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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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
3-(2-hydroxyethyl)-p-phenylenediammonium sulphate
EC Number:
298-995-1
EC Name:
3-(2-hydroxyethyl)-p-phenylenediammonium sulphate
Cas Number:
93841-25-9
Molecular formula:
C8H12N2O.H2O4S
IUPAC Name:
3-(2-hydroxyethyl)-p-phenylenediammonium sulphate
Specific details on test material used for the study:
Batch number : 36/37
Purity : 99.8a/a%

Method

Target gene:
Histidine locus
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9
Test concentrations with justification for top dose:
Pre-experiment : 3, 10, 33, 100, 333, 1000, 2500 and 5000 ug/plate
Experiment I and II : 33, 100, 333, 1000, 2500 and 5000 ug/plate
Experiment IIa : 1000, 1500, 2000, 2500, 3000, 3500, 4000 and 5000 ug/plate (TA100 without metabolic activation only)
In the pre-experiment the concentration range of the test item was 3-5000ug/plate. The pre-experiment is reported as part of experiment I since no relevant toxic effects were observed and 5000ug/plate was chosen as the maximum concentration. The concentration range included two logarithmic decades.
Vehicle / solvent:
Deionised water
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: (4-nitro-o-phenylenediamine), [2-aminoanthracene]
Details on test system and experimental conditions:
For each strain and dose level, including the controls three plates were used.

The following materials were mixed in a test tube and poured onto the selective agar plates
- 100uL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control),
- 500uL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
- 100uL bacteria suspension
- 2000uL overlay agar

In the pre-incubation assay 100uL test solution, 500uL S9 mix/S9 mix substitution buffer and 100uL bacterial suspension were mixed in a test tube and incubated at 37 deg C for 60 minutes. After pre-incubation 2.0mL overlay agar (45 deg C) was added to each tube. The mixture was poured onto agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37 deg c in the dark.

The colonies were counted using Petri Viewer Mk2 with the software program Ames Study Manager.
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA98, TA100, TA102) or thrice (strains TA1535, TA1537) the colony count of the corresponding solvent control is observed. A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration. An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment. A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Results and discussion

Test results
Species / strain:
S. typhimurium, other: TA1535, TA1537, TA98, TA100, TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Since the data of the controls in strain TA102 in experiment I were far above the historical control data of the laboratory, this part had to be repeated (reported as part of experiment I). Due to questionable results in experiment II in strain TA100 without metabolic activation, this part had to be repeated (reported as experiment IIa). Each concentration, including the controls, was tested in triplicate. The plates incubated with the test item showed normal background growth up to the highest concentration in all strains used with and without metabolic activation.

A reduction in the number of revertants occurred in experiment II with metabolic activation in strain TA1535 at 1000ug/plate and in strain TA1537 at 100ug/plate and 333ug/plate. Since this reduction is not dose dependent, it is not judged as a true toxic effect.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any concentration level, neither in the presence nor in the absence of metabolic activation. A single increase of the revertant colonies was observed in experiment II in strain TA100 without metabolic activation. The number of colonies exceeded the threshold of twice (strain TA100) the number of the corresponding solvent control at 2500ug/plate. Since there was no dose dependency observed, the isolated finding at 2500ug/plate was considered questionable. It was therefore decided to perform a confirmatory experiment under identical conditions (TA100, pre-incubation assay without metabolic activation). This additional experiment showed no biologically relevant increases in revertant colony numbers. Consequently, the isolated increase that was seen in TA100 in experiment II is considered not to be biologically relevant.

Appropriate reference mutagens were used as positive controls. These showed a distinct increase in induced revertant colonies. The historical control range was slightly exceeded in the solvent control (experiments I and II) of strain TA102 with metabolic activation.

The historical control range was slightly exceeded in the solvent control (experiments I and II) of strain TA102 with metabolic activation. In experiment I the historical control range was slightly exceeded in strain TA1535 (negative control) without metabolic activation. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies. Also in strains TA100 and TA102 with metabolic activation the historical control range was slightly exceeded at 100 and 333ug/plate (TA102 experiment I) at 100ug/plate (TA102 experiment II) and at 1500 and 4000 ug/plate (TA100 experiment IIa). Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies. Due to a new evaluation unit, new historical control data were evaluated from 80 experiments. Since this number of experiments is rather small, the range can be exceeded in some experiments.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Applicant's summary and conclusion

Conclusions:
Under the test conditions employed hydroxyethyl-p-phenylenediamine sulfate did not induce gene mutations in bacteria.
Executive summary:

The study was performed to investigate the potential og hydroxy-p-phenylenediamine sulfate to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, TA102. The ssay was performed with and without liver microsomal activation. Since the data of the controls in strain TA102 in experiment I were far above the historical control data of the laboratory, this part had to be repeated (reported as part of experiment I). Due to questionable results in experiment II in strain TA100 without metabolic activation, this part had to be repeated (reported as experiment IIa). Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations : Pre-experiment : 3, 10, 33, 100, 333, 1000, 2500 and 5000 ug/plate ; Experiment I and II : 33, 100, 333, 1000, 2500 and 5000 ug/plate ; Experiment IIa : 1000, 1500, 2000, 2500, 3000, 3500, 4000 and 5000 ug/plate. The plates incubated with the test item showed normal background growth up to the highest concentration in all strains used with and without metabolic activation. A reduction in the number of revertants occurred in experiment II with metabolic activation in strain TA1535 at 1000ug/plate and in strain TA1537 at 100ug/plate and 333ug/plate. Since this reduction is not dose dependent, it is not judged as a true toxic effect. No biologically relevant increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any concentration level, neither in the presence nor in the absence of metabolic activation. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, hydroxyethyl-p-phenylenediamine sulfate is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

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