant fraction of homogenised liver centrifuged at 9000 g/10 min (S9) was prepared from Sprague-Dawley rats pretreated with Aroclor 1254 (Ames et al., 1975). A single batch of homogenized liver S9 fraction was employed in this study. Each day fresh S9 mix was prepared consisting of the following ingredients: MgCl2 (0.4 M), 20 µL, KCl (1.65 M) 20 µL, Glucose-6-phosphate (0.5 M) 5 µL, NADP (0.05 M) 40 µL, phosphate buffer (0.2 M, pH 7.4) 815 µL, S9 fraction 100 µL.This mix was stored on ice until required, then incorporated together with the test material and each of the bacteria strains as follows: 100 µL bacterial broth culture, 100 µL test compound solution in DMSO at various concentrations, and 500 µL S9 mix as required were added to each 2 mL of top agar at 42 °C
Test concentrations with justification for top dose:
0, 32, 100, 320, and 1000 µg/plate
Vehicle / solvent:
DMSO
Controlsopen allclose all
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Aflatoxin B1
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: daunomycin
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate

METHOD OF TREATMENT/ EXPOSURE:
plate incorporation test; no further data available

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: Plates were incubated at 37 °C for 72 h before counting

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: no data available
Evaluation criteria:
(a) dose dependent response
(b) reproducibility of the result
Statistics:
no data

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
without S9 at 1000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
without S9 at 1000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
without S9 at 1000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
without S9 at 1000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
without S9 at 1000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not examined
Positive controls validity:
valid

Any other information on results incl. tables


Dose
[µg/plate]
TA98 TA100 TA1535 TA1537 TA1538
NA 20 %
RLI
NA 20 %
RLI
NA 20 %
RLI
NA 20 %
RLI
NA 20 %
RLI
  - - - - - - - - - -
1000 t 44 t 105 t 14 t 7 t 13
320 32 47 154 134 28 21 8 16 23 31
100 30 46 139 124 39 24 9 12 20 28
32 31 47 145 126 35 22 10 17 17 26
0 29 40 146 116 36 22 10 17 21 33

Abbreviations
NA, not activated
RLI, Aroclor 1254-induced rat liver S-9
t, indicates toxicity
- , nonmutagenic.

Applicant's summary and conclusion

Conclusions:
With and without addition of S9 mix as the external metabolizing system, the test item was not mutagenic under the experimental conditions described.
Executive summary:

The purpose of this assay was to provide information on the genotoxic potential of the test item. The investigations for the mutagenic potential of the test item were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 1538, TA 1535 and TA 1537. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254 -pretreated rats was used. Two independent experimental series were performed with and without S9 mix, respectively. The test item was dissolved in DMSO and tested at concentrations ranging from 32 to 1000 µg/plate. Toxicity to the bacteria was observed at 100 µg/plate without S9 mix. Daunomycin, 9 -aminoacridine, aflatoxin B1, 2 acetylaminofluorene, 4 -N-nitroquinoline-N-oxide, and methylmethansulfonate served as strain specific positive control test materials. Each treatment with the test materials used as positive controls led to a clear increase in revertant colonies, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used. In both series of experiments, each performed with and without the addition of rat liver S9 mix as the external metabolizing system, the test item showed no increase in the number of revertants of any bacterial strain. These published data clearly indicate that the test item was not mutagenic under the described experimental conditions.

With and without addition of S9 mix as the external metabolizing system, the test item was not mutagenic under the experimental conditions described.