2,4,6-tri-n-propyl-2,4,6-trioxo-1,3,5,2,4,6-trioxatriphosphorinane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test

The test compound is not mutagenic in the bacterial reverse mutation assay either with or without exogenous metabolic activation.

Chromosome aberration assay - 1st assay

The test substance induced chromosome mutations (=aberrations) in V79 Chinese hamster cells in the presence and in the absence of a metabolic activation system.

Chromosome aberration assay - 2nd assay

The test substance was not clastogenic in this chromosome aberration test in vitro with cells of the V79 Chinese hamster cell line with and without metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1995-05-16 to 1995-05-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

T

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes

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: PPA 4/94
- Expiration date of the lot/batch: September 1995

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: darkness, appr. 20 °C
- Stability under test conditions: stable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: On the day of the experiment the test substance was dissolved in DMSO at appropriate concentrations.

Target gene:

Salmonella typhimurium: Histidine operon
E. Coli: trpE gene, uvrA gene

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Salmonella typhimurium strains were obtained from B. N. Ames, Biochemistry Department, University of California, Berkeley, California 94720, USA, in April 1987. Escherichia coli strain was obtained from M. H. L. Green, MRC. Cell mutation, University of Sussex, Falmer Brighton, BNI 90G (England), in April 1987.

MEDIA USED
- Type and identity of media: Bacteria were grown overnight in nutrient broth (25 g Oxoid Nutrient Broth No. 2 /liter). Inoculation was performed with stock cultures which had been stored at approx. -80 °C.

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from rat liver, Aroclor 1254 induced

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 0, 4, 20, 100, 500, 2500, 5000 µg/plate
Concentration range in the main test (without metabolic activation): 0, 4, 20, 100, 500, 2500, 5000 µg/plate

Vehicle / solvent:

- Vehicle solvent used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: N-Methyl-N-nitro-N-nitrosoguanidine, (without metabolic activation, WP2uvrA); 2-Aminoanthracene (with metabolic activation, all strains)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

SELECTION AGENT (mutation assays): histidine (Salmonella strains) and tryptophan (E. coli)

NUMBER OF REPLICATIONS:2

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Rationale for test conditions:

The test compound did not precipitate on the plates up to the highest investigated dose of 5000 microgram/plate and cytotoxicity was observed at the two highest doses.

Evaluation criteria:

A test article is classified as mutagenic if it has either of the following effects:
a) a test article produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
b) a test article induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test article at complete bacterial background lawn.
The test results must be reproducible.

Statistics:

not applicable

Key result

Species / strain:

other: all strains tested

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in the experiment for cytotoxicity conducted with strain TA100 the number of revertants at the dose groups 2500 and 5000 µg/plate was reduced compared to the lower dose groups

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test compound did not precipitate on the plates up to the highest investigated dose of 5000 microgram/plate.



ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: number of revertants

For details on results, please refer to the attached document.

Conclusions:

The test substance was not mutagenic in bacterial cells with and without metabolic activation in this assay.

Executive summary:

The test substance was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537 and TA 98 of Salmonella typhimurium and Escherichia coli WP2uvrA.

The mutagenicity studies were conducted in the absence and in the presence of a metabolizing system derived from rat liver homogenate. The test substance was dissolved in DMSO and a dose range of 6 different doses from 4 microgram/plate to 5000 microgram/plate was used (0, 4, 20, 100, 500, 2500, 5000 µg/plate). An experiment for cytotoxicity conducted with strain TA100 revealed a decrease in the number of revertants at the two highest doses compared to the lower doses, indicating cytotoxicity. In the main test this was not confirmed.

Control plates without mutagen showed that the number of spontaneous revertant colonies was similiar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies.

In the absence of the metabolic activation system the test compound did not show a dose dependent increase in the number of revertants in any of the bacterial strains. Also in the presence of a metabolic activation system, treatment of the cells with the test compound did not result in relevant increases in the number of revertant colonies.

Summarizing, it can be stated that the test compound is not mutagenic in these bacterial test systems either with or without exogenous metabolic activation at the dose levels investigated.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1995-06-26 to 1995-10-10

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)

Version / remarks:

1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5375 (In Vitro Mammalian Chromosome Aberration)

Version / remarks:

1985

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Guideline for toxicity studies of drugs

Version / remarks:

1990

Deviations:

no

GLP compliance:

yes

Type of assay:

other: in vitro mammalian chromosome aberration assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Hoe CG 0501 0D ZD.. 0001
- Expiration date of the lot/batch: September 1995

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: darkness at approximately 20°C in a fume cupboard
- Stability under test conditions: stable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: On the day of the experiment the test compound was dissolved in DMSO at appropriate concentrations.

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: cell bank of "Genetic Toxicology", Hoechst AG

MEDIA USED
Seeding was carried out with about 1 x 106 cells per flask in 30 ml of MEM-medium supplement with approx. 10 % (v/v) FCS (foetal calf serum) containing approx. 2 mM L-glutamine and approx. 0.1 % (w/v) neomycinsulfate.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from rat liver, Aroclor 1254 induced

Test concentrations with justification for top dose:

Preparation time 20 h:
without metabolic activation: 0, 300, 1500, 2500, 3182 µg/mL
with metabolic activation: 0, 300, 1500, 3182 µg/mL

Preparation time 28 h:
without metabolic activation: 0, 300, 1500, 2500, 3182 µg/mL
with metabolic activation: 0, 300, 1500, 3182 µg/mL

Preparation time 48 h:
without metabolic activation: 0, 300, 1500, 2500, 3182 µg/mL

Higher concentrations were not applied because of the 10 mM limitation (OECD guideline)

Vehicle / solvent:

Vehicle/solvent used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Exposure period (with metabolic activation): 6 hours
Exposure period (without metabolic activation): 20 hours

Fixation time: 20 h

Rationale for test conditions:

A preliminary cytotoxicity test was undertaken in order to set appropriate dose levels for the cytogenetic assay. Cell cultures were subjected to the same treatment conditions as in the main experiment. Cytotoxic effects were determined by photometric measurement of V79 cell cultures bred in microwell plates and stained with crystal violet. The relative cell density in the microwell plates was nearly the same as in the Quadriperm® dishes.
The test included the following treatments:
Solvent control : the final concentration of organic solvents will not exceed approx. 1 % (v/v).
Test compound : the highest dose level will be determined by the solubility of the test compound up to the maximum of 10 mM.
Treatments were performed both in the presence and absence of S9 metabolic activation system; a single cell culture was used at each test point.
The concentrations for the cytogenetic assay were based on the results of the cytotoxicity experiment.
The highest dose level used is 10 mM unless limited by the solubility or the cytotoxicity of the test article.
In the case of toxic effects, the highest dose level should reduce the survival rate to approximately 20 - 50 % and/or the mitotic index to approximately 50 % compared with the corresponding solvent control.
According to the criteria described above, three adequately spaced dose levels extending over at least one decadic logarithm were evaluated at the 20 h fixation interval. At the 28 and 48 h fixation interval one appropriate dose level was selected to evaluate the metaphases.

Evaluation criteria:

The evaluation of the results was performed as follows:
The test compound is classified as mutagenic if it induces a reproducible statistically significant increase in the aberration rate (without gaps) as compared with the solvent controls with one of the concentrations tested. The test compound is classified as mutagenic if there is a reproducible concentration-related increase in the aberration rate (without gaps). The test compound is classified as non-mutagenic if it tests negatively both with and without metabolic activation.

Statistics:

The Biometry of the results was performed with a one-sided Fisher - Exact test.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(> 3182 µg/ml)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH-value of the treatment medium at all concentrations was influenced by the test compound. The treatment medium was re-adjusted to pH 7.3 with 5 M NaOH.
- Effects of osmolality: none
- Precipitation: no visible precipitation was observed

RANGE-FINDING/SCREENING STUDIES:
In the cytotoxicity experiment the test compound was slightly cytotoxic to the V79 cells in the absence of metabolic activation (S9-mix) at the dose level of 3182 µg/mL. In the presence of metabolic activation no indication of cytotoxicity was observed.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: survival (extinction in microwell plates)

For a summary of the results, please refer to the attached document.

Conclusions:

The test substance caused chromosome aberrations in vitro with and without metabolic activation.

Executive summary:

The study was performed to investigate the potential of the test substance to induce chromosome aberrations in V79 cells of the Chinese hamster in vitro in three independent experiments. For each experiment two cell cultures were used.

The test article was dissolved in DMSO and tested at the following concentrations:

 Without S9-mix:

20 h; 300, 1500, 2500+ and 3182* µg/mL

28 h: 300+, 1500+, 2500+ and 3182* µg/mL

48 h: 300+ 1500+, 2500+ and 3182* µg/mL

 With S9-mix:

20 h: 300, 1500 and 3182* µg/mL

28 h: 300+, 1500+ and 3182* µg/mL

* = 10 mM

+ = not evaluated

 

The concentration ranges were based on the results of preliminary testing for solubility and toxicity. The test substance produced a distinct lowering of the mitotic index. Higher concentrations were not applied because of the 10 mM limitation (OECD guideline).

The test compound induced a significant, but not always reproducible increase in the aberration rates with and without metabolic activation.

Appropriate reference mutagens used as positive controls showed a distinct increase in chromosome aberrations, thus indicating the sensitivity of the assay.

In conclusion, the test substance induced chromosome aberrations in V79 Chinese hamster cells in the presence and in the absence of a metabolic activation system, under the experimental conditions described.

The test substance is therefore considered to be clastogenic in this chromosome aberration assay.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Micronucleus assay

The test substance was not mutagenic in this in vivo micronucleus test.

 

UDS assay

The test article did not induce DNA-damage leading to increased repair synthesis in the hepatocytes of the treated rats and is therefore considered to be non-effective in the in vivo/in vitro UDS test system.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1995-10-16 to 1995-12-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

EEC Directive 92/69, L 383 A, Annex B. 12., p. 154 -156

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: PPA 4/94
- Expiration date of the lot/batch: June 1996
- Purity test date: March 6th, 1995

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: darkness at approximately 20°C in a fume cupboard
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: stability in the vehicle is guaranteed for 4 hours

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: On the day of the experiment the test substance was dissolved in DMSO (dried) at an appropriate concentration. A magnetic stirrer was used to keep the preparation homogeneous until dosing had been completed.

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Tierzucht Schonwalde GmbH i.G., Hauptstrafte 62, 16352 Schonwalde, Germany
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: males: mean 40.2 g+/- 2.73 g, females: mean 31.9 g +/- 2.93 g
- Assigned to test groups randomly: yes
- Housing: in fully air-conditioned rooms in makrolon cages type Type 3 (five animals per cage) on soft wood granulate
- Diet: rat/mice diet ssniff® R/M-H (V 1534), ad libitum ssniff® GmbH, Postbox 2039, 59480 Soest, Germany
- Water: tap water in plastic bottles, ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ±3°C
- Humidity (%): 50 ± 20 %
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle/solvent used: DMSO, dried
- Concentration of test material in vehicle: 0 (vehicle control) and 12.5 % (w/v)
- Amount of vehicle: total volume 10 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment the test substance was dissolved in DMSO (dried) at an appropriate concentration. A magnetic stirrer was used to keep the preparation homogeneous until dosing had been completed. DMSO (dried) was administered in the same way to the negative control groups. The study included a simultaneous positive control using Endoxan®, which was administered once orally at a dose of 50 mg per kg body weight.

Duration of treatment / exposure:

In conformity with the test procedure the animals were killed by carbon dioxide asphyxiation 12, 24 or 48 hours after administration.

Frequency of treatment:

single administration

Post exposure period:

12, 24 or 48 hours

Dose / conc.:

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

Remarks:

vehicle control, killing time 24 h p.a.

Dose / conc.:

1 250 mg/kg bw/day (actual dose received)

Remarks:

test group, killing time 24 h p.a.

Dose / conc.:

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

Remarks:

vehicle control, killing time 48 h

Dose / conc.:

1 250 mg/kg bw/day (actual dose received)

Remarks:

test group, killing time 48 h

Dose / conc.:

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

Remarks:

vehicle control, killing time 12 h

Dose / conc.:

1 250 mg/kg bw/day (actual dose received)

Remarks:

test group, killing time 12 h

No. of animals per sex per dose:

5 male anf 5 female animals per group per killing time

Control animals:

yes, concurrent vehicle

Positive control(s):

Endoxan containing cyclophosphamide
- Route of administration: oral by gavage
- Doses / concentrations: 50 mg/kg bw, killing time 24 h p.a.

Tissues and cell types examined:

bone marrow erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Oral administration of 1500 mg per kg body weight resulted in mortality in male mice. The highest sub lethal dose of 1250 mg per kg body weight was selected for the main study.

TREATMENT AND SAMPLING TIMES
In conformity with the test procedure the animals were killed by carbon dioxide asphyxiation 12, 24 or 48 hours after administration.

DETAILS OF SLIDE PREPARATION:
For each animal, about 3 mL foetal bovine serum was poured into a centrifuge tube. Both femora were removed and the bones freed of muscle tissue. The proximal ends of the femora were opened and the bone marrow flushed into the centrifuge tube. A suspension was formed. The mixture was then centrifuged for 5 minutes at approx. 1200 rpm, after which almost all the supernatant was discarded. One drop of the thoroughly mixed sediment was smeared onto a cleaned slide, identified by project code and animal number and air-dried for about 12 hours.
Staining was performed as follows:
- 5 minutes in methanol
- 5 minutes in May-Grunwald's solution
- brief rinsing twice in distilled water
- 10 minutes staining in 1 part Giemsa solution to 6 parts buffer solution, pH 7.2 (Weise)
- rinsing in distilled water
- drying
- coating with Entellan

METHOD OF ANALYSIS:
1000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded, not the number of individual micronuclei. As a control measure 1000 mature erythrocytes were also counted and examined for micronuclei. In addition, the ratio of polychromatic to normochromatic erythrocytes was determined. All bone marrow smears for evaluation were coded to ensure that the group from which they were taken remained unknown to the investigator. The number of polychromatic erythrocytes with micronuclei occurring in the 1000 polychromatic erythrocytes, and the number of normocytes with micronuclei occurring in the 1000 normocytes, were evaluated statistically.

Evaluation criteria:

Both biological and statistical significances were considered together for evaluation purposes.
A substance is considered positive if there is a significant increase in the number of micronucleated polychromatic erythrocytes for at least one of the time points compared with the concurrent negative control group. A test substance producing no significant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.
For the assay to be valid, individual and/or group mean values for the positiv control should exeed the laboratory's historical control range.

Statistics:

A Wilcoxon-Test (one-sided) was evaluated to check the validity of the study. This is done by comparing the number of polychromatic erythrocytes with micronuclei in the positive control with the negative control.
A Wiicoxon-Test (one-sided) was performed for each treatment interval (12h, 24h, 48h) and for polychromatic and normochromatic erythrocytes. These tests are performed sequentially with a multiple level of significance of 5%.
The following comparisons are performed only if there is a difference between the positive control and the negative control (24 h). This was performed with a Wilcoxon-Test (two-sided) with a 5 %-level of significance. Wilcoxon-Tests (two-sided) are performed sequentially for the ratio of polychromatic erythrocytes for each treatment interval (12h, 24h, 48h) at a multiple level of significance of 5 %. The data obtained were also compared with historical controls.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Doses producing toxicity: 1250 mg/kg, 48 hours after application all animals were free of clinical signs of toxicity.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: Oral administration of 2000 and 1500 mg per kg body weight resulted in mortality in 1 out of 3 males and 1 out of 3 females (2000 mg/kg bw) and 2 out of 3 males (1500 mg/kg bw), respectively. No mortality occured in the 1000 mg/kg bw dose group.
- Clinical signs of toxicity in test animals:
2000 mg/kg bw dose group:
stilted gait, squatting posture, palpebreal fissure narrow, coat bristling, spontaneous activity decreased, cyanosis
1250 mg/kg bw doese group:
spontaneous activity decreased, palpebreal fissure narrow, palpebreal fissure very narrow, squatting posture, stilted gait and coat bristling
1500 mg/kg bw dose group:
stilted gait, squatting posture, palpebreal fissure narrow, palpebreal fissure closed, coat bristling, spontaneous activity decreased, eyelids adhering and sonoures rales
1000 mg/kg bw dose group:
no clinical signs were observed

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: The incidence of micronucleated polychromatic and normochromatic erythrocytes in the test group was within the normal range of the negative control groups.
- Ratio of PCE/NCE: The ratio of polychromatic erythrocytes to normocytes remained essentially unaffected by the test compound.

For details on results, please refer to the attached document.

Conclusions:

The test substance did not lead to a substantial increase of micronucleated polychromatic erythrocytes and is not mutagenic in the micronucleus test.

Executive summary:

 

The micronucleus test was carried out with the test substance to assess its potential to cause

chromosomal damage (clastogenicity). The test compound was dissolved in DMSO (dried) and was given once as an orally dose of 1250 mg per kg body weight to male and female mice, based on the results of a previous dose range finding assay (see preliminary study). According to the test procedure the animals were killed 12, 24 or 48 hours after administration.

Endoxan® was used as positive control substance and was administered once orally at a dose of 50 mg per kg body weight.

The number of polychromatic and normochromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment with the test substanceand was statistically not different from the control values.

Endoxan®induced in both males and females a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the test system. The ratio of polychromatic erythrocytes to normocytes was not changed to a significant extent.

Under the conditions of the present study the results indicate that the test substanceis not mutagenic in the micronucleus test.

Reference 2

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1996-01-15 to 1996-03-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1991 (Draft)

Deviations:

no

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: PPA 4/94 of September 1994
- Expiration date of the lot/batch: June 1996

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, air- and moisture-protection
- Stability under test conditions: stable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: On the day of the experiment (immediately before treatment), the test article was formulated in dimethylsulfoxide (DMSO, free from water).

Species:

other: rat, Wistar, HanJbm: WIST (SPF)

Route of administration:

oral: unspecified

Vehicle:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The vehicle was chosen according to its relative non-toxicity for the animals.
- Amount of vehicle: total dose volume 5 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test article was formulated in dimethylsulfoxide (free from water).

Duration of treatment / exposure:

2 hous and 16 hours

Frequency of treatment:

one treatment

Post exposure period:

animals were sacrificed after treatment

Dose / conc.:

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

Remarks:

2 hour preparation interval

Dose / conc.:

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

Remarks:

16 hour preparation interval

Dose / conc.:

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

Remarks:

16 hour preparation interval

No. of animals per sex per dose:

Male: 2000 mg/kg; No. of animals: 5; Sacrifice time: 2 hours
Male: 2000 mg/kg; No. of animals: 5; Sacrifice time: 16 hours
Male: 200 mg/kg; No. of animals: 5; Sacrifice time: 16 hours

Control animals:

yes, concurrent vehicle

Positive control(s):

2-acetylaminofluorene
- Route of administration: orally
- Doses / concentrations: 100 mg/kg bw

Tissues and cell types examined:

primary hepatocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The highest dose recommended by the OECD-draft guideline to be used in this test system (2000 mg/kg b.w.) was determined by the pre-experiment to be suitable. The animals expressed slight toxic reactions. The low dose was one tenth of the high dose.

TREATMENT AND SAMPLING TIMES:
After anaesthetizing the rats with Na-pentobarbital (Narcoren) the liver was perfused through the vena portae with Hanks' balanced salt solution (HBSS, Gibco/BRL, D-76344 Eggenstein) supplemented with collagenase (0.05 % w/v, Boehringer Mannheim, D-68305 Mannheim) adjusted to pH 7.4 and maintained at 37° C (8). The isolated hepatocytes were washed twice with HBSS. The crude cell suspension was filtered through a stainless steel mesh to yield a single cell suspension. The washed hepatocytes were centrifuged and transferred into William's medium E (WME, Gibco/BRL, D-76344 Eggenstein) supplemented with Hepes, Penicillin, Streptomycin, Glutamin, Insulin and Fetal Calf Serum. This complete medium was adjusted to pH 7.6.

DETAILS OF SLIDE PREPARATION:
At least three cultures were established from each animal. Aliquotes of 2.5 mL with hepatocytes in complete culture medium (2.0 x 10^5 viable cells/mL) were added to 35 mm six-well dishes (Greiner, 72603 Niirtingen, Germany) containing one 25 mm round plastic coverslip (Thermanox, Nunc, 65203 Wiesbaden, Germany) per well coated with gelatine.
After an attachment period of approximately 1.5 h in a 95% air/ 5% C02 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, 63033 Dreieich, Germany) 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 detection. The cells on the coverslips were then fixed by three changes of methanohacetic acid (3+1 v/v) for 20 minutes each, rinsed with 96 % ethanol, and air-dried.

METHOD OF ANALYSIS:
Autoradiographic Processing
The cover slips were mounted the side carrying the cells up on glass slides and coated with KODAK NTB2 (Tecnomara, 35463 Fernwald, Germany) photographic emulsion in the dark. The coated slides were stored in light-proof boxes in the presence of a drying agent for 12 -14 days at 4° C. The photographic emulsion was then developed with KODAK Dektol Developer (Tecnomara, 35463 Fernwald, Germany) at room temperature, fixed in TETENAL (Tetenal, 22844 Norderstedt, Germany) and stained with hematoxylin/eosin.

Quantification of UDS
Evaluation was performed microscopically on coded slides using NIKON microscopes with oil immersion objectives. The cells for scoring were randomly selected according to a fixed scheme. The number of silver grains in the nuclear area was counted automatically using the ARTEK 880 or 982 counter. In addition, the number of grains of one nuclear-sized cytoplasm area adjacent to the nucleus was counted. At least two slides per animal and 50 cells per slide were evaluated. Heavily labelled S-phase cells were excluded from counting.

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 information to confirm a positive response with less than 5 net grains.
Statistical significance may give further evidence for a positive evaluation. Statistical significance can be evaluated by means of the non-parametric Mann-Whitney test.
A test article producing net grains not greater than 0 at anyone of the test points is considered non-effective in this system.

Statistics:

A statistical evaluation of the results was not necessary to perform, since the number of nuclear grain counts of the groups treated with the test article were in the range of the controls and net grain values obtained were consistently negative.

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

no induction of DNA repair

Toxicity:

yes

Remarks:

Doses producing toxicity: 2000 mg/kg bw

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 200 mg/kg bw
- Solubility: the test article was solved in the vehicle, no precipitation reported
- Clinical signs of toxicity in test animals: reduction of sponataneous acitivity, apathy

RESULTS OF DEFINITIVE STUDY
The viability of the hepatocytes was not affected by the in vivo treatment with the test article at any of the treatment periods or dose groups.
No dose level of the test article revealed UDS induction in the hepatocytes of the treated animals as compared to the current vehicle controls. Neither the nuclear grains nor the resulting net grains were distinctly enhanced due to the in vivo treatment of the animals with the test article for 2 hours or 16 hours, respectively. Therefore, the net grain values obtained after treatment with the test article were consistently negative. in addition, no substantial shift to higher values was obtained in the percentage distribution of the nuclear grain counts.

For details on results, please refer to the attached document.

Conclusions:

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

Executive summary:

The test article was assessed in the in vivo/in vitro UDS assay for its potential to induce DNA repair (UDS) in the hepatocytes of rats.

The test article was formulated in dimethylsulfoxide (free from water). This suspending agent was used as vehicle control. The volume administered orally was 5 mL/kg body weight (b.w ). After a treatment period of 2 and 16 hours, respectively, the animals were anaesthetized and sacrificed by enzymatic 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:

2hour preparation interval: 2000 mg/kg bw

16hour preparation interval: 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,

The highest dose (maximum OECD-draft guideline-recommended dose) was determined by a pre-experiment to be suitable. The animals expressed slight toxic reactions.

The viability of the hepatocytes was not affected by the in vivo 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.

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 increased repair synthesis in the hepatocytes of the treated rats. Therefore it is considered to be non-effective in this in vivo/in vitro UDS test system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

The test material was not mutagenic in bacterial cells but clastogenic (caused chromosome aberrations) in one of two in vitro tests in mammalian cells. In the second chromosome aberrations study the result was clearly negative (not clastogenic).

The in vivo/in vitro UDS assay proved the test substance also not being mutagenic to mammalian cells in vivo. The in vivo micronucleus assay was also negative, overruling the ambiguous results from the in vitro chromosome aberration studies.

As a conclusion, the test substance was considered not to be mutagenic or clastogenic in vivo.

 

The available experimental test data is reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified for acute oral toxicity under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.