Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24th May 2012 to 11th September 2012.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted in accordance with relevant testing guidelines, with no deviations.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 487: In Vitro Mammalian Cell Micronucleus Test
Deviations:
no
Principles of method if other than guideline:
Not applicable.
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: Liquid
Details on test material:
- Name of test material (as cited in study report): TOFA_DimerFA_TETA_PAA
- Physical state: Brown liquid
- Analytical purity: 100%
- Lot/batch No.: BB001030V1
- Expiration date of the lot/batch: 30 May 2013
- Storage condition of test material: Stored at 15-25°C and protected from light
- Other: Preliminary solubility data indicated that TOFA_DimerFA_TETA_PAA was soluble in acetone at a concentration of at least 534.7 mg/mL.

Method

Target gene:
Not applicable - chromosome aberration study.
Species / strain
Species / strain / cell type:
lymphocytes: Human lymphocyte cultures
Details on mammalian cell type (if applicable):
The human lymphocyte cultures were prepared from the pooled blood of two female donors in a single experiment.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Range finding test, with and without metabolic activation: 5.442 to 1500 μg/mL
Micronucleus Experiment with metabolic activation: 5.000 to 140 μg/mL
Micronucleus Experiment without metabolic activation (3+21 hour treatment): 2.5 to 120 μg/mL
Micronucleus Experiment without metabolic activation (24 hour treatment): 1.25 to 40 μg/mL

Vehicle / solvent:
- Vehicle used: acetone
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
no
Remarks:
0.6 and 0.8 μg/mL
Positive control substance:
mitomycin C
Remarks:
In the absence of S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
6.25 and 12.50 μg/mL
Positive control substance:
cyclophosphamide
Remarks:
In the presence of S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
0.02, 0.03 and 0.04 μg/mL
Positive control substance:
other: Vinblastine
Remarks:
In the absence of S9 mix
Details on test system and experimental conditions:
In a Cytotoxicity range-finder test, which was performed initially prior to all treatment and in the micronucleus test, all 24 hour test groups without S9 mix had 0.1 mL culture medium removed to give a final pre-treatment volume of 9.3 mL. S9 mix or KCl (0.5 mL/culture) was added appropriately. Cultures were treated with the test article, vehicle or untreated controls (0.1 mL/culture). Positive control treatments were not included. Cyto-B, formulated in DMSO, was added directly (0.1 mL/culture) to all 24+0 hour -S9 cultures at the time of treatment to give a final concentration of 6 μg/mL per culture. The final culture volume was 10 mL. Cultures were incubated at 37 ± 1°C for the designated exposure time.

In the micronucleus test, for the removal of the test article, cells were pelleted (approximately 300 g, 10 minutes), washed twice with sterile saline (pre-warmed in an incubator set to 37 ± 1°C), and resuspended in fresh pre-warmed medium containing foetal calf serum and penicillin / streptomycin. At the appropriate times, Cyto-B (formulated in DMSO) was added to post wash-off culture medium to give a final concentration of 6 μg/mL per culture. Changes in osmolality of more than 50 mOsm/kg and fluctuations in pH of more than one unit may be responsible for an increase in chromosome aberrations.
At the defined sampling time, cultures were centrifuged at approximately 300 g for 10 minutes, the supernatant removed and discarded and cells resuspended in 4 mL (hypotonic) 0.075 M KCl at 37 ± 1°C for 4 minutes to allow cell swelling to occur. Cells were fixed by dropping the KCl suspension into fresh, cold methanol/glacial acetic acid (3:1, v/v). The fixative was changed by centrifugation (approximately 300 g, 10 minutes) and resuspension. This procedure was repeated as necessary (centrifuging at approximately 1250 g, 2-3 minutes) until the cell pellets were clean.

For the cytotoxicity range-finder test, a minimum of 200 cells per concentration were analysed to determine the replication index. For the micronucleus test, a minimum of 500 cells pe culture were examined when possible. The highest concentration selected for micronucleus analysis under all treatment conditions was limited by the observation of precipitate (by eye) at the end of the respective treatment incubation periods.
Slides from the highest selected concentration and two or three lower concentrations were taken for microscopic analysis, such that a range of cytotoxicity from maximum to little was covered.

For each treatment regime, two vehicle control cultures were analysed for micronuclei. As the vehicle control data were considered acceptable, the untreated control cultures were not analysed. Positive control concentrations, which gave satisfactory responses in terms of quality and quantity of binucleated cells and numbers of micronuclei, were analysed.
Evaluation criteria:
For valid data, the test article was considered to induce clastogenic and/or aneugenic events if:
1. A statistically significant increase in the frequency of MNBN cells at one or more concentrations was observed.
2. An incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates was observed.
3. A concentration-related increase in the proportion of MNBN cells was observed.
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.
Statistics:
After completion of scoring and decoding of slides, the numbers of binucleate cells with micronuclei (MNBN cells) in each culture were obtained. The proporations of MNBN cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test (Richardson et al, 1989). The proportion of MNBN cells for each treatment condition were comparated with the proportion in vehicle controls by using Fisher's exact test. Values of p ≤ 0.05 were considered significant.

Results and discussion

Test results
Species / strain:
lymphocytes: Human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No marked changes in pH were observed at the highest concentration tested in the Range-Finder (1500 μg/mL), compared to the concurrent vehicle controls.

- Effects of osmolality: No marked changes in osmolality, compared to the concurrent vehicle controls, were observed at 1500 μg/mL following 3+21 hour treatments in the absence and presence of S-9 in the Range-Finder. For 24+0 hour –S9 treatments, increases in osmolality of >50 mOsm/kg over the concurrent vehicle controls were observed at all three concentrations analysed (540.0, 900.0 and 1500 μg/mL) but there was no concentration-related increase (individual data not reported). Furthermore, the osmolality value of the untreated control was 114 mOsm/kg higher than the vehicle (acetone) control. Further osmolality measurements were therefore made for the 24+0 hour –S9 treatments in the Micronucleus Experiment.

For the 3+21 hour +S9 and 24+0 hour –S9 treatments, the highest concentrations analysed for micronuclei were the lowest at which post-treatment precipitate was observed. For the 3+21 hour –S9 treatment, two precipitating concentrations (15.00 and 20.00 μg/mL) were analysed in addition to one non-precipitating concentration (10.00 μg/mL).

Treatment of cells with TOFA_DimerFA_TETA_PAA for 3+21 hours in the absence of S9 resulted in frequencies of MNBN cells that were generally similar to (and not significantly higher than) those observed in concurrent vehicle controls at all concentrations analysed. The MNBN cell frequency exceeded the normal range (0.1% to 1.0%) in one culture at the lowest concentration analysed (10.00 μg/mL) but the MNBN cell frequency in the replicate culture at this concentration fell within the normal range. This isolated observation was considered not biologically relevant.

Treatment of cells for 3+21 hours in the presence of S9 resulted in frequencies of MNBN cells that were significantly higher (p ≤ 0.01) than those observed in concurrent vehicle controls at the lowest and highest concentrations analysed (10.00 and 30.00 μg/mL). However, the MNBN cell frequencies exceeded the normal range (0.1% to 1.1%) only in single cultures at both concentrations, with no evidence of a concentration-related response. The mean MNBN cell frequencies at 10.00 and 30.00 μg/mL marginally exceeded the 95th percentile of the observed range but fell within the observed range (0% to 1.2%). These data did not fulfil the evaluation criteria for a positive response.

Treatment of cells for 24+0 hours in the absence of S9 resulted in frequencies of MNBN cells that were similar to (and not significantly different) from those observed in concurrent vehicle controls at all concentrations analysed. The MNBN cell frequencies fell within the normal range (0.1% to 1.4%) at all concentrations analysed.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

The binomial dispersion test demonstrated acceptable heterogeneity (in terms of MNBN cell frequency) between replicate cultures for the 3+21 hour and 24+0 hour treatments in the absence of S9. For the 3+21 hour treatment in the presence of S9, significant heterogeneity (p ≤ 0.05) was observed, primarily due to a marked difference in MNBN cell frequencies between replicate cultures at the highest concentration analysed (30.00 μg/mL). At this concentration, the MNBN cell frequency exceeded the normal range in one of the two cultures.

The positive control chemicals induced statistically significant increases in the proportion of cells with micronuclei. Both replicate cultures at the positive control concentration analysed under each treatment condition demonstrated MNBN cell frequencies that clearly exceeded the current historical vehicle control ranges.

A minimum of 50% of cells had gone through at least one cell division (as measured by binucleate + multinucleate cell counts) in vehicle control cultures at the time of harvest.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

Under the conditions of this study, TOFA_DimerFA_TETA_PAA did not induce biologically relevant increases in the frequency of micronuclei in cultured human peripheral blood lymphocytes when tested up to the limit of solubility for 3+21 hours in the absence and presence of a rat liver metabolic activation system (S9) and for 24+0 hours in the absence of S9.
Executive summary:

The ability of TOFA_DimerFA_TETA_PAA to cause mutagenicity was tested in an in vitro micronucleus assay, conducted in accordance with OECD Test Guideline 487 and GLP. Duplicate human lymphocyte cultures were prepared and the assayy were performed in the presence and absence of metabolic activation (S9 mix). A preliminary cytotoxicity range-finder experiment was conducted to determine the concentrations for use in the micronucleus assay. The concentrations used were:

Micronucleus Experiment with metabolic activation: 5.000 to 140 μg/mL

Micronucleus Experiment without metabolic activation (3+21 hour treatment): 2.5 to 120 μg/mL

Micronucleus Experiment without metabolic activation (24 hour treatment): 1.25 to 40 μg/mL

Appropriate vehicle and positive controls were also used.

Treatment of cells with TOFA_DimerFA_TETA_PAA for 3+21 hours in the absence of S9 resulted in frequencies of MNBN cells that were generally similar to (and not significantly higher than) those observed in concurrent vehicle controls at all concentrations analysed. The MNBN cell frequency exceeded the normal range (0.1% to 1.0%) in one culture at the lowest concentration analysed (10.00 μg/mL) but the MNBN cell frequency in the replicate culture at this concentration fell within the normal range. This isolated observation was considered not biologically relevant.

Treatment of cells for 3+21 hours in the presence of S9 resulted in frequencies of MNBN cells that were significantly higher (p ≤ 0.01) than those observed in concurrent vehicle controls at the lowest and highest concentrations analysed (10.00 and 30.00 μg/mL). However, the MNBN cell frequencies exceeded the normal range (0.1% to 1.1%) only in single cultures at both concentrations, with no evidence of a concentration-related response. The mean MNBN cell frequencies at 10.00 and 30.00 μg/mL marginally exceeded the 95th percentile of the observed range but fell within the observed range (0% to 1.2%). These data did not fulfil the evaluation criteria for a positive response.

Treatment of cells for 24+0 hours in the absence of S9 resulted in frequencies of MNBN cells that were similar to (and not significantly different from) those observed in concurrent vehicle controls at all concentrations analysed. The MNBN cell frequencies fell within the normal range (0.1% to 1.4%) at all concentrations analysed.

Under the conditions of this study, TOFA_DimerFA_TETA_PAA did not induce biologically relevant increases in the frequency of micronuclei in cultured human peripheral blood lymphocytes when tested up to the limit of solubility for 3+21 hours in the absence and presence of a rat liver metabolic activation system (S9) and for 24+0 hours in the absence of S9.