REAKTIVE GELB F 97494

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance is not genotoxic in in-vitro assays for mutagenicity or for  cytogenicity at non-toxic concentrations.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

QSAR Toolbox estimation

Type of information:

(Q)SAR

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE
QSAR Toolbox

2. MODEL (incl. version number)
QSAR Toolbox 4.5
Database version: 4.5

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CAS#: 477795-15-6

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
- Defined endpoint: gene mutation
see attachment

5. APPLICABILITY DOMAIN
The substance lies well within the applicability domains


6. ADEQUACY OF THE RESULT
see attachment

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

HPRT

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Prediction summary
Predicted endpoint: in vitro gene mutation study in mammalian cells; No effect specified; No species
specified; No duration specified; No guideline specified
Predicted value: Negative
Unit/scale: Gene mutation I
Data gap filling method: Read-across analysis


Prediction details
redicted value: Negative
Predicted endpoint (OECD Principle 1 - Defined endpoint): Human Health Hazards -> Genetic Toxicity -> in vitro gene mutation study in mammalian cells
Calculation approach (OECD principle 2 - Unambiguous algorithm): takes the highest mode value from the nearest 5 neighbours
Active descriptor: log Kow (calculated): 4.54
Data usage: All values (when multiple values are available for the same chemical, all of them are taken individually in prediction calculations)
Uncertainty of the prediction (OECD principle 4 - Uncertainty of the prediction):
The prediction is based on 7 values, 7 of them (100%) equal to predicted value
Prediction confidence is measured by the p-value: 0.000457

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

Conclusions:

Based on the QSAR prediction results, the test substance has no mutagenic properties

Executive summary:

Predicted endpoint: in vitro gene mutation study in mammalian cells; No effect specified; Chinese hamster lung fibroblasts (V79); No duration specified; No guideline specified
Predicted value: negative
Unit/scale: IUCLID6 Picklist T143 - v2.0
Data gap filling method: Read-across analysis
Summary: manually editable field
The test substance in not mutagen in mammalian cells based on the results of structurally similar substances

Predicted value: negative
Predicted endpoint (OECD Principle 1 - Defined endpoint): Human Health Hazards -> Genetic Toxicity -> in Vitro -> Mammalian Cell Gene Mutation Assay -> in vitro gene mutation study in mammalian cells -> Chinese hamster lung fibroblasts (V79)
Calculation approach (OECD principle 2 - Unambiguous algorithm): takes the mode value from the 5 nearest neighbours
Active descriptor: log Kow (calculated)
Data usage: All values

Uncertainty of the prediction (OECD principle 4 - Uncertainty of the prediction):
The prediction is based on 5 values, 5 of them (100%) equal to predicted value
Prediction confidence is measured by the p-value: 5,95E-05
Mechanistic interpretation: manually editable field
The assessment is made based on structural similarity of substances that react via the same priciple during the dyeing process. This means all substanes also show the same physico-chemical properties and reactivity profiles.
Adequacy of the prediction: manually editable field
The prediction is considered to be valid based on the close similarity in functionality of the substances

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 22, 2001 to November 29, 2001

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

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from rat liver

Test concentrations with justification for top dose:

First experiment with 3 h treatment time:

without S9-mix: 500*, 1000, 1500*, 2500, 3000*, 3500* and 5000§ µg/mL
with S9-mix: 500*, 1000, 1500*, 2500, 3000*, 3500* and 5000§ µg/mL

§ = not evaluated because of high toxicity
* = evaluated concentrations

Second experiment with 20 h treatment time:$
without S9-mix: 125, 250, 500, 1000, 1500 and 2000 µg/mL

$=because of mutagenicity in the first experiment analysis of the second experiment was not conducted

Vehicle / solvent:

cell culture medium

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

(without metabolic activation)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

(with metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION: The cells were treated for 3 h in both the presence and absence of S9-mix. Then, the cells were sampled 20 h after the start of treatment as were the concurrent solvent and positive control cultures. Colcemide was added to each culture 2 h before sampling in order to arrest cell division. Chromosome preparations were made, fixed, stained and examined.

Before treatment, the pH values and osmolality of the treatment medium were determined. If necessary the pH was adjusted to pH 7.3 with NaOH or HCl. Exponentially growing cultures which were more than 50% confluent were trypsinated by an approximately 0.25% (v/v) trypsin solution ready for use. A single cell suspension (culture) was prepared. The trypsin concentration was approxiamtely 0.25% (v/v) in Ca-Mg-free salt solution. Two slides were placed in Quadriperm® dishes which were then seeded with cells to yield 3-4x104 cells/slide. Thus for each dose level and treatment time, duplicate cultures slides were used. The Quadriperm® dishes contained 6 mL MEM with approximately 10% (v/v) FCS. After 48 h, the medium was replaced with one containing approximately 10 % (v/v) FCS and the test substance, or positive control, or solvent and in the presence of metabolic activation additionally 2% (v/v) S9-mix. For the 3 h treatment time, the medium was replaced by normal medium following two rinses. In the second experiment the cells were exposed to the treatment medium without S9-mix for 20 h. 18 h after the start of the treatment, Colcemide was added (approximately 0.05 µg/mL/culture medium) to the cultures to arrest mitosis and 2 h later (20 h after the start of treatment) metaphase spreads were prepared.

Evaluation criteria:

Analysis of metaphases
The slides were coded and 25-100 metaphases per experimental group and cell culture were examined. The set of chromosomes was examined for completeness and the various chromosomal aberrations were assessed and classified. Only metaphases with 22+/-2 chromosomes are included in the analysis. The metaphases were examined for the following aberrations: chromatid gap, chromosome gap, chromatid break, chromosome break, minute, double minute, chromatid deletion, chromosome deletion, chromatid exchanges including intrachanges, chromosome exchanges including intrachanges, dicentrics, pulverization and ring formation. Further the incidence of polyploid metaphases was determined in 1000 cells of each cell culture.
Additionally the mitotic index was determined by counting the number of cells undergoing mitosis in a total of 1000 cells. The mitotic index is expressed as a percentage. After the metaphases had been evaluated, the code was broken. For each experiment the results from the dose groups were compared with thoseof the control group and the positive control at each sampling time.

Criteria for a valid assay
The assay is considered valid if the solvent control data are within the laboratory's normal control range for the spontaneous mutant frequency and positive control induce increases in the mutation frequency which are statistically significant and within the laboratory's normal range
Criteria for clastogenicity
The test substance is classified as clastogenic if it induces a statistically significant increase in the number of phases with aberrations (without gaps) with one or more of the concentrations tested as compared with the solvent controls.
The test substance is classified as clastogenic if there is a concentration-related increase in the number of phases with aberrations (without gaps). The test substance is classified as nonclastogenic if the tests are negative both with and without metabolic activation.

Statistics:

The Biometry of the results was performed with a one-sided Fisher's exact test.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

clastogenicity was observed only at cytotoxic concentration

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 3000 µg/mL

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the main experiment cytotoxicity was also evaluated by treatment of cells seeded in microwell plates. Survival was reduced in a dose-related manner reaching 21.8% of the solvent control value with and without S9-mix at the 3 h treatment time at the highest concentration tested, 5000 µg/mL. At the 20 h treatment time survival was reduced in a dose-related manner reaching 1.2% of the solvent control value at the highest concentration tested, 2000 µg/mL. After 3 h treatment the mitotic index was reduced (indication of toxicity) significantly after treatment with the highest dose levels with and without S9-mix. After treatment with the test substance there was no relevant increase in the number of polyploidy cells as compared with the solvent controls. The test substance was assessed for its clastogenic potential to induce chromosome aberrations in vitro after short-term treatment (3 h). An enhancement of the aberration rates was observed with and without S9-mix at concentrations of 3000 and 3500 µg/mL inclusive and exclusive gaps. These data were found significantly enhanced in the Fisher's exact-test. This is an indication of heavy chromosomal damage. An enhancement was first observed at a toxic concentration of 3500 µg/mL and therefore an additionally lower toxic concentration of 3000 µg/ml was evaluated. The mutagenic effect was confirmed. Both doses (3000 and 3500 µg/mL) caused a distinct reduction in cell survival and moreover a moderate to distinct decrease in the mitotic index. In summary the test substance induded structural chromosome aberrations at cytotoxic concentrations. On the basis of these findings the evaluation of slides of the longer treatment time (20 h) is not necessary. The sensitivity of the test system was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control compounds.

SOLUBILITY AND PRELIMINARY TOXICITY TESTING: Test substance was dissolved in cell culture medium. Evaluation of the solubility of that solution in MEM Hank's cell culture medium showed that 5000 µg/mL was the highest practicable concentration and produced no precipitate. Accordingly, the preliminary toxicity study was carried out using a maximum concentration of 5000 µg/mL and a range of lower dose levels down to 10 µg/L. These results show that test substance was toxic to the V79 cells in the absence and in the presence of metabolic activation (S9-mix) after 3 and 20 h treatment time. Following treatment for 3 h in the absence and in the presence of S9 metabolic activation, high toxicity was observed at 5000 µg/mL. Survival declined in a dose-related manner reaching 38% of the solvent control value without S9-mix and 35.5% of the solvent control value with S9-mix at the highest dose level, 5000 µg/mL. After 20 h treatment survival was reduced to 64.6% of the solvent control value at a concentration of 1000 µg/mL, while at higher dose levels survival was reduced below the limit of detection. Before treatment, the pH values and osmolality of the treatment media were determined. The addition of test substance solutions did not have a significant effect on these parameters.

Conclusions:

Under the study conditions, the test substance was not clastogenic in the absence and presence of metabolic activation at non-cytotoxic concentrations. However, the substance did induce structural chromosome aberrations in V79 Chinese hamster cells at cytotoxic concentrations in the presence and in the absence of a metabolic activation system.

Executive summary:

A study was conducted to investigate the potential of the test substance to induce chromosome aberrations in V79 cells of the Chinese hamster lung according to OECD Guideline 473, EPA OPPTS 870.5375 and EU Method B.10, in compliance with GLP.

 

The substance was dissolved in cell culture medium and tested at the concentrations ranging from 500-5,000 µg/mL with and without metabolic activation, based on the results of preliminary testing for solubility and toxicity.

Positive controls showed a significant increase in chromosome aberrations, thus indicating the sensitivity of the assay and the efficacy of the S9-mix. The test substance induced a significant and dose-dependent increase in the number of aberrant metaphases and in the number of aberrations inclusive and exclusive gaps with and without metabolic activation at 3 h treatment time at doses of 3,000 and 3,500 µg/mL. However, doses of 3,000 and 3,500 µg/mL caused distinct toxicity in the cell survival test and a moderate to distinct reduction of the mitotic index. Further, the highest concentration of 5,000 µg/mL was not evaluated because of high toxicity.

 

Under the study conditions, the test substance was not clastogenic in the absence and presence of metabolic activation at non-cytotoxic concentrations. However, the substance did induce structural chromosome aberrations in V79 Chinese hamster cells at cytotoxic concentrations in the presence and in the absence of a metabolic activation system.

Reference 3

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:

From October 31, 2001 to January 25, 2002

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines for toxicity studies of drugs, 1990 MITI Guidelines for toxicity testing of chemicals

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from rat liver (plate incorporation test) and S9-mix from hamster liver (preincubation test:)

Test concentrations with justification for top dose:

plate incorporation test:
with metabolic activation (10% rat liver):
50, 160, 500, 1600 and 5000 µg/plate
without metabolic activation:
50, 160, 500, 1600 and 5000 µg/plate

Preincubation test:
with metabolic activation (30% hamster liver):
50, 160, 500, 1600 and 5000 µg/plate
without metabolic activation:
50, 160, 500, 1600 and 5000 µg/plate

Vehicle / solvent:

Deionized water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without metabolic activation for strain TA 100 and TA 1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without metabolic activation for strain TA 1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without metabolic activation for strain T A 98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without metabolic activation for strain WP2uvrA

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation (10% rat liver) for all strains

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation (30% syrian golden hamster liver) for strain TA 100, TA 1535, TA 1537 and WP2uvrA

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

congo red

Remarks:

with metabolic activation (30% syrian golden hamster liver) for strain TA 98

Details on test system and experimental conditions:

ASSAY PROCEDURE:

Each test was performed in both the presence and absence of S9-mix using all bacterial tester strains and a range of concentrations of the test substance. Positive and negative controls as well as solvent controls were included in each test. Triplicate plates were used. The highest concentration in the first mutation experiment was 50 mg/mL of the test substance in the chosen solvent, which provided a final concentration of 5000 µg/plate. Further dilutions of 1600, 500, 160 and 50 µg/plate were also used. Dose levels used in the second experiment were based on findings, including toxicity, in the first experiment. Toxicity was assessed after microscopic thinning of the bacterial lawn and/or reduction of the number of spontaneously occurring mutants compared to the corresponding solvent control value.

In both tests top agar was prepared which, for the Salmonella strains, contained 100 mL agar (0.6% (w/v) agar, 0.5% (w/v) NaCI) with 10 mL of a 0.5 mM histidine-biotin solution. For E. coli histidine was replaced by tryptophan (2.5 mL, 2 mM).
The following ingredients were added (in the following order) to 2 mL of molten top agar at approx. 48°C:
0.5 mL S9-mix (if required) or buffer
0.1 mL of an overnight nutrient broth culture of the bacterial tester strain
0.1 mL test compound solution (dissolved in deionized water)

In the second mutagenicity test if appropriate these top-agar ingredients were preincubated by shaking for approximately 20 to 30 minutes at approximately 30°C. After mixing, and preincubation if appropriate, the liquid was poured into a petri dish containing a 25 mL layer of minimal agar (1.5% (w/v) agar, Vogel-BonnerE medium with 2% (w/v) glucose). After incubation for approximately 48 h at approximately 37°C in the dark, colonies (his+ or trp+ revertants) were counted by hand or by a suitable automatic colony counter. The counter was calibrated for each test by reading a test pattern plate to verify the manufacturer'srequirements for sensitivity.

Evaluation criteria:

Criteria for a valid assay
The assay is considered valid if the following criteria are met:
- the solvent control data are within the laboratory's normal control range for the spontaneous mutant frequency
- the positive controls induce increases in the mutation frequency which are significant and within the laboratory's normal range

Criteria for a positive response
A test substance is classified as mutagenic if it has either of the following effects:
a) it 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) it 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 compound at complete bacterial background lawn

If the test substance does not achieve either of the above criteria, it is considered to show no evidence of mutagenic activity in this system.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(in plate incorporation test and peincubation test)

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(in plate incorporation test and peincubation test)

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

SOLUBILITY AND TOXICITY: Test substance was dissolved in deionized water and a stock solution of 50 mg/mL was prepared for the highest concentration, which provided a final concentration of 5000 µg/plate. This stock solution was filtrated through filter pore size 0.2 11M. Further dilutions of 1600, 500, 160 and 50 µg/plate were used in both experiments. Test substance did not precipitate on the plates up to the highest investigated dose of 5000 µg/plate. In the plate incorporation test toxicity was not observed with and without metabolic activation in the plate incorporation test and in the preincubation test.

MUTAGENICITY: In both independent mutation tests test substance was tested for mutagenicity with the same concentrations as described above. The number of colonies per plate with each strain as well as mean values of 3 plates were given.
Plate incorporation test: In the absence of the metabolic activation system the test substance did not result in relevant increases in the number of revertants in any of the bacterial strains. Also in the presence of rat liver activation system (10% (v/v)), treatment of the cells with test substance did not result in relevant increases in the number of revertant colonies.
Preincubation test: In the absence and in the presence of hamster liver S9-mix (30% (v/v)) using the preincubation method according to Prival test substance did not result in relevant increases in the number of revertant colonies with any of the tester strains.

All positive controls produced significant increases in the number of revertant colonies. Thus the sensitivity of the assay and the efficacy of the exogenous metabolic activation system were demonstrated

STERILITY CHECKS AND CONTROL PLATES:

Sterility of S9-mix and the test substance were indicated by the absence of contamination on the test substance and S9-mix sterility check plates. Control plates (background control and positive controls) gave the expected number of colonies, i.e. values were within the laboratory's historical control range. Only the number of revertant colonies of the negative controls with the strain WP2uvrA (plate incorporation test and Prival test) in the presence of S9-mix and the solvent controls (Prival test with and without S9-mix) were slightly below of the historical control data range. The positive controls with WP2uvr A were slightly above the historical control data range in the prival test. Additionally, with the tester strains TA 100 and TA 1537 both with S9 in the Prival test the number of revertant colonies of the solvent controls were slightly out of the historical control data range but the criteria for the positive response were fulfilled.

Conclusions:

Under the study conditions, the test substance was found to be non-mutagenic in the bacterial reverse mutation assay.

Executive summary:

An in vitro study was performed to investigate the potential of the test substance to induce gene mutations according to OECD Guideline 471, EPA OPPTS 870.5100, EU Method B.14 and Japan: Guidelines for Screening Mutagenicity Testing of Chemicals, in compliance with GLP.

Two independent mutagenicity studies were conducted, one as the standard plate test with the plate incorporation method and the other as a modified preincubation test (Prival test). The studies were performed in the absence and in the presence of a metabolizing system derived from a rat liver homogenate or a hamster liver homogenate. The test substance was tested for mutagenic effects without and with metabolic activation at five concentrations in the range of 50 to 5,000 µg/plate in both assays.

In the plate incorporation test, the test substance did not result in relevant increases in the number of revertants in any of the bacterial strains in the absence and presence of the metabolic activation (rat liver S9-mix (10% (v/v)). Also, in preincubation test no relevant increases in the number of revertants was observed in any of the bacterial strains in the absence and presence of the metabolic activation (hamster liver S9-mix (30% (v/v)).

Under the study conditions, the test substance was found to be non-mutagenic in the bacterial reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The substance did not induce micronuclei in bone marrow cells of the rat. Therefore, the test substance was not considered to be clastogenic in this micronucleus assay.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 14 January, 2002 to 25 January, 2002

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

rat

Strain:

other: Hsd:Sprague Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Gartenstrasse 27, 33178 Borchcn
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: male animals-mean = 187.8 g (= 100 %) (min = 179 g (-4.7 %), max = 198 g (+5.4 %)); female animals- mean = 149.8 g (=100 %) (min = 143 g (-4.5%), max = 159 g (+6.1%))
- Assigned to test groups randomly: [yes, under following basis: randomization schemes 2002.00 16 and 2002.0017]
- Housing: five animals per cage in transparent macrolon cages (type IV) on soft wood granulate in an air conditioned room.
- Diet (e.g. ad libitum): rat/mice diet ssniff R/M-H (V 1534), ad libitum, ssniff® GmbH, Postbox 2039, 59480 Soest
- Water (e.g. ad libitum): tap water in plastic bottles, ad libitum
- Acclimation period: 5 d under study conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C (except short lasting deviations due to disturbances of air condition)
- Humidity (%): 50% (except short lasting deviations due to disturbances of air condition)
- Photoperiod (hrs dark / hrs light): 12 h light/dark cycle

IN-LIFE DATES: From 14 January, 2002 to 16 January, 2002

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: deionized water
- Concentration of test material in vehicle: 200 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: the test substance was dissolved in deionized water at an appropriate concentration. A magnetic stirrer was used to keep the preparation homogeneous until dosing had been completed.

Duration of treatment / exposure:

2 d

Frequency of treatment:

twice at an interval of 24 h

Remarks:

Doses / Concentrations:
2000 mg/kg bw /d
Basis:
actual ingested

No. of animals per sex per dose:

5/sex/group

Control animals:

yes, concurrent vehicle

Positive control(s):

Positive control: Cyclophosphamide
Dissolved in: distilled water
Dose: 40 mg/kg bw
Route and frequency of administration: Oral (gavage), once
Volume Administered: 10 mL/kg bw

Tissues and cell types examined:

-2000 polychromatic erythrocytes were counted for each animal.
-The number of cells with micronuclei was recorded, not the number of individual micronuclei.
- The ratio of polychromatic erythrocytes to 200 normochromatic erythrocytes was determined.
-Main parameter for the statistical analysis, i.e. validity assessment of the study and mutagenicity of the test substance, was the proportion of polychromatic erythrocytes with micronuclei out of the 2000 counted erythrocytes.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Based on the results of the acute oral toxicity study the dose of 2000 mg/kg bw was selected as the limit dose, for the main study.

-Extraction of the bone marrow: Animals were killed by carbon dioxide asphyxiation 24 h after dosing. One femora was removed and the bone freed of muscle tissue. The proximal end of the femora was opened, the bone marrow flushed into a centrifuge tube containing about 3 mL of fetal bovine serum and a suspension was prepared. The mixture was then centrifuged for 5 minutes at approximately 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 h.

-Staining procedure: The slides were stained 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

Evaluation criteria:

Both biological and statistical significances were considered together for evaluation purposes. A test substance is considered as positive if there is a significant dose- related increase in the number of micronucleated polychromatic erythrocytes compared with the concurrent negative control group. A test substance producing no significant dose-related increase in the number of micronucleated polychromatic erythrocytes is considered non-clastogenic in this system.

Statistics:

Assuming the study is valid based on a monotone-dose-relationship, one-sided Wilcoxon tests were performed initially comparing control values with those of the highest dose group. A significance level of 5% is adopted for all tests.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

-All animals survived after treatment. No signs of toxicity were observed in the main study.
-The dissection of the animals revealed an ocher colored content of the gastro-intestinal tract.

Conclusions:

Under the study conditions, the test substance did not induce micronuclei in bone marrow cells of the rat. Therefore, the test substance was not considered to be clastogenic in this micronucleus assay.

Executive summary:

A study was conducted to investigate the potential of the test substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the rat according to OECD Guideline 474, EPA OPPTS 870.5395 and EU Method B.12 in compliance with GLP.

 

The substance was dissolved in deionised water and was given twice at an interval of 24 h as a oral dose of 2000 mg/kg bw/d to male and female rats. The dose was selected based on the results of a previous rat acute oral toxicity study. The animals were killed 24 h after the last administration and bone marrow cells were collected for micronuclei analysis. After treatment with the test substance, the number of polychromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic erythrocytes to total erythrocytes in both male and female animals remained unaffected and differed less than 20% from the control value.

 

Under the study conditions, the test substance did not induce micronuclei in bone marrow cells of the rat. Therefore, the test substance was not considered to be clastogenic in this micronucleus assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

An in vitro study was performed to investigate the potential of the test substance to induce gene mutations according to OECD Guideline 471, EPA OPPTS 870.5100, EU Method B.14 and Japan: Guidelines for Screening Mutagenicity Testing of Chemicals, in compliance with GLP. Two independent mutagenicity studies were conducted, one as the standard plate test with the plate incorporation method and the other as a modified preincubation test (Prival test). The studies were performed in the absence and in the presence of a metabolizing system derived from a rat liver homogenate or a hamster liver homogenate. The test substance was tested for mutagenic effects without and with metabolic activation at five concentrations in the range of 50 to 5,000 µg/plate in both assays. In the plate incorporation test, the test substance did not result in relevant increases in the number of revertants in any of the bacterial strains in the absence and presence of the metabolic activation (rat liver S9-mix (10% (v/v)). Also, in preincubation test no relevant increases in the number of revertants was observed in any of the bacterial strains in the absence and presence of the metabolic activation (hamster liver S9-mix (30% (v/v)). Under the study conditions, the test substance was found to be non-mutagenic in the bacterial reverse mutation assay.

The substance was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster in an QSAR assessment from five structurally similar substances. Based on the results of the structurally similar substances, the substance does not induce mutagenic effects in mammalian cells with or without an external metabolising system.

A study was conducted to investigate the potential of the test substance to induce chromosome aberrations in V79 cells of the Chinese hamster lung according to OECD Guideline 473, EPA OPPTS 870.5375 and EU Method B.10, in compliance with GLP. The substance was dissolved in cell culture medium and tested at the concentrations ranging from 500-5,000 µg/mL with and without metabolic activation, based on the results of preliminary testing for solubility and toxicity. Positive controls showed a significant increase in chromosome aberrations, thus indicating the sensitivity of the assay and the efficacy of the S9-mix.The test substance induced a significant and dose-dependent increase in the number of aberrant metaphases and in the number of aberrations inclusive and exclusive gaps with and without metabolic activation at 3 h treatment time at doses of 3,000 and 3,500 µg/mL. However, doses of 3,000 and 3,500 µg/mL caused distinct toxicity in the cell survival test and a moderate to distinct reduction of the mitotic index. Further, the highest concentration of 5,000 µg/mL was not evaluated because of high toxicity. Under the study conditions, the test substance was not clastogenic in the absence and presence of metabolic activation at non-cytotoxic concentrations. However, the substance did induce structural chromosome aberrations in V79 Chinese hamster cells at cytotoxic concentrations in the presence and in the absence of a metabolic activation system. Based on these results, the substance was not considered genotoxic under the conditions of this assay.

A study was conducted to investigate the potential of the test substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the rat according to OECD Guideline 474, EPA OPPTS 870.5395 and EU Method B.12 in compliance with GLP.

The substance was dissolved in deionised water and was given twice at an interval of 24 h as a oral dose of 2000 mg/kg bw/d to male and female rats. The dose was selected based on the results of a previous rat acute oral toxicity study. The animals were killed 24 h after the last administration and bone marrow cells were collected for micronuclei analysis. After treatment with the test substance, the number of polychromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic erythrocytes to total erythrocytes in both male and female animals remained unaffected and differed less than 20% from the control value.

Under the study conditions, the test substance did not induce micronuclei in bone marrow cells of the rat. Therefore, the test substance was not considered to be clastogenic in this micronucleus assay.