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Genetic toxicity in vitro

Description of key information

Genetic toxicity was evaluated in seven in vitro tests and in one in vivo test. The in vitro tests comprise three bacterial reverse mutation tests (Sokolowski, 2011 (K1); Brusick, 1977 (K2); Zeiger et al. (K2)), an in vitro mammalian chromosomal aberration test (Morris, 2011 (K1)), an in vitro mammalian cell gene mutation test (Godek, 1985 (K1)), an unscheduled DNA synthesis test (Steinmetz, 1985 (K1)) and a mitotic recombination assay with Saccharomyces cerevisiae (Brusick, 1977 (K2)). Negative results were determined in all in vitro tests.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study carriet out according to to internationally accepted guidelines. No deviations.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
9th addendum
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese guidelines: Kanpoan No.287, Eisei No.127, Heisei 09/10/31 Kikyoku No.2
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Salmonella typhimurium histidine (his)
Escherichia coli tryptophan (trp)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Mammalian Microsomal Fraction S9 Mix
Test concentrations with justification for top dose:
In the pre-experiment (reported as experiment I), the concentration range of the test item was 3-5000 ug/plate.
Since toxic effects were observed, 8 concentrations were tested in experiment II and 5000 ug/plate was chosen as maximal concentration:

3 - 10 - 33 - 100 - 333 - 1000 - 2500 and 5000 ug/plate
Vehicle / solvent:
DMSO - the solvent was chosen because of its solubility properties and it relative non-toxicity to the bacteria.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
with and without metabolic activation
Positive control substance:
other: multiple positive controls - see "details on test system and conditions"
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- Experiment I: plate incorporation
- Experiment II: pre-incubation

POSITIVE CONTROL SUBSTANCES:
- Without metabolic activation:
* sodium azide -- TA 1535, TA 100
* 4-nitro-o-phenylene-diamine -- TA 1537, TA 98
* methyl methane sulfonate -- WP2 uvrA
-With metabolic activation:
* 2-aminoanthracene -- all strains

DETERMINATION OF TOXICITY:
To evaluate the toxicity of the test item a pre-experiment was performed with strains TA 1535, TA 1537, TA 98, TA 100 and WP2 uvrA.
Eight concentrations were tested for toxicity and mutation induction with three plates each. The experimental conditions in this pre-experiment were the same as described for experiment I (plate incorporation test).
Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

NUMBER OF REPLICATIONS: for each strain and dose level including the controls, 3 plates were used.

PRECULTURES:
From the thawed ampoules of the strains 0,5 mL suspension was transferred into 250 mL Erlenmeyer flasks containing 20 mL nutrient medium. A solution of 20 uL ampicillin (25 ug/mL) was added to the strains TA 98 and TA 100. This nutrient medium contains per litre: 8g nutrient broth (MERCK, 64293 Darmstadt/Germany) and 5 g NaCl (MERCK, 64293 Darmstadt/Germany).
The bacterial cultures were incubated in a shaking water bath for 4 hours at 37°C The optical density of the bacteria was determined by absorption measurement and the obtained values indicated that the bacteria were harvested at the late exponential or early stationary phase (10E08 - 10E09 cells/mL).

PLATE INCORPORATION TEST (EXPERIMENT I):
The following materials were mixed in a test tube and poured onto the selective agar plates:
* 100 uL test solution at each dose level (solvent or reference mutagen solution)
* 500 uL S9 mix (for tests with metabolic activation) or S9 mix substitution buffer (for tests without metabolic activation)
* 100 uL bacteria suspension (cfr. precultures)

PRE-INCUBATION ASSAY (EXPERIMENT II):
In the pre-incubation assay 100 uL test solution, 500 uL S9 mix / S9 mix substitution buffer and 100 uL bacterial suspension were mixed in a test tube and shaken at 37°C for 60 minutes. After pre-incubation 2,0 mL overlay agar (45°C) was added to each tube. The mixture was poured on selective agar plates.
Evaluation criteria:
ACCEPTABILITY OF THE ASSAY:
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of the lab's historical data
- the positive control substance should produce a significant increase in mutant colony frequencies

EVALUATION OF RESULTS:
A test item is conisdered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (for TA 98, TA 100 and WP2 uvrA) or thrice (for TA 1535 and TA 1537) 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.
Statistics:
/
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
for TA 1537 (as from 1000 ug/plate) and TA 100 (as from 2500 ug/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
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
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

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.
Executive summary:

An Ames study was performed to investigate the potential of dibutyl phenyl phosphate (DBPP) reaction mass to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent exerpiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations in both experiment I and experiment II: 3 - 10 - 33 - 100 - 333 - 1000 - 2500 and 5000 ug/plate.

The plates incubated with the test item showed reduced background growth in all strains at higher concentrations.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0,5), occurred in nearly all strains with and without activation.

No substantial increase in revertant colony numbers of any of the 5 tester strains was observed following treatment with dibutyl phenyl phosphate (DBPP) reaction mass at any dose level, neither in the presence nor absence of metabolic activation mix (S9).

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study carriet out according to to internationally accepted guidelines. No deviations.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: Japanese New Chemical Susbtance Law (METI)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
lymphocytes: human, whole blood
Details on mammalian cell type (if applicable):
- Type and identity of media: Eagle's minimal essential medium with HEPES buffer (MEM), supplemented "in-house" with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum
Metabolic activation:
with and without
Metabolic activation system:
Microsomal enzyme fraction (S9)
Test concentrations with justification for top dose:
Experiment I:
* 4(20)-hour without S9: 10 - 20 - 40 - 60 - 80 - 120 ug/mL
* 4(20)-hour with S9 (2%): 20 - 40 - 80 - 100 - 120 - 160 ug/mL

Experiment II:
* 24 -hour without S9: 10 - 20 - 30 - 40 - 60 - 80 ug/mL
* 4(20)-hour with S9 (1%): 10 - 20 - 40 - 80 - 100 - 120 ug/mL
Vehicle / solvent:
DMSO
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: mitomycin C (without metabolic activation), cyclophosphamide (with metabolic activation)
Details on test system and experimental conditions:
CELLS:
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitability. The volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. The cell-cycle time for the lymphocytes form the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT)? The AGT for the regular donors used in this laboratory has been determined to be approximately 16 hours under typical experimental conditions.

PREPARATION OF TEST ITEMS:
The test item was accurately weighed, dissolved in DMSO and serial dillutions were prepared. There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm at the dose levels investigated. The test item was formulated within 2 hours of it being applied to the test system and it is assumed that the formulation was stable during this period. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regartd to CLP.

METHOD OF APPLICATION: in medium

CULTURE CONDITIONS:
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:
- 9,05 mL MEM, 10% (FBS)
- 0,1 mL Li-heparin
- 0,1 mL phytohaemagglutinin
0,75 mL heparinised whole blood

a) With metabolic activation (S9) treatment:

After approximately 48 hours incubation at 37°C, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approx. 9 mL of the culture medium was removed, reserved and replaced with the required volume of MEM (including serum) and 0.1 mL of the appropriate solution of vehicle control or test item was added to the culture. 1 mL of 20% S9-mix was added to the cultures of the preliminary toxicity test and of experiment 1. In experiment 2, 1 mL of 10% S9-mix was added. All cultures were then returned to the incubator. The nominal final volume of each culture was 10 mL.
After 4 hours of incubation in humidified air, the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a furhter centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 30 hours at 37°C in 5% CO2 in humidified air.

b) Without metabolic activation (S9) treatment:

In experiment 1, after approximately 48 hours incubation at 37°C, 5% CO2 in humidified air, the cultures were decanted into tubes and centrifuged. Approx. 9 mL of the culture medium was removed, reserved. The cells were then re-suspended in the required volume of fresh MEM (including serum) and 0.1 mL of the appropriate vehicle control, test item solution or positive control solution was added to the culture. The nominal final volume of each culture was 10 mL.
After 4 hours of incubation in humidified air, the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a furhter centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 30 hours at 37°C in 5% CO2 in humidified air.

In experiment 2, without metabolic activation, the exposure was continuous for 24 hours. Therefore, when the cultures were established the culture volume was a nominal 9,9 mL. After approx. 48 hours incubation the cultures were removed from the incubator and dosed with 0,1 mL of vehicle control, test item solution or positive control solution. The nominal values of each culture was 10 mL. The cultures were then incubated at 37°C in 5% CO2 in humidified air for 24 hours.

CELL HARVEST:
Mitosis was arrested by addition of demecolcine (Colcemid 0,1 ug/mL) two hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and sicarded, and the cells re-suspended in 0,075M hypotonic KCl. After approx. 14 minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1, v/v). The fixative was changed at least 3 times and the cells stored at approx. 4°C for at least 4 hours to ensure complete fixation.

The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and resuspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Subsequently, the slides were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
Evaluation criteria:
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there were approx. 30 to 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing. Cells with crhomosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.

In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) reported. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid calls were compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.
Species / strain:
lymphocytes: human, whole blood
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
PRELIMINARY TOXICITY TEST:
The test item showed evidence of toxicity in all three exposure groups with no methaphases present at and above 156,25 ug/mL. A precipitate of test item was observed in the parallel blood-free cultures at the end of the exposure period, at and above 625 ug/mL in the 4(20)-hour pulse exposure group and at and above 312,5 ug/mL in the continuous exposure group. Haemolysis was also noted in the blood cultures at the end of the exposure period at and above 156,25 in the 4(20)-hour and the 24-hour exposure groups in the absence of S9. In the 4(20)-hour exposure group in the presence of S9 haemolysis was noted at and above 312,5 ug/L at the end of the exposure period. Microscopic assessment of the slies prepared from the treatment cultures showed that metaphase cells were present up to 78,13 ug/mL in all three exposure groups.

EXPERIMENT 1:
The qualitative assessment of the slides determined that the toxicity was similar to that observed in preliminary toxicity test and that there were metaphases suitable for scoring present at 80 ug/mL in the absence of S9. In the presence of S9 the maximum test item dose level with metaphases suitable for scoring was 120 ug/mL.
In the absence of S9 the toxicity curve was steep with no toxicity at 60ug/mL and 65% mitotic inhibition at 80 ug/mL. The toxicity in the presence of S9 was more gradual with near optimum mitotic inhibition of 44% being achieved at 100 ug/mL.
All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control items induces statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.
The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of S9 at dose levels which did not exceed optimum toxicity. The dose levels of 80 ug/mL in the absence of S9 and 120 ug/mL in the presence of S9 both induced modest but statistically significant increases in the frequency of cells with aberrations but at dose levels which achieved 65% mitotic inhibition and could therefore be excluded from the study. In both cases the response only marginally exceeded the upper limit of the historical vehicle control ranges. The aberrations seen at these doses were simple break type aberrations which is indicative of a cytotoxic mechanism rather than a true mutagenic response were considered to have no toxicological significance.
The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

EXPERIMENT 2:
The qualitative assessment of the slides determined that there were metaphases suitable for scoring present at 100 ug/mL in the absence of S9. In the absence of S9 the maximum test item dose level with metaphases suitable for scoring was 80 ug/mL, however, due to the obvious toxicity observed the maximum dose selected for mitotic index analysis was 60 ug/mL.
The results of the mitotic indices (MI) from the cultures after their respective treatments show a 44% and 74% growth inhibition was achieved at 40 and 60 ug/mL respectively in the absence of S9. In the presence of S9 29% mitotic inhibition was achieved at 100 ug/mL with the higher dose level of 120 ug/mL having no metaphases suitable for scoring.
All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control items induces statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.
The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of S9.
The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

The test item did not induce any toxicologically significant increases in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments at dose levels which achieved less than or near optimum toxicity. The test item was therefore considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

The in vitro study for the detection of structural chromosome aberrations in cultured mammalian cells was carried out according to internationally accepted protocols. Duplicate cultures of human lymphocytes, treated with test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, i.e.:

* In experiment 1, a 4 -hour exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20 -hour expression period and a 4 -hour exposure in the absence of metabolic activation (S9) with a 20-hour expression period.

* In experiment 2, the 4 -hour exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

The dose levels used in experiment 1 and experiment 2 were selected using data from the preliminary toxicity test and were as follows:

* 4(20)-hour without S9: 10 - 20 - 40 - 60 - 80 - 120 ug/mL

* 4(20)-hour with S9 (2%): 20 - 40 - 80 - 100 - 120 - 160 ug/mL

* 24 -hour without S9: 10 - 20 - 30 - 40 - 60 - 80 ug/mL

* 4(20)-hour with S9 (1%): 10 - 20 - 40 - 80 - 100 - 120 ug/mL

All vehicle (solvent) control groups had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items included statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test item did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using dose levels that induced approximately 50% mitotic inhibition or less. The test item had a steep toxicity curve which made achieving optimum toxicity of 50% difficult. In some cases dose groups where greater than optimum toxicity was achieved were scored and achieved statistically significant increases in the frequency of aberrations, predominantly breaks. However, since these dose levels exceeded the optimum limit for toxicity (50%) and the response was considered to be as a result of a cytotoxic mechanism rather than a true mutagenic response this data can be excluded for the purpose of this study and is considered to have no toxicological significance.

Conclusion: the test item was considered to be non-clastogenic to human lymphocytes in vitro.

Endpoint:
in vitro DNA damage and/or repair study
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study. Although no internationally accepted guideline is mentioned, the study protocol is robust and both the methodology and results sections are described in detail.
Qualifier:
no guideline followed
Principles of method if other than guideline:
As described by Williams, 1977: Detection of chemical carcinogens by unscheduled DNA synthesis in rat liver primary cell cultures. Cancer Res. 37, 1845-1851.
GLP compliance:
yes
Type of assay:
DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro
Target gene:
not applicable
Species / strain / cell type:
hepatocytes:
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
not applicable
Test concentrations with justification for top dose:
0,1 - 0,5 - 1 - 5 - 10 - 25 - 50 - 100 - 500 - 1000 - 5000 µg/mL
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
Details on test system and experimental conditions:
PRIMARY CELL CULTURES:
Primary rat liver cell cultures derived from the livers of 2 adult male Fischer-344 rats weighing 293 and 214g were used for the preliminary and replicate experiments, respectively. Each rat was anesthetized with 0,2 cc of Nembutal / 100g body weight prior to each experiment. Hepatocytes were isolated by liver perfusion with a collagenase solution according to the procedure of Williams (1977) and inoculated into culture dishes containing coverslips in Williams' Medium E supplemented with 2mM glutamine, 50 µg/mL gentamicin and 10% fetal bovine serum. After 1,5 - 2,0 hours of incubation, the nonviable cells (those not attached to the coverslips) were washed out of the cultures and the viable cells were used immediately for the UDS assay. All subsequent steps were performed in serum-free medium.

PRELIMINARY UDS ASSAY:
For the preliminary UDS assay, three cultures were used for each of 10 dilutions of DBPP, for the positive control, the negative control and the untreated medium control. The maximum concentration tested (5000 Ug/mL) was found to be the limit of solubility of this compound. Cutlures were exposed simultaneously to the test material and to 10 µCi/mL 3H-thymidine (specific activity approx. 80 Ci/mmole) for 19 to 20 hours. After exposure, all cultures were washed with medium, swelled in a hypotonic solution, fixed and washed with water. The coverslips were mounted on slides, dipped in Kodak NTB-2 emulsion and exposed at -20°C for 7 days prior to development. Cells were stained by methy-green Pyronin Y. The UDS assay was repeated at 3 non-cytotoxic concentrations of DBPP. The highest concentration tested in the replicate experiment was 50 µg/mL.

MEASUREMENT OF UDS:
Quantitative autoradiographic grain-counting was accomplished using an ARTEK Model 880 or 980 colony counter interfaced with a Zeiss Universal Microscope via an ARTEK TV camera.
Fifty morphological unaltered cells on a randomly selected area of the slide were counted. The highest count from 2 nuclear-size areas over the most heavily labeled cytoplasmic areas adjacent to the nucleus was subtracted from the nuclear count to give the net grains/nucleus (NG). The percentage of cell repair was calculated as the percentage of cells with at least 5 NG. 150 Cells were scored for each concentration reported for each experiment.
Evaluation criteria:
Positive:
A test article is considered positive if UDS (amount of incorporated 3H-thymidine) is markedly elevated above that in the solvent control. The presence of a dose-response, a change in the frequency distribution of cellular responses, an increase in the percentage of cells in repair and reproducibility of data are all considered in classifying the test article as "positive" or "negative". If the data warrant, a test article is classified as a "weak positive".

Negative:
A material is considered negative if testing has been performed to the limits of solubility or cytotoxicity, or at 5000 µg/mL, and if UDS is not significantly elevated above that in the solvent control.
Key result
Species / strain:
hepatocytes:
Metabolic activation:
not applicable
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The replicate experiment was performed twice, using the same concentrations in both experiments. In the first replicate experiment, DBPP was cytotoxic at all concentrations. Because this level of cytotoxicity occurred only in this experiment, it was attributed to a technical error rather than to an effect of DBPP.
In the preliminary experiment, cytotoxicity was observed in the preliminary experiment at 50, 100, 500, 1000 and 5000 µg/mL, and in the replicate experiment at 10, 25 and 50 µg/mL. An oily precipitate was observed at 5000 µg/mL.
UDS was measured at DBPP concentrations between 0,1 and 10 µg/mL in the preliminary experiment and between 0,5 and 5 µg/mL in the replicate assay. The net grain counts were negative at each concentration of the test compound and in the solvent control, in contrast to the strong positive response produced in both experiments by the positive control.
Remarks on result:
not determinable
Conclusions:
Interpretation of results (migrated information):
negative

The results of the UDS assay indicate that DBPP is not a genotoxic agent in the in vitro rat hepatocyte DNA repair assay.
Executive summary:

The study report describes an unscheduled DNA synthesis (UDS) test with primary cultures of rat hepatocytes. No appreciable increase in UDS above that of the solvent control level was observed after treatment of the hepatocytes with DBPP. The study was conducted in compliance with the GLP standards.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The in vivo study report comprises an acute in vivo rat bone marrow cytogenetics assay (Blazak, 1986 (K2)). A negative results was determined in the in vivo test.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: No internationally accepted guideline followed but study under GLP, well documented and robust protocol.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Test substance was administered by intraperitoneal injection to induce chromosomal damage in bone marrow cells of Fischer-344 rats. In the definitive stuy, male rats were given DBPP at doses of 0, 40, 200, and 400 mg/kg bw; female rats were given DBPP at doses of 0, 60, 300, and 600 mg/kg bw. Groups of animals were sacrificed 6, 12, and 24 hr after treatment. A concurrent positive control group received triethylenemelamine.
Cells from males exposed to 0 and 400 mg/kg bw and from females exposed to 0 and 600 mg/kg bw DPP and from animals in the positive control groups were evaluated microscopically for mitotic indec and chromosomal abnormalities.
GLP compliance:
yes
Type of assay:
mammalian germ cell cytogenetic assay
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Simonsen Laboratories Inc., 1180C Day road, Gilroy, CA 95020
- Age at study initiation: not specified
- Weight at study initiation: males: 203 g, females: 191 g.
- Assigned to test groups randomly: yes
- Housing: 2 or 3 per polycarbonate cage with wood shavings as bedding
- Diet (e.g. ad libitum): Certified Purina Lab Chow, ad libidum
- Water (e.g. ad libitum): fresh purified water, ad libidum
- Acclimation period: 5 day quarantaine

ENVIRONMENTAL CONDITIONS
- Temperature (°C): not specified
- Humidity (%): not specified
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): not specified
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
Details on exposure:
ADMINISTRATION OF TEST ARTICLE
DBPP was diluted with corn oil. Fresh dilutions were made for each pilot and definitive study. All animals were treated with a single intraperitoneal injection. Dosing injection volumes were 1 to 5 mg/kg bw depending on the study.
Duration of treatment / exposure:
Single injection
Frequency of treatment:
1x
Post exposure period:
Dose range-finding study: 14 days.
Pilot studies: 24 hours or 14 days.
Definitive study: 6, 12 and 24 hours
Remarks:
Doses / Concentrations:
8, 40, 200, 1000 and 5000 mg/kg
Basis:
other: injected (dose range-finding)
Remarks:
Doses / Concentrations:
312, 625, 1250, 2500 and 5000 mg/kg
Basis:
other: injected (pilot)
Remarks:
Doses / Concentrations:
40, 60, 200, 300, 400 and 600 mg/kg
Basis:
other: injected (definitive)
No. of animals per sex per dose:
dose range-finding: 2 rats per sex per dose
pilot: 7 rats per sex per dose (2 sacrificed after 24 hrs, 5 after 14 days)
definitive: 6 rats of one sex per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
- yes: triethylenemelamine (TEM)
- Route of administration: ip injection, solution in Hanks' balanced salt solution (HBSS)
- Doses / concentrations: 0,2 mg/kg
Tissues and cell types examined:
bone marrow cells
Details of tissue and slide preparation:
DETAILS OF SLIDE PREPARATION:
Femoral bone marrow cells were prepared from each rat according to a modification of the approach specified by Nichols et al. (1972). Approximately 2 to 3 hrs prior to sacrifice, each rat was injected ip with colchicine dissolved in HBSS. The animals were sacrificed by CO2 asphyxiation followed by cervical dislocation. Skin and muscle tissue were carefully removed from the femur immediately after sacrifice. The femur, exposed knee to hip, was cut just above the knee, and cells were aspirated from the distal end of the femur through an 18-gauge needle into a 5-mL syringe containing 2 mL of warm HBSS. The procedure was then repeated for the other leg, and HBSS and bone marrow cells were dispensed along the inner side of a 15-mL centrifuge tube. The suspension was mixed by aspirating it up and down at least 5 times through the 18-gauge needle to break the fibrin and ensure cell dissociation. Care was taken to avoid pushing HBSS into the femur.
The tubes were centrifuged at 700 rpm for 10 minutes and the supernatant was carefully discharged so as not to disturb the cell pellet. Then 0,55% KCl (warmed to 37°C) was added to each tube, the pellet was gently disrupted and gross debris was removed. Next the tubes were incubated in a 37°C water bath for 20 minutes. Then 1 mL of fixative (methanol : glacial acetic acid 3:1) was added and mixed with the hypotonic solution. The tubes were cetrifuged, the supernatant discarded and fixative was added and mixed with the pellet. Tubes were centrifuged, supernatant discarded and fresh fixative was added. Subsequently, tubes were allowed to stand for 10 to 30 min at room temperature and the fixation was repeated.

Slides were prepared by dropping cell suspensions onto water-dipped slides, passing the slides through the flame of an alcohol lamp several times, and checking the cell density using a phase-contrast microscope. If cell density was too thick, more fixative was added to dilute the suspension. Based on the amount of cell suspension, six or fewer slides were prepared from each tube.
The slides were placed in 7% Giemsa for 5 to 20 minutes, rinsed in distilled water, soaked in xylene and mounted with Permount to make permanent slides for scoring under bright-field or phase-contrast optics.

METHOD OF ANALYSIS:
Prepared slides from males and females exposed to 0 mg/kg (negative controls), from males exposed to 400 mg/kg, from females exposed to 60 mg/kg and from all animals exposed to TEM (positive controls) were divided into identical groups and coded by an individual not involved in the microscopic evaluation. Slides from 5 animals (if available) in each of the above treatment groups per sex per sacrifice period were coded after choosing the animals randomly or from the results of initial evaluation of the quality of the slides. The slides were decoded only after all slides in each group had been analyzed completely.
For this stuy, two individuals separately analyzed coded slides from each animal. Slides were evaluated for mitotic index (based on at least 1000 cells/animal), and 60 cells per animal, when possible, were evaluated for chromosomal aberrations. Because a sufficient number of cells (60 or nearly 60) was analyzed for the majority of animals in each treatment group, the chromosomal preparations from the additional animals were not evaluated.
For analysis of the slides, score sheets were used, in which was noted, i.a., quality of the slide, vernier settings, MI, chromosome number and numbers of various categories of chromatid and chromosomal aberrations for each cell scored. Chromatid and isochromatid gaps were recorded for each cell but were not considered chromosomal aberrations in the analysis of the data. After analysis was completed, the slides were decoded and the score sheets were summarized.
Evaluation criteria:
Positive:
A test article was considered to have elicited a positive response in the in vivo bone marrow cytogenetic assay if either the mean aberrant cell frequency or the mean chromosomal aberration frequency per cell, or both, was significantly greater (p<0,05) in the test article-treated animals than in the negative control animals.

Negative:
A test article was considered to have alicited a negative response if the results obtained by each cytogeneticist were in general agreement and the criteria for a positive response were not met.

Inconclusive:
The results of this assay were considered inconclusive if there was reason to believe that the concentrations of the test article selected for evaluation were inappropriate (i.e. excessive toxicity or lack of any toxicity)
Statistics:
The mean number of chromosomal aberrations per cell, the mean frequency of aberrant cells, the mean MI, the mean frequency of cells with structurally aberrant chromosomes and the mean frequency of aneuploid (hyperploid) cells were calculated for each treatment group x sex x sacrifice-time subclass from the individual animal means. For the first 3 of these values, Bartlett's test was employed to investigate homogeneity among treatment variances. A one-way analysis of variance was used to analyze the data from each sex x sacrifice time subclass. If a significant (i.e. p<0,05) F-value was obtained, Dunnett's test was used to compare the test article treatment means with the appropriate negative control mean. Student's t-test was used to evaluate the significance of differences between the negative control and positive control groups.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
at > 625 mg/kg
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
24h PILOT STUDY
- No significant deleterious effect of treatment with DBPP on mitotic indices.
- All rats treated with DBPP lost more weight than did the negative control rats.
- Toxicity in male rats:
* 5000 mg/kg (2/2): mortality
* 2500 mg/kg (2/2) and 1250 mg/kg (1/2): exudate anround the penis and a lower abdominal region:
* 625 mg/kg or less: no toxic symptoms
- Toxicity in female rats:
* 5000 mg/kg: animal 1: moderately humped back; animal 2: no toxicity
* 2500 mg/kg (2/2): no toxic symptoms
* 1250 mg/kg (1/2): marked hyperpnea and lack of activity + marked diarrhea at time of sacrifice
* 625 mg/kg (1/2): lack of activity, slight hyperpnea, exudate from both eyes + moderate diarrhea, slight red-brown exudate around the anus and left eye and a moderate humped back at time of sacrifice.
* 312 mg/kg or less: no toxic symptoms

14d PILOT STUDY
- Toxicity in male rats:
* 5000 mg/kg: (5/5) mortality, no surviving animals by day 7.
* 2500 mg/kg: (4/5) mortality, 1 animal surviving by day 7. Remaining animal showed the following symptoms: an exudate arount he penis, lower abdomen, a midly humped back, mildly to moderately rough fur and an abdominal lump around the site of injection.
* 1250 mg/kg: (1/5) mortality, 4 animals surviving by day 7. Remaining animals showed one or more of the following symptoms: exudate from the eye(s), exudate from the mouth, exudate around the penis, exudate from the anus, lower abdominal region, mild diarrhea, mild hyperpnea, mildly humped back, mildly rough fur. One animal in this dose group exhibited no toxic symptoms.
* 625 mg/kg: (1/5) mortality, 4 animals surviving by day 7. Remaining animals showed one or more of the following symptoms: slight ataxia, decreased activity, sligh rough fur, slight exudate from the eye(s), slight exudate from the mouth, slight exudate from the nose, mild diarrhea. Two animals in this dose group exhibited no toxic symptoms.
* 312 mg/kg: no clinical signs of toxicity
- Toxicity in female rats:
* 5000 mg/kg: (3/5) mortality, 2 animals surviving by day 7. Remaining animals showed one or more of the following symptoms: exudate around the vagina, lower abdomen, mildly humped back, mildly rough fur, abdominal lumps around the site of injection.
* 2500 mg/kg: (3/5) mortality, 2 animals survivyng by day 7. Remaining animals showed one or more of the following symptoms: exudate around the vagina, lower abdomen, mildly humped back, abdominal lumps at site of injection.
* 1250 mg/kg: (3/5) mortality, 2 animals surviving by day 7. Remaining animals showed one or more of the following symptoms: mildlly rough fur, mild exudate around the vagina and lower abdomen, lack of activity, mild to marked hyperpnea, marked serous descharge from one eye, moderate wheezing.
* 625 mg/kg: no mortality. The animals showed one or more of the following symptoms: mild to moderate hyperpnea, mildly humped back, decreased activity, exudate around the eyes, exudate around the vagina and abdomen, mildly rough fur, marked diarrhea, mild exudate from the mouth and nose, paler eyes. One animals showed no toxic symptoms.
* 312 mg/kg or less: one animal had a mild exudate around the eye, another one favored its left hind leg. The other animals showed no symptoms of toxicity.

DEFINITIVE STUDY - CLINICAL OBSERVATIONS
Males:
* 400 mg/kg - 6h dose group: decreased activity (1/5), marked ataxia (1/5)
* 400 mg/kg - 12h dose group: mild to moderate diarrhea (5/5), decreased activity (3/5)
* 200 mg/kg - 6h dose group: slight discharge from the nose (1/5)
Females:
* 600 mg/kg - 6h dose group: decreased activity (5/5), marked ataxia (4/5), slight exudate from the eye (1/5)
* 600 mg/kg - 12h dose group: decreased activity (5/5), moderately humped back (3/5),
* 600 mg/kg - 24h dose group: slight to moderate ataxia (4/5), decreased activity (3/5), slightly humped back (4/5)
The rats treated with the test substance lost weight when compared to their concurrent negative controls.

DEFINITIVE STUY - CYTOLOGICAL OBSERVATIONS
The 3 measures of cytological damage that were statistically evaluated were the MI, the percentage of chromosomally aberrant cells and the frequency of chromosomal aberrations per cell. Prior to analysis of variance, Bartlett's test was conducted on these variables to investigate differences in treatment-group variances. The variances among treatment groups were found to be homogeneous.
At 6, 12 and 24h post treatment, no significant differences in the % of chromosomally aberrant cells or the frequency of chromosomal aberrations per cell were observed between the negative control and DBPP-treatment groups in male or female rats? The only significant difference in MI was between the female negative control and DBPP-treated groups 24h after treatment: a higher MI was noted for females treated with DBPP.
Animals in the concurrent 24h-post treatment positive control groups had significantly higher percentages of aberrant cells and significantly higher frequencies of aberrations per cell than did animals in the negative control groups.

From the results of these studies, it is concluded that DBPP does not induce chromosomal damage in male or female Fischer-344 rats under the conditions used in this study.
Conclusions:
Interpretation of results (migrated information): negative
From the results of these studies, it is concluded that DBPP does not induce chromosomal damage in male or female Fischer-344 rats under the conditions used in this study.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In the bacterial reverse mutation study considered as the key study (Sokolowski, 2011), the potential of DBBP was investigated to induce gene mutations in a plate incorporation test and a pre-incubation test using Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537, and the Escherichia coli strain WP2 uvrA. The four tested S. typhimurium have GC base pairs at the primary reversion site and it is known that they may not detect certain oxidizing mutagens, cross-linking agents and hydrazines. Such substances may be detected by the E. coli WP2 strain. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations in the incorporation and pre-incubation test: 3 - 10 - 33 - 100 - 333 - 1000 - 2500 and 5000 ug/plate. The plates incubated with the test item showed reduced background growth in all strains at higher concentrations. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in nearly all strains with and without activation. No substantial increase in revertant colony numbers of any of the 5 tester strains was observed following treatment with DBPP at any dose level, neither in the presence nor absence of metabolic activation mix (S9). Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

 

In a supporting bacterial reverse mutation study by Brusick (2011), DBPP was investigated to induce gene mutations in a plate test using S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538. The compound was tested over a series of concentrations such that there was either quantitative or qualitative evidence of some chemically-induced physiological effects at the high dose level. The low dose in all cases was below a concentration that demonstrated any toxic effect. The dose range employed for the evaluation of this compound was from 0.001 uL to 5 uL per plate. The compound was slightly toxic to TA-1537 at 5 uL dose level in the activation assay. The results of the test conducted on the compound in the absence or presence of a metabolic system were all negative. The test compound did not demonstrate mutagenic activity in any of the assays conducted in this evaluation and was considered as not mutagenic under these test conditions.

 

In another supporting bacterial reverse mutation study (Zeiger et al. 1988), 300 chemicals including DBPP were tested using a preincubation protocol. S. typhimurium strains were tested with concentrations ranging from 1000 to 200.000 µg/plate both with and without metabolic activation. DBPP was non mutagenic in this assay.

An in vitro mammalian chromosome aberration test (Morris, 2011) was performed with human lymphocytes. Duplicate cultures were evaluated at up to four dose levels of the test compound. Four treatment conditions were used;

-      4 h exposure in the presence of a final concentration of 2% of a metabolising system (S9), with a cell harvest after a 20 h expression period

-      4 h exposure without S9 with a 20 h expression period

-      4 h exposure with 1% S9 with a 20h expression period

-      24 h exposure without S9

All vehicle (solvent) control groups had frequencies of cells with aberrations within the range expected for normal human lymphocytes. The appropriate positive controls, cyclophosphamide and mitomycin C, respectively for with and without metabolic activation, scored positive in the assay.

The test item did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using dose levels that induced approximately 50% mitotic inhibition or less. The test item had a steep toxicity curve which made achieving optimum toxicity of 50% difficult. In some cases dose groups where greater than optimum toxicity was achieved were scored and achieved statistically significant increases in the frequency of aberrations, predominantly breaks. However, since these dose levels exceeded the optimum limit for toxicity (50%) and the response was considered to be as a result of a cytotoxic mechanism rather than a true mutagenic response this data can be excluded for the purpose of this study and is considered to have no toxicological significance.

Conclusion: the test item was considered to be non-clastogenic to human lymphocytesin vitro.

An in vitro mammalian cell gene mutation assay (Godek, 1985) was evaluated which used Chinese Hamster Ovary (CHO) cells to check the ability of DBPP to induce mutations in the HPRT1 gene. Based on the results of preliminary cytotoxicity assays and a preliminary CHO/HGPRT assay, DBPP was evaluated at dose levels of 10, 15, 20, 30 and 50 μg/mL without metabolic S9 activation preparation and at 25, 75, 150 and 175 μg/mL with a 5% concentration of S9.

Without metabolic activation the mean relative cell survivals were 103, 90, 69, 51 and 20% at the 10, 15, 20, 30 and 50 μg/mL dose levels, respectively. Mean relative cell survivals with the 5% concentration of metabolic activation were 112, 87, 61 and 13% at the 25, 75, 100, 150 and 175 μg/mL dose levels, respectively. Statistical analysis of the mutation data produced no statistically significant increases in the frequencies of the DBPP treated cultures when compared to the negative solvent controls. Positive controls, N-dimethylnitrosamine and ethylmethanesulphonate, respectively for with and without metabolic activation, were used and evaluated positive in the test. Conclusion: the test item was evaluated negative in this HGPRT test.

An unscheduled DNA synthesis (UDS) test (Steinmetz, 1985) was performed with primary cultures of rat hepatocytes. No appreciable increase in UDS above that of the solvent control level was observed after treatment of the hepatocytes with DBPP. The positive control, 2-acetylaminofluorene, produced a strong positive response in the experiment.

The results of the UDS assay indicate that DBPP is not a genotoxic agent in thein vitrohepatocyte DNA repair assay.

A mitotic recombination assay with S. cerevisiae (Brusick, 1977) was performed to measure mitotic recombination. This assay used the D4 strain of S. cerevisiae, which is suitable for the detection of mitotic gene conversion. DBPP was tested in the presence and absence of metabolic activation (S9). The compound was tested over a series of concentrations such there was either quantitative or qualitative evidence of some chemically-induced physiological effects at the high dose level. The low dose in all cases was below a concentration that demonstrated any toxic effects. The dose range employed for the evaluation of this compound was from 0.001μL to 5 μL per plate. The test compound did not demonstrate mutagenic activity in this assay and was considered as not mutagenic under these conditions.

 

The in vivo genetic toxicity study performed is an acutein vivorat bone marrow cytogenetics assay (Blazak, 1986). This study assessed the ability of DBPP administered by intraperitoneal injection to induce chromosomal damage in bone marrow cells of Fischer-344 rats. In the definitive study, male rats were given DBPP at doses of 0, 40, 200 and 400 mg/kg bw and female rats were given DBPP at doses of 0, 60, 300 and 600 mg/kg bw. Groups of animals were sacrificed 6, 12 and 24 h after treatment. The positive control groups received triethylenemelamine (0.2 mg/kg bw) by intraperitoneal injection and were sacrificed 24 h after treatment. Cells from males exposed to 0 and 400 mg/kg DBPP, from females exposed to 0 and 600 mg/kg DBPP, and from animals in the positive control groups were evaluated microscopically for mitotic index and chromosomal abnormalities.

On the basis of the results, we conclude that DBPP does not induce chromosomal damage in male or female Fischer-344 rats under the conditions used in this study.


Justification for classification or non-classification

Since DBPP was evaluated negative in all the performed in vitro tests and the in vivo test, the substance is not classified according to the CLP Regulation as a germ cell mutagen.