Reaction mass of Dimethyl [3-[(hydroxymethyl) amino]-3-oxopropyl] phosphonate and Dimethyl (3-amino-3-oxopropyl)phosphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

DMPPA_701-402 -5 was not mutagenic in the bacterial reverse mutation assay, while it had positive outcomes in the mouse lymphoma assay and in vitro chromosomal aberration assay.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP (The Netherlands)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 137244407
- Expiration date of the lot/batch: July 2004

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-mix

Test concentrations with justification for top dose:

Trial 1: 0.3125, 0.625, 1.25, 2.5 and 5.0 µL/plate
Trial 2: 0.213, 0.470, 1.033, 2.272 and 5.0 µL/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA 1537; In absence of metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA1535 and TA100; in absence of metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA98; in absence of metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cumene hydroperoxide

Remarks:

TA102; in absence of metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminofluorene

Remarks:

TA1537, TA 1535, TA98, TA100, TA102; in presence of metabolic activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; in agar (plate incorporation);

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:
- Exposure duration/duration of treatment: Overnight

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, relative survival (RS)
- Any supplementary information relevant to cytotoxicity:

Statistics:

Simple linear regression

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Solubility and Precipitation Tests

Solubility and precipitation tests were performed prior to cytotoxicity test. The solubility of Aflammit KWB was tested in distilled water and found to be a suitable vehicle for treatment as it completely dissolves in distilled water. Stock A (50 gL/mL) was prepared by addition of 50 ul of test substance to 950 PL of water. A volume of 100 ul of this stock solution was added to 2 mL of molten top agar to assess the precipitation. Precipitation was not observed at this concentration. Therefore 5 uL/plate has been selected as the highest concentration for cytotoxicity assay.

Cell Viability Test

Fresh cultures for the test were prepared by inoculating frozen permanent cultures to a flask containing 10 ml of sterile nutrient broth N^O2 (Oxoid). The flasks were then incubated at 37 ± 1 °C in an orbital shaking incubator for approximately 16 h up to early stationary or late exponential phase (approximately 10⁸to 10⁹cells/mL). After incubation period, the culture flasks were removed from the orbital shaking incubator. Aseptically, the cultures were diluted in distilled water and the optical density was measured at 660 nm using a Photoelectric Colorimeter (Erection & Instrumentation Engineerings India), Nutrient broth diluted in the same manner was used as control blank to set "0", Cell viability of tester strain was determined prior to treatment. The optical density was found to be in the acceptable range (Approx 0.4) and used for the study.

Genotype Confirmation Test

Fresh cultures for the test were prepared by inoculating frozen permanent cultures to a flask containing 10 mL of sterile nutrient broth N^O2 (Oxoid). The flasks were then incubated at 37 ± 1^oC in an orbital shaking incubator for approximately 16 h up to early stationary or late phase.

Cytotoxicity Test

Before commencing the mutagenicity study, Aflammit KWB was assessed for cytotoxicity to Salmonella typhimurium tester strain TA100. The experiment was conducted both in the presence and absence of metabolic activation (5 % v/v S9). A stock solution (50 µg/mL) of Aflammit KWB was prepared by addition of 75 µL of Aflammit KWB to 1.425 mL sterile distilled water (Stock A). Further stock solution of concentrations viz, 10.0, 2.0, 0.4, 0.08 and 0.016 µL/mL were prepared from the first stock solution. Six different concentrations viz., 0.0016, 0.008, 0.04, 0.2, 1.0 and 5.0 µL/plate were tested for cytotoxicity. Tubes containing 2 mL of molten top agar with 0.5 rnM histidine/biotine were maintained at 45 °C. A volume of 500 µL of 5% VIV S9 mix was added to one ofthe sets and 500 µL of 0.2 M Phosphate buffer was added to the second set. 100 uL of relevant stock solution of test substance or distilled water was to the tubes for treatment and negative control respectively. Finally 100 µl. of bacterial culture was added to the tubes and mixed. Details of culture preparation are provided under "Cell Viability Test" (cultures used were checked for cell viability prior to testing). This treatment mixture was poured on minimal glucose agar plates and allowed to solidify. Duplicate sets were used for each concentration and controls. The petriplates were incubated at 37 ± 1 °C for 48 hours and then examined to assess the state of the background bacterial growth. Cytotoxicity was assessed by clearance of background lawn. Background lawn inhibition was not observed up to 5.0 µl/plate in presence and absence of metabolic activation. Hence 5.0 uL/plate has been selected as the highest test concentration for mutagenicity test.

Mutagenicity Test

Mutagenicity test was conducted as two independent experiments. In both the trials, the treatment was given in the presence and absence of metabolic activation (5 % and 10 % v/v S9 mix in first and second trial respectively). The treatments were performed by plate in corporation technique as in cytotoxicity test. Duplicate plates were maintained for each test concentration of Aflammit KWB, negative and positive controls.

In the first trial the strains were exposed at 0.3125, 0.625, 1 .25, 2.5 and 5.0 µL/pIate (factor 2). First stock solution (Stock A) of test substance was prepared by addition of 300 µL Aflammit K WB in 5.7 mL of sterile distilled water (50.0 µL/mL). Further stock solutions viz 25 (Stock B), 12.5 (Stock C), 6.25 (Stock D) and 3.125 (Stock E) were prepared from the first stock solution by dilution. Volumes of 100 µLof these stock solutions (A-E) were used to obtain the required test concentrations. The number of revenant colonies was recorded after 48 h incubation period. Similarly a second trail was conducted to confirm the negative results obtained in the first trial. In the second trial, the concentration spacing was modified using a factor 2.2. The highest concentration being 5.0 µL/plate, four lower doses viz 0.213, 0.470, 1.033 and 2.272 µL/plate were tested. In the second trial the first stock solution of the test substance (Stock A) was prepared by addition of 300 µL Aflammit KWB in 5.7 mL of sterile distilled water (50.0 uL/mL). Further stock solutions viz 22.72 (Stock B), 10.33 (Stock C), 4.70 (Stock D) and 2.13 (Stock E) µL/mL were prepared from stock A by dilution. Volumes of 100 µL of these stock solutions (A-E) were used to obtain the required test concentrations. The number of revertant colonies was recorded after 48 h incubation period.

Treatment with 2-aminofluorene in the absence of metabolic activation was performed along with both the trials to confirm the efficiency of S9 fraction used in the study.

Evaluation Criteria

A positive result is defined as a statistically significant, dose-dependent increase in the numb histidine independent revertants with at least one dose level inducing a revertant frequency that is at least two-fold of the spontaneous solvent control value. If the test substance does not induce a statistically significant, dose-dependent increase in revertant frequency in both the replicates, but does induce revertant frequency at one dose level that is at least two-fold the spontaneous control value, the result is considered equivocal. A negative result is defined as the absence of statistically significant or dose-dependent increase in the number of histidine independent revenant.

Conclusions:

DMPPA_701-402-5 is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of DMPPA_701-402-5 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 TA102 according to OECD 471 guideline. 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 both with and without metabolic activation. Based on the study result, DMPPA_701-402-5 is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

V79

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Name: FAT 80'001/I
Batch No: EN 746916/1991
Aggregate State at RT: Solid
Colour: White
Molecular Weight: 211.16
Stability: Pure: see expiration date. In solvent: >2 hours in water, ethanol, methanol, acetone, DMSO, DMF
Storage: room temperature
Expiration Date: September 1996

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: supplied by LMP; Technical University Darmstadt, D-6100 Darmstadt.

Large stocks of the V79 cell line were stored in liquid nitrogen in the cell bank of CCR allowing the repeated use of the same cell culture batch in experiments. Before freezing, each batch was screened for mycoplasma contamination and checked for karyotype stability. Consequently, the parameters of the experiments remained similar because of the reproducible characteristics of the cells.

Thawed stock cultures were propagated at 37°C in 80 sq.cm plastic flasks (GREINER, D-7443 Frickenhausen, F.R.G.). Seeding was done with about 5 x 10^5 cells per flask in 15 mL of MEM (minimal essential medium; SEROMED; D-1000 Berlin, F.R.G.) supplemented with 10% fetal calf serum (FCS; SEROMED). The cells were subcultured twice weekly. The cell cultures were incubated at 37°C and 4.5 % carbon dioxide atmosphere.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Without S9 mix:
18 h: 0.03; 0.1; 0.3 mg/mL
28 h: 0.3 mg/mL

with S9 mix
18 h: 0.1; 0.3; 0.6 mg/mL
28 h: 0.6 mg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: culture medium MEM (Minimal Essential Medium).
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties and its non-toxcicity for the cells.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 500000

Evaluation criteria:

A test article is classified as mutagenic if it induces either a concentration-related increase in the number of structural chromosomal aberrations or a significant positive response for at least one of the test points.
A test article producing neither a concentration-related increase in the number of structural chromosomal aberrations nor a significant positive response at any one of the test points is considered non-mutagenic in this system. This can be confirmed by means of the chi-square test. However, both biological and statistical significance should be considered together.

Statistics:

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the chi-square test. Evaluation was performed only for cells carrying aberrations exclusive gaps.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without S9 mix: 0.30 mg/ml; with S9 mix: 0.60 mg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

In the pre-experiment the plating efficiency (PE) was clearly reduced after treatment with concentrations of 0.3 mg/mL and higher in presence and absence of S9 mix. Also, in the main experiment a reduction of mitotic index could be observed after treatment with the highest concentrations scored in both fixation intervals (without S9 mix: 0.3 mg/mL; with S9 mix: 0.6 mg/mL).
In the pre-experiment as well as in the main experiment very strong toxic effects of the test article were found after treatment with concentrations higher than 0.6 mg/mL at both fixation intervals in presence and absence of S9 mix.

There was a statistically significant and biologically relevant increase in cells with structural aberrations after treatment with 0.3 mg/mL of the test article without metabolic activation and after treatment with 0.6 mg/mL with metabolic activation by S9 mix at both fixation intervals. Treatment with lower concentrations showed no relevant increases in cells with structural aberrations in any fixation interval either with or without metabolic activation by S9 mix.

The relevant increase in the number of polyploid metaphases in comparison to the control data (found 28 h after treatment with 0.3 mg/mL in the absence of S9 mix) give some indications of the potential of the test article to induce numeric aberrations.
Appropriate reference mutagens were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Conclusions:

DMPPA_701-402-5 induced structural chromosome aberrations in the V79 Chinese hamster cell line after treatment with a concentration of 0.3 mg/mL in the absence of metabolic activation and 0.6 mg/mL with metabolic activation by S9 mix.

Executive summary:

The test article DMPPA_701-402-5 was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro. This test was conducted in accordance with OECD TG 473 in a GLP certified laboratory. Preparation of chromosomes was done 18 h (low, medium and high concentration range), and 28 h (high concentration range) after start of treatment with the test article which was dissolved in the culture medium (MEM). The treatment interval was 4 h. In each experimental group two parallel cultures were used. Per culture 100 metaphases were scored for structural chromosomal aberrations except for the positive control culture where at least 25 metaphases were scored. The following concentrations were evaluated:

without S9 mix:

18 h: 0.03; 0.10; 0.30 mg/mL

28 h: 0.3 mg/mL

with S9 mix: 18 h: 0.10; 0.30; 0.60 mg/mL

28 h: 0.6 mg/mL

According to recommendations in several guidelines 5 mg/mL FAT 80'001/I was chosen as highest concentration in the pre-experiment and in the main experiment. In the pre-experiment the plating efficiency (PE) was clearly reduced after treatment with concentrations of 0.3 mg/mL and higher in presence and absence of S9 mix. Also, in the main experiment a reduction of mitotic index could be observed after treatment with the highest concentrations scored in both fixation intervals (without S9 mix: 0.3 mg/mL; with S9 mix: 0.6 mg/mL). In the pre-experiment as well as in the main experiment very strong toxic effects of the test article were found after treatment with concentrations higher than 0.6 mg/mL at both fixation intervals in presence and absence of S9 mix. There was a statistically significant and biologically relevant increase in cells with structural aberrations after treatment with 0.3 mg/mL of the test article without metabolic activation and after treatment with 0.6 mg/mL with metabolic activation by S9 mix at both fixation intervals. Treatment with lower concentrations showed no relevant increases in cells with structural aberrations in any fixation interval either with or without metabolic activation by S9 mix. The relevant increase in the number of polyploid metaphases in comparison to the control data (found 28 h after treatment with 0.3 mg/mL in the absence of S9 mix) give some indications of the potential of the test article to induce numeric aberrations. Appropriate reference mutagens were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

CONCLUSION

In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article induced structural chromosome aberrations after treatment with 0.3 mg/mL (in the absence of metabolic activation) and 0.6 mg/mL (in the presence of metabolic activation) as determined by the chromosomal aberration test in the V79 Chinese hamster cell line. Therefore, DMPPA_701-402-5 is considered to be clastogenic in this chromosomal aberration test.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: European Economic Community (EEC), Directive 87/302/EEC. Annex V o f the EEC Directive 67/548/EEC

Version / remarks:

Part B: Methods for the Determination of Toxicity; "Other Effects - Mutagenicity: InVitro Mammalian Cell Gene Mutation Test". EEC Publication no. L133 (adopted May 30, 1988).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

Identification: PYROVATEX CP neu
Description: Clear colourless liquid
Batch: 65978
Test substance storage: At room temperature in the dark
Stability under storage conditions: Stable
Expiry date: October 10, 1996

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Source: Dr. A.G.A.C. Knaap, Department of, Radiation Genetics and Chemical Mutagenesis of the State University of Leiden, The Netherlands (1981).
This mouse lymphoma cell line was originally derived from the Fischer L5178Y line, isolated by Clive (1975).

L5178Y mouse lymphoma cells were cultured in F10 complete culture medium. Cultures were incubated in a humid atmosphere containing 5% CO2 in air at 37°C. Cell density was preferably kept below 7 x 10^5 cells/ mL.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Rat liver microsomal enzymes were routinely prepared from adult male Wistar rats, which were obtained from Charles River Wiga, Sulzfeld, Germany.
- method of preparation of S9 mix : The animals were housed at NOTOX in a special room under standard laboratory conditions, as described in the SOP's. The rats were injected intraperitoneal with a solution (20% w/v) of Aroclor 1254 (500 mg/kg body weight) in corn oil. Five days later, they were killed by decapitation; (they were denied access to food for at least 12 hours preceding sacrifice). The livers of the rats were removed aseptically, and washed in cold (0°C) sterile 0.1 M sodium phosphate buffer (pH 7.4) containing 0.1 mM Disodium EDTA. Subsequently the livers were minced in able nder and homogenized in 3 volumes of phosphate buffer with a Potter homogenizer. The-homogenate was centrifuged for 15 min at 9000 g. The supernatant (S9) was transferred into sterile ampules, which were stored in liquid nitrogen (-196 °C). S9-mix was prepared immediately before use and kept on ice during the test. S9-mix contained per mL: 1.02 mg MgCl2. 6H20; 2.46 mg KCl; 1.7 mg glucose-6-phosphate; 3.4 mg NADP; 4 µmoL HEPES and 0.5 mL S9 (experiment 1) and 0.7 mL S9 (experiment 2). The above solutions were mixed and filter (0.22 µm)- sterilized (apart from the S9-fraction, which was added after filter - sterilization of the S9-mix components). Metabolic activation was achieved by adding 0.2 mL liver S9-mix per mL of cell suspension. The S9-batch used was no. 95-2 (final concentration of Cytochrome P-450 in the S9-mix in the cytotoxicity test and in experiment 1 was 24.4 nmoL/mL and in experiment 2 was 34.1 nmoL/mL). The concentration of the S9 - fraction in the exposition medium was 8- and 12% (v/v) in the first and second experiment respectively.

Vehicle / solvent:

The test substance was dissolved in exposition medium (F10-medium buffered with 20 mM HEPES) and filter (0.22 µm)- sterilized.
Test substance concentrations were prepared directly prior to use.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: single
- Number of independent experiments : Two

Evaluation criteria:

A test substance was considered positive (mutagenic) in the mutation assay if:
a) It induced at least a 3-fold increase in the mutant frequency compared to the solvent control in a dose-dependent manner; and
b) The results were reproducible in an independently repeated test.

A test substance was considered negative (not mutagenic) in the mutation assay if:
a) None o f the tested concentrations showed a mutant frequency of at least three-fold compared to the solvent control.
b) The results were confirmed in an independently repeated test.

Statistics:

Not applicable

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE FINDING TEST: In the dose range finding test L5178Y mouse lymphoma cells were treated with a test substance concentration range of 33 to 5000 µg/ml with and without S9- mix. The pH and osmolarity of a concentration of 5000 µg/ml were 7.26 and 320 mOsm/kg respectively (compared to 7.34 and 290 mOsm/kg in the solvent control).

Both in the absence and presence of S9-mix, no reduction was observed in the cell count of the remaining cells directly after treatment. In the absence of S9-mix at a test substance concentration of 1000 µg/mL, the initially surviving cells showed a reduction of 75 % in the cloning efficiency compared to the cloning efficiency of the solvent control. At the test substance concentrations of 3330 and 5000 µg/plate almost or total cell killing was observed in the cloning efficiency of the remaining cells. In the presence o f S9-mix at a test substance concenctration of 3330 µg/mL, the initially surviving cells showed a reduction of 94 % in the cloning efficiency compared to the cloning efficiency of the solvent control. At a test substance concentration of 5000 µg/ml almost total cell killing was observed in the cloning efficiency of the remaining cells.

Based on the results of the cytotoxicity test, the following dose range was selected for mutagenicity testing:
Experiment 1
Without S9-mix: 237, 333, 562, 750, 1000 and 1334 µg/ml exposition medium
With S9-mix : 562, 750, 1000, 1334, 1778 and 2371 µg/ml exposition medium


MAIN TEST
In both experiments, in the absence o f S9-mix PYROVATEX CP NEU induced dose-related, 9-fold, increases in the mutant frequency at the TK-locus. In the presence of S9-mix the test substance induced also dose-related, 4-and 26-fold increases in the mutant frequency at the TK-locus, in the first and second experiment respectively. As well as in the absence as in the presence of S9-mix the number of small and large colonies were increased. The increase in small colonies was 3-and 6 - fold in the absence of S9-mix and 5- and 23-fold in the presence of S9-mix, in the first and second experiment respectively. The increase in large colonies was 3-and 6 -fold in the absence of S9-mix and 3-and 10-fold in the presence of S9-mix, in the first and second experiment respectively.

Conclusions:

PYROVATEX CP NEU is mutagenic in the TK mutation test system under the experimental conditions described in this report.

Executive summary:

This report describes the effects of PYROVATEX CP NEU on the induction of forward mutations at the TK-locus in L5178Y mouse lymphoma cells in the presence and absence of S9-mix. This test was conducted on basis of OECD TG 476 and European Economic Community (EEC), Directive 87/302/EEC. Annex V of the EEC Directive 67/548/EEC, Part B in a GLP compliant lab. The test substance was tested from 237 to 1000 µg/mL in the absence of S9 -mix and from 562 to 3162 µg/mL in the presence of S9-mix. In both experiments, in the absence of S9-mix PYROVATEX CP NEU induced dose related 9-fold increase in the mutant frequency at the TK-locus. In the presence of S9-mix the test substance induced also dose-related, 4- and 26-fold Increases in the mutant frequency at the TK-locus, in the first and second experiment respectively. As well as in the absence as in the presence of S9-mix the number of small and large colonies were increased. The increase in small colonies was 3-and 6-fold in the absence of S9-mix and 5-and 23-fold in the presence of S9-mix, in the first and second experiment respectively. The increase in large colonies was 3 -and 6 -fold in the absence of S9-mix and 3-and 10-fold in the presence of S9-mix, in the first and second experiment respectively. The increases were reproducible in two independently repeated experiments. Therefore, these increases were considered to be biologically significant. The large colonies were the result of mutants with single gene mutations (substitutions, deletions of base-pairs) affecting the TK gene. The small colonies can be associated with the induction of chromosomal mutations. Mutant frequencies induced by positive control chemicals were increased by 6-and12-fold for EMS, in the first and second experiment respectively, and by 12- and 42-fold for DMN, in the first and second experiment respectively. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were suitable for the detection of a mutagenic response and that the metabolic activation system (S9-mix) functioned properly. It is concluded that PYROVATEX CPN EU is mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

DMPPA_701-402-5 had negative outcomes in the unscheduled synthesis assay and the micronucleus assay.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Micronucleus test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Specific details on test material used for the study:

Name: FAT 80'001/I
Batch No: EN 746916/1991
Aggregate State at RT: solid
Colour: white
Stability: Pure: years. In vehicle: in water, DMSO and DMF > 2 hours
Storage: at room temperature
Expiration Date: September 1996

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga GmbH, D-8741 Sulzfeld 1, Germany.
- Age at study initiation: minimum 10 weeks
- Weight at study initiation: approximately 30 g
- Fasting period before study: 18 hours
- Housing: single. Housed in Makrolon Type I, with wire mesh top (EHRET GmbH, D-7830 Emmendingen). Bedding provided was granulated soft wood.
(ALTROMIN, D-4937 Lage/Lippe)
- Diet (e.g. ad libitum): pelleted standard diet (ALTROMIN 1324, D-4937 Lage/Lippe)
- Water (e.g. ad libitum): tap water, ad libitum (Gemeindewerke, D-6101 Roßdorf)
- Acclimation period: minimum 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 6 am to 5 pm artificial light.

Route of administration:

oral: gavage

Vehicle:

Deionised water

Details on exposure:

Single oral gavage

Duration of treatment / exposure:

single oral gavage

Frequency of treatment:

single treatment

Dose / conc.:

5 000 mg/kg bw/day (actual dose received)

Remarks:

The maximum tolerated dose level was determined to be the dose that caused toxic reactions without having major effects on survival within 72 hours.

No. of animals per sex per dose:

Six males and six females were assigned to each test group. The animals were identified by their cage number as shown below in the table.

Control animals:

yes, concurrent vehicle

Positive control(s):

Name: CPA; Cyclophosphamide
Supplier: SERVA, D-6900 Heidelberg
Catalogue no: 17681
Dissolved in: physiological saline
Dosing: 30 mg/kg b.w.
Route and Frequency of Administration: orally, once
Volume Administered: 10 mL/kg b.w.

Tissues and cell types examined:

Bone marrow cells

Details of tissue and slide preparation:

The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a 5 ml syringe. The cell suspension was centrifuged at 1,500 rpm for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (MERCK, D-6100 Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-7800 Freiburg). At least one slide was made from each bone marrow sample.

Evaluation criteria:

A test article is classified as mutagenic if it induces a statistically significant increase in the number of micronucleated polychromatic erythrocytes at for at least one of the test points. A test article producing no statistically significant increase in the number of micronucleated polychromatic erythrocytes at any of the test points is considered non-mutagenic in this system.

Statistics:

Non-parametric Mann-Whitney test

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

PRE-EXPERIMENT FOR TOXICITY: In several pre-experiments 4 animals (2 males, 2 females) per dose group received orally a single dose of 2000, 3000, 4000 or 5000 mg/kg bw, respectively. DMPPA_701-402-5 was dissolved in aqua deionized. The volume administered was 20 ml/kg b.w. In pre-experiments this dose level was estimated to be the maximum attainable dose. The animals expressed toxic reactions - reduction of spontaneous activity, eye lid closure and apathy was observed at all doses. The mean number of normochromatic erythrocytes was not substantially increased after treatment with the test article as compared to the mean values of NCEs of the corresponding negative controls, indicating that FAT 80'001/I had no cytotoxic properties.

Conclusions:

Based on the findings of the study, the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse. Therefore, DMPPA_701-402-5 is considered to be non-mutagenic in this micronucleus assay.

Executive summary:

A study was performed to investigate the potential of DMPPA_701-402-5 to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. This test was conducted in accordance with OECD TG 474 and EEC Directive 84/449 in a GLP certified laboratory. The test article was dissolved in aqua deionized. This solvent was used as negative control. The volume administered orally was 20 mL/kg bw. 24 h, 48 h and 72 h after a single application of the test article the bone marrow cells were collected for micronuclei analysis. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 PCE. The following dose level of the test article was investigated: 24 h, 48 h, and 72 h preparation interval: 5000 mg/kg bw. In pre-experiments, this dose level was estimated to be the maximum attainable dose. The animals expressed toxic reactions. After treatment with the test article the ratio between PCEs and NCEs was not affected as compared to the corresponding negative controls thus indicating no cytotoxic effects. In comparison to the corresponding negative controls there was no statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. An appropriate reference mutagen was used as positive control (Cyclophosphamide) which showed a distinct increase of induced micronucleus frequency. Based on the findings of the study, the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse. Therefore, DMPPA_701-402-5 is considered to be non-mutagenic in this micronucleus assay.

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Deviations:

yes

Remarks:

no effect on study outcome was seen

GLP compliance:

yes (incl. QA statement)

Type of assay:

unscheduled DNA synthesis

Specific details on test material used for the study:

Name: FAT 80'001/I
Batch No: EN 746916/1991
Aggregate State at RT: solid
Colour: white
Stability: Pure: years. In vehicle: in water, DMSO and DMF >2 hours
Storage: at room temperature
Expiration Date: September 1996

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga GmbH, D-8741 Sulzfeld 1, Germany.
- Age at study initiation: minimum 6-8 weeks
- Weight at study initiation: 204 - 289 g
- Fasting period before study: The animals were starved overnight (2 hours treatment) or approximately 6 hours (16 hours treatment) before receiving the test article.
- Housing: single. Housed in Makrolon Type I, with wire mesh top (EHRET GmbH, D-7830 Emmendingen). Bedding provided was granulated soft wood. (ALTROMIN, D-4937 Lage/Lippe)
- Diet (e.g. ad libitum): pelleted standard diet (ALTROMIN 1324, D-4937 Lage/Lippe)
- Water (e.g. ad libitum): tap water, ad libitum (Gemeindewerke, D-6101 Roßdorf)
- Acclimation period: minimum 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 6 am to 5 pm artificial light.

Route of administration:

oral: gavage

Vehicle:

Deionised water

Details on exposure:

Route and frequency of administration: singly, orally
Volume administered: 10 mL/kg bw

Duration of treatment / exposure:

2 hour treatment period: 2000 mg/kg b.w.
16 hour treatment period: 200 and 2000 mg/kg b.w.

Frequency of treatment:

Once

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Five male rats were assigned to each test group. The animals were identified by their cage numbers as shown below in the table.

Control animals:

yes, concurrent vehicle

Positive control(s):

Positive control substance used:
Name: 2-AAF; 2-Acetylaminofluorene
Supplier: FLUKA FEINCHEMIKALIEN GmbH, D-7910 Neu-Ulm, F.R.G.
Catalogue no: 00300
Purity: >99 %
Dissolved in: dimethyl sulfoxide/polyethylene glycol 400 ( 1 + 9)
Dosing: 100 mg/kg bw
Route and frequency of administration: singly, orally.
Volume administered: 10 mL/kg bw.

Tissues and cell types examined:

Hepatocytes

Details of tissue and slide preparation:

Dose Selection
The maximum tolerated dose is determined to be the dose that causes toxic reactions (e.g. reduced spontaneous activity, eyelid closure, apathy, etc.) without having major effects on survival within 24 hours. If no toxic reactions are observed the highest dose to be used should be 2000 mg/kg bw. For the low dose one tenth of the high dose level is tested.

Isolation of the Primary Hepatocytes
The animals were sacrificed by liver perfusion. After anaesthetising the rats with Nembutal (Sanofi/Ceva, D-4000 Düsseldorf, F.R.G.) the liver was perfused through the vena portae with Hanks' balanced salt solution (HBSS, Gibco/BRL, D-7514 Eggenstein, F.R.G.) supplemented with collagenase (0.05 % w/v, Boehringer Mannheim, D-6800 Mannheim, F.R.G.) adjusted to pH 7.4 and maintained at 37 °C. The hepatocytes were isolated from the liver and washed twice with HBSS. The crude cell suspension was filtered through a 94 µm stainless steel mesh to yield a single cell suspension. The quality of the actual performed perfusion was determined by the trypan blue dye exclusion method. In addition, the number of the isolated cells was determined. The washed hepatocytes were centrifuged and transferred into Williams medium E. The medium without the cells was adjusted to pH 7.6. At least three cultures were established for each animal. Aliquotes of 2.5 ml with freshly isolated hepatocytes in complete culture medium (1.0 x 10^ living cells/ml) were added to 35 mm six-well cluster dishes (Greiner, D-7440-Nürtingen, F.R.G.) containing one gelatinised 25 mm round plastic coverslip (Thermanox. Flow Laboratories, D-5309 Meckenheim, F.R.G.) per well. After an attachment period of approximately 1.5 h in a 95 % air/ 5 % CO2 humidified incubator at 37 °C the culture medium was discarded. Then the cell layer was rinsed once with PBS to remove non-adherent cells. Subsequently ³HTdR (5 µCi/mL, specific activity 20 Ci/mmol; New England Nuclear, D-6072 Dreieich, F.R.G.) in 2.0 ml culture medium (WME, 1 % FCS) was added to the cultures. After a labelling time of 4 h the cells were washed twice with WME supplemented with 1 % FCS and 0.25 mM unlabelled thymidine.
Cultures were incubated overnight using the same medium. To prepare for autoradiography the medium was replaced by a hypotonic solution of 1 % sodium citrate for 10 minutes to swell the nuclei for better grain quantification. The cells on the coverslips were then fixed by three changes of methanol:acetic acid (3+1 v/v) for 20 minutes each, rinsed with 96 % ethanol, and air dried.

Evaluation criteria:

Nuclear and net grain counts are estimated together. Increased net grains should be based on enhanced nuclear grain counts rather than on decreased cytoplasmic grain counts. A test article is classified as positive if the mean number of net grains is higher than five per nucleus at one of the test points. A group average between 0 and 5 net grains is considered as a marginal response. A dose-related increase in nuclear and net grains and/or a substantial shift of the percentage distribution of the nuclear grain counts to higher values provide additional informations to confirm a positive response with less than 5 net grains. Statistical significance may give further evidence for a positive evaluation. A test article producing net grains not greater than 0 at anyone of the test points is considered non-effective in this system.

Statistics:

Statistical significance can be evaluated by means of the nonparametric Mann-Whitney test

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

In a pre-experiment two animals were treated with 2000 mg/kg bw yielding no signs of toxicity.

Conclusions:

Under the experimental conditions reported, the test article did not induce DNA-damage leading to repair synthesis in the hepatocytes of the treated rats. Therefore, DMPPA_701-402-5 is considered to be non-effective in this in vivo/in vitro UDS test system.

Executive summary:

The test article DMPPA_701-402-5 was assessed in the in vivo/ in vitro UDS assay for its potential to induce DNA repair (UDS) in the hepatocytes of rats. This test was conducted in accordance method similar or equivalent to OECD TG 486. The test article was formulated in aqua deionised. This suspending agent was used as vehicle control. The volume administered orally was 10 mL/kg body weight (bw). After a treatment period of 2 and 16 hours, respectively, the animals were narcotised and sacrificed by liver perfusion. Primary hepatocyte cultures were established and exposed for 4 hours to ³HTdR (methyl-³H-thymidine) which is incorporated if UDS occurs.

The test article was tested at the following dose levels:

2-hour treatment period: 2000 mg/kg bw.

16-hour treatment period: 200 and 2000 mg/kg bw.

For each dose level, including the controls, hepatocytes from four treated animals were assessed for the occurrence of UDS. No toxic reactions of the animals occurred at any of the treatment periods or dose groups. In addition, the viability of the hepatocytes was not substantially affected due to the in vivo pre-treatment with the test article. No dose level of the test article revealed UDS induction in the hepatocytes of the treated animals as compared to the current vehicle controls. An appropriate reference mutagen (2-AAF, 100 mg/kg bw) was used as positive control. Treatment with 2-AAF revealed distinct increases in the number of nuclear and net grain counts. Under the experimental conditions reported, the test article did not induce DNA-damage leading to repair synthesis in the hepatocytes of the treated rats. Therefore, DMPPA_701-402-5 is considered to be non-effective in this in vivo/in vitro UDS test system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

- in vitro data

Bacterial reverse gene mutation assay:

A bacterial reverse gene mutation assay was performed to investigate the potential of DMPPA_701-402-5 to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100. The assay was performed in two independent experiments, using identical procedures, both with and without metabolic activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations:

Exp. I: 10; 33; 100; 333; 1000; and 5000 µg/plate

Exp. II: 10; 33; 100; 1000; 2500; and 5000 µg/plate

Toxic effects, evidenced by a reduction in the number of revertants occurred in the strains TA 1535 (exp. I), TA 1538 (exp. I and exp. II), and TA 98 (exp. I and exp. II) all at 5000 µg/plate without metabolic activation. The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with DMPPA_701-402-5 at any dose level, either in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of significance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

 

CONCLUSION

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce point mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, DMPPA_701-402-5 is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

 

Mammalian gene mutation assay:

The potential of PYROVATEX CP NEU to induce forward mutations was assessed at the TK-locus in L5178Y mouse lymphoma cells in the presence and absence of S9-mix. This test was conducted on basis of OECD TG 476 and European Economic Community (EEC), Directive 87/302/EEC. Annex V of the EEC Directive 67/548/EEC, Part B in a GLP compliant lab. The test substance was tested from 237 to 1000 µg/mL in the absence of S9- mix and from 562 to 3162 µg/mL in the presence of S9-mix. In both experiments, in the absence of S9-mix PYROVATEX CP NEU induced dose related 9-fold increase in the mutant frequency at the TK-locus. In the presence of S9-mix the test substance induced also dose-related, 4- and 26-fold increases in the mutant frequency at the TK-locus, in the first and second experiment respectively. In the absence and in the presence of S9-mix the number of small and large colonies were increased. The increase in small colonies was 3-and 6-fold in the absence of S9-mix and 5-and 23-fold in the presence of S9-mix, in the first and second experiment respectively. The increase in large colonies was 3 -and 6 -fold in the absence of S9-mix and 3-and 10-fold in the presence of S9-mix, in the first and second experiment respectively. The increases were reproducible in two independently repeated experiments. Therefore, these increases were considered to be biologically significant. The large colonies were the result of mutants with single gene mutations (substitutions, deletions of base-pairs) affecting the TK gene. The small colonies can be associated with the induction of chromosomal mutations. Mutant frequencies induced by positive control chemicals were increased by 6-and12-fold for EMS, in the first and second experiment respectively, and by 12-and 42-fold for DMN, in the first and second experiment respectively. Therefore, it was concluded that the test conditions, both in the absence and presence of S9-mix, were suitable for the detection of a mutagenic response and that the metabolic activation system (S9-mix) functioned properly. It is further concluded that PYROVATEX CPN EU is mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described.

 

mammalian chromosome aberration assay:

The test article DMPPA_701-402-5 was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro. Preparation of chromosomes was done 18 h (low, medium and high concentration range), and 28 h (high concentration range) after start of treatment with the test article which was dissolved in the culture medium (MEM). The treatment interval was 4 h. In each experimental group two parallel cultures were used. Per culture 100 metaphases were scored for structural chromosomal aberrations except for the positive control culture where at least 25 metaphases were scored. The following concentrations were evaluated: without S9 mix and with S9 mix: According to recommendations in several guidelines 5 mg/ml

DMPPA_701-402-5 was chosen as highest concentration in the pre-experiment and in the main experiment. In the pre-experiment the plating efficiency (PE) was clearly reduced after treatment with concentrations of 0.3 mg/ml and higher in presence and absence of S9 mix. Also, in the main experiment a reduction of mitotic index could be observed after treatment with the highest concentrations scored in both fixation intervals (without S9 mix: 0.3 mg/ml; with S9 mix: 0.6 mg/ml). In the pre-experiment as well as in the main experiment very strong toxic effects of the test article were found after treatment with concentrations higher than 0.6 mg/ml at both fixation intervals in presence and absence of S9 mix. There was a statistically significant and biologically relevant increase in cells with structural aberrations after treatment with 0.3 mg/ml of the test article without metabolic activation and after treatment with 0.6 mg/ml with metabolic activation by S9 mix at both fixation intervals. Treatment with lower concentrations showed no relevant increases in cells with structural aberrations in any fixation interval either with or without metabolic activation by S9 mix.

18 h: 0.03; 0.1; 0.3 mg/ml

28 h: 0.3 mg/ml

18 h: 0.1; 0.3; 0.6 mg/ml

28 h: 0.6 mg/ml

The relevant increase in the number of polyploid metaphases in comparison to the control data (found 28 h after treatment with 0.3 mg/ml in the absence of S9 mix) give some indications of the potential of the test article to induce numeric aberrations. Appropriate reference mutagens were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

CONCLUSION

The test article induced structural chromosome aberrations after treatment with 0.3 mg/ml (in the absence of metabolic activation) and 0.6 mg/ml (in the presence of metabolic activation) as determined by the chromosomal aberration test in the V79 Chinese hamster cell line. Therefore, DMPPA_701-402 -5 is considered to be mutagenic in this chromosomal aberration test.

 

In vivo data

UDS test:

The test article DMPPA_701-402-5 was assessed in the in vivo UDS assay for its potential to induce DNA repair (UDS) in the hepatocytes of rats. The test article was formulated in aqua deionised. This suspending agent was used as vehicle control. The volume administered orally was 10 ml/kg body weight (bw). After a treatment period of 2 and 16 hours, respectively, the animals were narcotised and sacrificed by liver perfusion. Primary hepatocyte cultures were established and exposed for 4 hours to -%TdR (methyl-³H-thymidine) which is incorporated if UDS occurs. The test article was tested at the following dose levels:

2-hour treatment period: 2000 mg/kg b.w.

16-hour treatment period: 200 and 2000 mg/kg b.w.

For each dose level, including the controls, hepatocytes from four treated animals were assessed for the occurrence of UDS. No toxic reactions of the animals occurred at any of the treatment periods or dose groups. In addition, the viability of the hepatocytes was not substantially affected due to the in vivo pre-treatment with the test article. No dose level of the test article revealed UDS induction in the hepatocytes of the treated animals as compared to the current vehicle controls. An appropriate reference mutagen (2-AAF, 100 mg/kg b.w.) was used as positive control. Treatment with 2-AAF revealed distinct increases in the number of nuclear and net grain counts.

 

CONCLUSION

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce DNA-damage leading to repair synthesis in the hepatocytes of the treated rats. Therefore, DMPPA_701-402-5 is considered to be not genotoxic in this in vivo/in vitro UDS test system.

 

Micronucleuas Assay:

The potential of DMPPA_701-402-5 to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse was assessed in a micronucleus assay. The test article was dissolved in aqua deionized. This solvent was used as negative control. The volume administered orally was 20 ml/kg b.w.. 24 h, 48 h and 72 h after a single application of the test article the bone marrow cells were collected for micronuclei analysis.

Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 PCE. The following dose level of the test article was investigated: 24 h, 48 h, and 72 h preparation interval: 5000 mg/kg b.w. In pre-experiments this dose level was estimated to be the maximum attainable dose. The animals expressed toxic reactions. After treatment with the test article the ratio between PCEs and NCEs was not affected as compared to the corresponding negative controls thus indicating no cytotoxic effects. In comparison to the corresponding negative controls there was no statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. An appropriate reference mutagen was used as positive control which showed a distinct increase of induced micronucleus frequency.

 

CONCLUSION

The test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, DMPPA_701-402-5 is considered to be non-clastogenic in this micronucleus assay.

 

Overall summary:

The negative result in the in vivo UDS and micronucleus assay overrules the positive result from the in vitro mammalian cell gene mutation assay and in vitro chromosome aberration test. Therefore, it can be concluded that DMPPA_701-402-5 is not genotoxic.

Justification for classification or non-classification

Based on the above stated assessment of the genotoxic potential DMPPA_701-402-5 a(Ames test negative, in vitro mammalian chromosome aberration test positive, in vivo mammalian micronucleus test in bone marrow cells negative and in vivo UDS test negative) the substance is deemed non-genotoxic and accordingly does not need to be classified according to CLP (Regulation (EC) No 1272/2008 of the European Parliament and of the Council) as implementation of UN-GHS in the EU.