Registration Dossier

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labeling and/or risk assessment. p-Phenylenediamine hydrochloride will dissociate in situ, to the chloride ion and its corresponding cation. p-Phenylenediamine will also dissociate in situ to form the corresponding cation. In view of the species being the same regardless of whether the test substance is in its salt form or not, p-phenylenediamine hydrochloride is therefore representative of the substance.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Remarks:
Conducted according to guideline in effect at the time of study conduct.
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Remarks:
Conducted according to guideline in effect at the time of study conduct.
Qualifier:
according to guideline
Guideline:
other: UKEMS Guidelines (1990)
Deviations:
no
Remarks:
Conducted according to guideline in effect at the time of study conduct.
Qualifier:
according to guideline
Guideline:
other: ICH Harmonised Tripartite Guideline (1997)
Deviations:
no
Remarks:
Conducted according to guideline in effect at the time of study conduct.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Details on test material:
- Purity: >99%

Method

Species / strain
Species / strain / cell type:
other: TA98, TA100, TA1535, TA1537, TA102
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254- induced rat liver metabolic activation system (S-9)
Test concentrations with justification for top dose:
Range-finder Experiment and Mutation Experiment 1 Final concentration (μg/plate): 1.6, 8, 40, 200, 1000, 5000
Mutation Experiment 2 Final concentration (μg/plate): 156.3, 312.5, 625, 1250, 2500, 5000
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: purified water
- Justification for choice of solvent/vehicle: test substance is a solid; dilution needed and very soluble in vehicle
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-nitrofluorene (TA98 -S9), sodium azide (TA100 and TA1535 -S9), 9-aminoacridine (TA1537 -S9), Mitomycin C (TA102 -S9), benzo[a]pyrene (TA98 +S9), 2- aminoanthracene (TA100, TA1535, TA1537, and TA102 +S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; For all assays, bacteria were cultured for 10 hours at 37±1°C in nutrient broth (containing ampicillin for strains TA98 and TA100 and ampicillin and tetracycline for strain TA102).

DURATION
- Preincubation period: 2 hours
- Exposure duration: 1 hour at 37±1°C
- Expression time (cells in growth medium): Not reported
- Selection time (if incubation with a selection agent): Not reported
- Fixation time (start of exposure up to fixation or harvest of cells): Not reported

SELECTION AGENT (mutation assays): The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was prepared from male Sprague Dawley rats induced with Aroclor 1254.

NUMBER OF REPLICATIONS: triplicate plates without and with S-9; negative (solvent) controls were included in each assay, in quintuplicate without and with S-9. In each experiment, bacterial strains were treated with diagnostic mutagens in triplicate in the absence of S-9.

NUMBER OF CELLS EVALUATED: Colonies were counted electronically using a Seescan Colony Counter (Seescan plc) or manually where confounding factors such as split agar affected the accuracy of the automated counter.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
The test article was considered to be mutagenic if: 1) the assay was valid, 2) Dunnett's test gave a significant response (p ≤ 0.01) and the data set(s) showed a significant dose correlation, 3) the positive responses described above were reproducible.
Statistics:
The m-statistic was calculated to check that the data were Poisson distributed, and Dunnett's test was used to compare the counts of each dose with the control. The presence or otherwise of a dose response was checked by linear regression analysis.

Results and discussion

Test resultsopen allclose all
Species / strain:
other: TA100, TA1535, TA1537, TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
A slight thinning in the background bacterial lawn was observed at 2500 and 5000 µg/plate (TA102 +S9) and 5000 µg/plate (TA102 -S9).
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
A slight thinning in the background bacterial lawn was observed at 5000 µg/plate (TA98 +S9).
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive

This study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability). The test substance induced mutation in Salmonella typhimurium strain TA98 in the presence of a rat liver metabolic activation system (S-9) when tested under the conditions employed in this study. These conditions included treatments of five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium at concentrations up to 5000 mg/plate, in the absence and in the presence of S-9.
Executive summary:

The test substance was assayed for mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation by an Aroclor 1254-induced rat liver postmitochondrial fraction (S-9), in two separate experiments.

 

An initial toxicity range-finder experiment was carried out in the absence and in the presence of S-9 in strain TA100 only, using final concentrations of the test substance at 1.6, 8, 40, 200, 1000 and 5000 μg/plate, plus negative (solvent) and positive controls. Following these treatments, no evidence of toxicity was observed. Experiment 1 treatments of the remaining test strains were performed in the absence and in the presence of S-9 and retained the same test doses as employed for the range-finder experiment. Following these treatments, evidence of toxicity was observed, but was limited to 5000 µg/plate treatments in strain TA102 in the absence and in the presence of S-9. Experiment 2 treatments of all the tester strains were performed in the absence and in the presence of S-9 with the maximum test dose of 5000 μg/plate retained. A narrowed dose range was employed (156.3 - 5000 µg/plate), in order to examine more closely those concentrations of the test substance approaching the maximum test dose and therefore, considered most likely to provide evidence of any mutagenic activity. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a preincubation step. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected using this assay system. Following an increase in revertants observed in strain TA98 in the presence of S-9 in Experiment 1, plate incorporation treatments of strain TA98 in the presence of S-9 were also performed in Experiment 2. Following the Experiment 2 treatments, evidence of toxicity was again observed in strain TA102, occurring at 2500 and 5000 µg/plate in this strain in the presence of S-9, but only at 5000 µg/plate in the absence of S-9. Similar evidence of toxicity was also observed following plate incorporation treatments of strain TA98 in the presence of S-9 at the maximum test dose of 5000 μg/plate.

 

The test article was completely soluble in the aqueous assay system at all concentrations treated, in each of the experiments performed. Negative (solvent) and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies on negative control plates all fell within acceptable ranges, and were significantly elevated by positive control treatments. Following the test substance treatments of all the tester strains, in the absence and in the presence of S-9, increases in revertant numbers were observed in strain TA98 in the presence of S-9 which were statistically significant when the data were analysed at the 1% level using Dunnett's test. These increases were dose-related and reproducible over two independent experiments, and were therefore considered to be indicative of the test substance’s mutagenic activity in this strain following metabolic activation. No comparable increases were seen in strain TA98 in the absence of S-9, and no other increases sufficient to be considered as indicative of mutagenic activity were observed in any of the other tester strains.