5,12-dihydro-2,9-dimethylquino[2,3-b]acridine-7,14-dione

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

During the described Ames mutagenicity tests in bacteria, the test item did not induce gene mutations by frameshifts or base-pair substitutions in the genome of the strains used. Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

The test item (Pigment Red 122) was not mutagenic in the HGPRT-test with cells of the V79 Chinese hamster cell line both in the presence and absence of rat liver S-9mix metabolic activation.

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:

12 December 2000 to 22 February 2001

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

GLP compliance:

yes

Remarks:

Study in compliance with GLP based on Art.19a of German Chemical Act and OECD principles

Type of assay:

bacterial reverse mutation assay

Target gene:

his- for S. typhimurium strains

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 102

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced S9 fraction (liver homogenate fraction from male rats

Test concentrations with justification for top dose:

Two independent mutagenicity studies were conducted (one plate incorporation test and one preincubation test), each in the absence and in the presence of a metabolizing system: 50, 160, 500, 1600 and 5000 ug/plate in both studies
The test compound was suspended in DMSO and a stock suspension of 50 mg/ml was prepared for the highest concentration, which provided a final concentration of 5000 ug/plate.

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: aminoanthracene (all strains with S9)

Evaluation criteria:

A test compound 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.

Statistics:

not required

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

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

Positive controls validity:

valid

Additional information on results:

The test compound was suspended in DMSO and a stock suspension of 50 mg/ml was prepared for the highest concentration, which provided a final concentration of 5000 ug/plate. Further dilutions of 1600, 500, 160 and 50 ug/plate were used in all experiments.
Visible precipitation of the test compound on the plates was observed at concentrations of 160 ug/plate and above in the plate incorporation test and in the preincubation test at dose levels of 500 ug/plate and above.
Because of heavy precipitation of the test compound the bacterial lawn could only be evaluated at the dose level of 500 ug/plate and lower concentrations in the plate incorporation test and in the preincubation test at dose level of 1600 ug/plate down to the lowest concentration of 50 ug/plate.
The test compound proved to be not toxic to the bacterial strains in the plate incorporation test and in the preincubation experiment. In the preincubation test with the strain TA 1535 in the presence of S9-mix the number of revertant colonies was slightly decreased.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Mean mutant number ratios treated/solvent control

Exp. I: plate incorporation method without S9 mix

Concentrations given in µg/plate

Strain -- 50 -- 160 -- 500 -- 1600 -- 5000

TA100 -- 0.9 -- 1.0 -- 1.0 -- 0.9 -- 0.9

TA1535 -- 0.9 -- 0.8 -- 0.8 -- 0.9 -- 0.6

TA1537 -- 1.5 -- 1.3 -- 1.5 -- 1.1 -- 1.5

TA98 -- 1.1 -- 1.4 --1.1 -- 1.1 -- 0.7

TA102 -- 1.0 -- 0.9 -- 1.2 -- 1.1 -- 1.1

Exp. I: plate incorporation method with rat S9 mix

Concentrations given in µg/plate

Strain -- 50 -- 160 -- 500 -- 1600 -- 5000

TA100 -- 1.0 -- 1.0 -- 1.1 -- 0.9 -- 1.2

TA1535 -- 1.4 -- 1.4 -- 1.3 -- 1.4 -- 1.3

TA1537 -- 0.9 -- 1.2 -- 0.8 -- 1.2 -- 0.7

TA98 -- 0.9 -- 0.6 --1.1 -- 0.9 -- 0.8

TA102 -- 1.0 -- 1.1 -- 1.3 -- 1.1 -- 1.0

Exp. II: pre-incubation method without S9 mix

Concentrations given in µg/plate

Strain -- 50 -- 160 -- 500 -- 1600 -- 5000

TA100 -- 1.2 -- 1.1 -- 1.2 -- 1.2 -- 1.1

TA1535 -- 1.1 -- 0.9 -- 1.2 -- 0.9 -- 0.9

TA1537 -- 0.8 -- 0.7 -- 0.8 -- 0.7 -- 0.6

TA98 -- 1.0 -- 0.8 --0.9 -- 0.6 -- 0.6

TA102 -- 1.0 -- 1.0 -- 0.9 -- 0.9 -- 0.9

Exp. II: pre-incubation method with rat S9 mix

Concentrations given in µg/plate

Strain -- 50 -- 160 -- 500 -- 1600 -- 5000

TA100 -- 1.0 -- 1.0 -- 1.2 -- 1.0 -- 0.9

TA1535 -- 0.6 -- 0.4 -- 0.5 -- 0.6 -- 0.6

TA1537 -- 1.2 -- 1.7 -- 1.3 -- 1.2 -- 1.1

TA98 -- 1.2 -- 1.2 --1.4 -- 1.3 -- 1.3

TA102 -- 1.0 -- 1.0 -- 1.0 -- 0.9 -- 1.1

Conclusions:

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

During the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test item was considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

The test item was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537, TA 98 and TA 102 of Salmonella typhimurium.

Two independent mutagenicity studies were conducted (one plate incorporation test and one preincubation test), each in the absence and in the presence of a metabolizing system derived from a rat liver homogenate.

For both studies, the compound was suspended in DMSO, and each bacterial strain was exposed to 5 dose levels.

Concentrations for both studies were 50, 160, 500, 1600 and 5000 ug/plate.

Visible precipitation of the test compound on the plates was observed at concentrations of 160 ug/plate and above in the plate incorporation test and in the preincubation test at dose levels of 500 ug/plate and above. Because of heavy precipitation of the test compound the bacterial lawn could only be

evaluated at the dose level of 500 ug/plate and lower concentrations in the plate incorporation test and in the preincubation test at dose level of 1600 ug/plate down to the lowest concentration of 50 ug/plate.

Control plates without mutagen showed that the number of spontaneous revertant colonies was within the laboratory's historical control range.

All the positive control compounds showed the expected increase in the number of revertant colonies. The number of revertant colonies of the positive compounds with the strains TA 1535 in the absence and with the strain TA 102 in the presence of S9 -mix were slightly out of the historical control data range, but the criteria for the positive response were succeeded.

Toxicity: In the plate incorporation test and in the preincubation experiment toxicity was not observed with and without metabolic activation.

Mutagenicity: In the absence and in the presence of the metabolic activation system the test item did not result in relevant increases in the number of revertants in any of the bacterial strains.

Summarizing, it can be stated that the test item was not mutagenic in this bacterial mutation test at any dose level either in the absence or presence of an exogenous metabolic activation.

Reference 2

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:

24 May 2007 to 17 August 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP according to German "Chemikaliengesetz" and OECD Guideline

Type of assay:

bacterial reverse mutation assay

Target gene:

his- for S. typhimurium strains
trp- for E. coli WP2 uvr A

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (phenobarbital/ß-naphthoflavone-induced rat liver S9)

Test concentrations with justification for top dose:

Experiment I (plate incorporation): 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate both with and without metabolic activation
Experiment II (pre-incubation): 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate both with and without metabolic activation

Vehicle / solvent:

DMSO, purity <99% (Merck, Darmstadt, Germany). The solvent was chosen because of its solubility properties and its relative non-toxicity to bacteria

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4 -nitro-o-phenylene diamine (Salmonella typhimurium TA 1537, TA 98) ; Aminoanthracene (al strain with S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
plate incorporation (experiment I); preincubation (experiment II). Since experiment I gave a negative result, experiment II was performed as a
preincubation assay.

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY: existence of evaluable plates (> 0 colonies) at five concentrations or more

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

Not mandatory according to OECD guideline 471

Key result

Species / strain:

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

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

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

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see below for details

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at and above 1000 µg/plate (experiment I), at and above 2500 µg/plate (experiment II), undissolved particles had no influence on data recording

RANGE-FINDING/SCREENING STUDIES:
Pre-experiment was reported as experiment I because the criterion (evaluable plates (>0 colonies) at five concentrations or more in all strains are used) was met.

COMPARISON WITH HISTORICAL CONTROL DATA:
yes

ADDITIONAL INFORMATION ON CYTOTOXICITY:
see below

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments. Slight toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed in the absence of metabolic activation in strain TA 1535 at 5000 µg/plate and in strain TA 1537 at 2500 and 5000 µg/plate in experiment I, and in strain TA 1537 from 1000 - 5000 µg/plate in experiment II. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor 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 biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant cololonies.

Mean mutant number ratios treated/solvent control

Exp. I: plate incorporation method without S9 mix

Concentrations given in µg/plate

Strain -- 3 -- 10 -- 33 -- 100 -- 333 -- 1000 -- 2500 -- 5000

TA1535 -- 1.1 -- 0.9 -- 0.9 -- 1.0 -- 0.8 -- 1.0 -- 0.5 -- 0.4

TA1537 -- 1.1 -- 1.8 -- 1.3 -- 1.4 -- 1.0 -- 0.9 -- 0.4 -- 0.2

TA98 -- 1.1 -- 0.9 -- 1.0 -- 1.3 -- 1.3 -- 1.0 -- 0.8 -- 0.6

TA100 -- 0.9 -- 0.8 --0.9 -- 1.0 -- 0.9 -- 0.9 -- 0.8 -- 0.8

WP2uvrA -- 1.2 -- 1.3 -- 1.4 -- 1.2 -- 1.1 -- 0.9 -- 0.9 -- 0.7

Exp. I: plate incorporation method with rat S9 mix

Concentrations given in µg/plate

Strain -- 3 -- 10 -- 33 -- 100 -- 333 -- 1000 -- 2500 -- 5000

TA1535 -- 0.9 -- 1.1 -- 1.0 -- 0.9 -- 0.6 -- 0.7 -- 0.6 -- 0.6

TA1537 -- 0.9 -- 0.8 -- 0.9 -- 1.3 -- 1.0 -- 0.9 -- 0.8 -- 0.8

TA98 -- 1.0 -- 1.0 -- 1.0 -- 1.0 -- 0.8 -- 0.8 -- 0.8 -- 0.8

TA100 -- 1.0 -- 0.9 --1.0 -- 1.1 -- 0.9 -- 0.8 -- 0.9 -- 0.8

WP2uvrA -- 0.9 -- 1.1 -- 0.9 -- 1.0 -- 0.9 -- 0.7 -- 0.8 -- 0.9

Exp. II: pre-incubation method without S9 mix

Concentrations given in µg/plate

Strain -- 10 -- 33 -- 100 -- 333 -- 1000 -- 2500 -- 5000

TA1535 -- 1.3 -- 1.1 -- 1.6 -- 0.9 -- 1.3 -- 0.6 -- 0.5

TA1537 -- 0.3 -- 0.8 -- 1.1 -- 0.7 -- 0.4 -- 0.3 -- 03

TA98 -- 1.1 -- 1.3 -- 1.3 -- 1.2 -- 0.8 -- 0.8 -- 0.7

TA100 --0.9 -- 1.0 -- 1.0 -- 0.9 -- 0.9 -- 1.0 -- 1.1

WP2uvrA -- 1.0 -- 0.9 -- 1.1 -- 0.8 -- 0.9 -- 0.8 -- 0.8

Exp. II: pre-incubation method with rat S9 mix

Concentrations given in µg/plate

Strain -- 10 -- 33 -- 100 -- 333 -- 1000 -- 2500 -- 5000

TA1535 -- 0.9 -- 0.7 -- 0.8 -- 0.8 -- 0.9 -- 0.5 -- 0.6

TA1537 -- 1.0 -- 0.9 -- 0.7 -- 0.9 -- 0.6 -- 0.6 -- 0.6

TA98 -- 1.0 -- 1.0 -- 1.1 -- 1.0 -- 0.8 -- 0.6 -- 0.7

TA100 --0.9 -- 0.9 -- 0.9 -- 1.0 -- 0.7 -- 0.9 -- 0.8

WP2uvrA -- 1.0 -- 0.9 -- 0.9 -- 1.0 -- 0.7 -- 0.6 -- 0.6

Conclusions:

Interpretation of results:
negative both with and without metabolic activation

During the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by frameshifts or base-pair substitutions in the genome of the strains used. Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of the test item to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate Experiment II: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments. Slight toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed in the absence of metabolic activation in strain TA 1535 at 5000 µg/plate and in strain TA 1537 at 2500 and 5000 µg/plate in experiment I, and in strain TA 1537 from 1000 - 5000 µg/plate in experiment II. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor 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 biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, 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.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 May 1991 to 8 August 1991

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

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

HGPRT locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM medium supplemented with 10% fetal calf serum containing 2 mM L-glutamine and 0.01% neomycinsulfate, for the selection of mutants
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes (mycoplasma-free cell line was stored in liquid nitrogen in the cell bank of "Genetic Toxicology")

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced S9 fraction (liver homogenate fraction from male rats)

Test concentrations with justification for top dose:

Preliminary toxicity test:
0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 5.0, 10.0, 15.0 and 20.0 µg/ml without metabolic activation
1.0, 5.0, 10.0, 15.0 and 20.0 µg/ml with metabolic activation

Mutation test.
2.0, 5.0, 10.0 and 20.0 µg/ml both with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Positive control in test with metabolic activation: 7.7 µg/ml DMBA Migrated to IUCLID6: 1 mg/ml EMS in test without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: two days
- Exposure duration: 4 h (on day 2)
- Expression time (cells in growth medium): day 2 (treatment) to day 9 (mutant selection)
- Selection time (if incubation with a selection agent): on day 9
- Fixation time (start of exposure up to fixation or harvest of cells): on day 16

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: subculturing in 5 flasks for selection on day 9 for the mutagenicity test

NUMBER OF CELLS EVALUATED: about 400000 cells/flask during selection

DETERMINATION OF CYTOTOXICITY
- Method: plating efficiency

Evaluation criteria:

- The test substance is classified as mutagenic if it reproducibly induces with one of the test substance concentrations a mutation frequency that is three times higher than the spontaneous mutant frequency in this experiment.
- The test substance is classified as mutagenic if there is a reproducible concentration-related increase in the mutation frequency. Such an evaluation may be considered independently from the enhancement factor for induced mutants. However, in a case by case evaluation both decisions depend on the level of the corresponding negative control data.

Statistics:

Biometry was not necessary to perform.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No cytotoxic effect with and without metabolic activation was observed in the main experiment and in the repeat.
The test substance was assessed for its mutagenic potential in vitro in the HGPRT-test in two independent experiments without metabolic activation and two independent experiments with metabolic activation. No relevant reproducible enhancement of the mutant colonies or mutant frequency over the range of the negative control was found with any of the concentrations used, either with or without metabolic activation by S9-mix. Biometry was not necessary to perform. The sensitivity of the test system was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control substances.

RANGE-FINDING/SCREENING STUDIES:
In a preliminary experiment the test item was assayed with respect to its solubility in cell culture medium. The highest concentration at which no visible precipitation was observed, was found to be 20 µg/ml.
Accordingly, the preliminary study was carried out using a maxium concentration of 20 µg/ml and a wide range of lower dose levels. The cytotoxicity experiment proved the test item was not toxic to the V79 cell s in the absence of metabolic activation (S9-mix). In the presence of metabolic activation (S9-mix) there was also no indication of cytotoxicity up to the limit of solubility.

Summary of results

Condition -- Conc. (µg/ml) -- Rel. plating efficiency (%) -- Mutant colonies per 10e6 cells

Exp. I, 4 hour treatment without S9 mix, mean of 5 flasks

Negative control -- -- 100.0 -- 32.8

Solvent control with water -- -- 100.0 -- 32.5

Positive control with EMS -- 1000 -- 88.2 -- 613.9

Test item -- 2.0 -- 127.3 -- 7.1

Test item -- 5.0 -- 114.8 -- 26.5

Test item -- 10.0 -- 121.0 -- 30.6

Test item -- 20.0 -- 110.7 -- 35.5

Exp. I, 4 hour treatment with S9 mix, mean of 5 flasks  

Negative control -- -- 100.0 -- 23.3

Solvent control with water -- -- 100.0 -- 19.6

Positive control with DMBA -- 7.7 -- 68.5 -- 527.1

Test item -- 2.0 -- 125.5 -- 25.6

Test item -- 5.0 -- 105.4 -- 35.1

Test item -- 10.0 -- 101.7 -- 12.7

Test item -- 20.0 -- 98.7 -- 17.7

Exp.II, 4 hour treatment without S9 mix, mean of 5 flasks

Negative control -- -- 100.0 -- 100.0 -- 60.6

Solvent control with water -- -- 100.0 -- 72.9

Positive control with EMS -- 1000 -- 84.9 -- 1157.3

Test item -- 2.0 -- 87.2 -- 51.8

Test item -- 5.0 -- 101.5 -- 42.3

Test item -- 10.0 -- 96.1 -- 39.4

Test item -- 20.0 -- 117.1 -- 50.3

Exp.II, 4 hour treatment with S9 mix, mean of 5 flasks

Negative control -- -- 100.0 -- 100.0 -- 30.4

Solvent control with water -- -- 100.0 -- 37.9

Positive control with DMBA -- 7.7 -- 75.4 -- 687.9

Test item -- 2.0 -- 92.0 -- 51.9

Test item -- 5.0 -- 99.8 -- 29.3

Test item -- 10.0 -- 96.8 -- 49.0

Test item -- 20.0 -- 96.8 -- 53.8

Conclusions:

Interpretation of results: negative

The test item (Pigment Red 122) was not mutagenic in the HGPRT-test with cells of the V79 Chinese hamster cell line both in the presence and absence of rat liver S-9mix metabolic activation.

Executive summary:

The study was performed to investigate the potential of the test item to induce gene mutations at the HGPRT locus in V 79 cells of the Chinese hamster in vitro.

The assay was performed in two independent experiments, using identical procedures, both with and without rat liver microsomal activation.

The test article was tested with the following concentrations:

without S9-mix:       2, 5, 10 and 20 µg/ml

with S9-mix:        2, 5, 10 and 20 µg/ml

According to the preliminary experiment for toxicity the concentration ranges were selected. The highest concentration produced no distinct decrease of the plating efficiency.

Up to the highest investigated dose no increase in mutant colony numbers was obtained in two independent experiments.

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

In conclusion, the test item does not induce gene mutations in the HGPRT-test with V79 Chinese hamster cells, either in the presence or in the absence of a metabolic activation system, under the experimental conditions described.

Therefore the test item is considered to be non-mutagenic in this HGPRT assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The test item was tested in the micronucleus test. The test compound was suspended in sesame oil and dosed once oral at 2500 mg per kg bodyweight to male and female mice. According to the test procedure the animals were killed 24, 48 or 72 hours after administration.

Endoxan^Rwas used as positive control substance and was administered orally at a dose of 50 mg per kg bodyweight.

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 unaffectedby the treatment withthe test itemand was statistically not different from the control values.

Endoxan^Rinduced in both males and females a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity af the system.

The results indicate that, under the conditions of the present study,the test itemis not mutagenic in the micronucleus test.

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:

10 June 1991 to 8 November 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study followed OECD Guideline 474

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain specifics: Hoe: NMRKf (SPF71)
- Source: Hoechst AG, Kastengrund, SPF breeding colony
- Age at study initiation: 7 weeks
- Weight at study initiation: males: 25-35 g, mean 29.7 g, females: 22-28 g, mean 24.9 g
- Housing: in groups of five in Macrolon type 3 cages in fully air-conditioned room, softwood granulate
- Diet (e.g. ad libitum): rat/mouse diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 55 ± 10 %
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: sesame oil
- Concentration of test material in vehicle: 25% (w/v)
- Amount of vehicle (if gavage or dermal): 10 ml/kg body weight
- Purity: Oleum sesame Ph. Eur. III, Fa. Pharm. Fabrik GmbH, Frankfurt/Main, Germany

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
25% was the highest concentration achievable in sesame oil

Duration of treatment / exposure:

One single oral administration

Frequency of treatment:

See above

Post exposure period:

24 hours (dosed group, negative control and positive control), 48 hours (dosed group and negative control) and 72 hours (dosed group and negative control)

Remarks:

Doses / Concentrations:
2500
Basis:
nominal conc.
mg/kg body weight

No. of animals per sex per dose:

5 males and 5 females (10 animals) per dose group

Control animals:

yes, concurrent vehicle

Positive control(s):

50 mg/kg body weight cyclophosphamide (endoxan, 5 males and 5 females)

Tissues and cell types examined:

1000 polychromatic erythrocytes from the femoral bone marrow of each animal

Details of tissue and slide preparation:

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Animals were killed 24, 48 or 72 h after application

DETAILS OF SLIDE PREPARATION:
Removal of femoral bones and bone marrow from the proximal ends flushed into centrifuge tube containing about 3 ml foetal bovine serum, centrifugation (5 min at 1200 rpm), one drop of thoroughly mixed sediment smeared on a cleaned slide, air-dried for approx. 24 h, staining (methanol, May-Grünwalds solution, Giemsa) and coating with Entellan

METHOD OF ANALYSIS:
Number of cells with micronuclei and ratio of polychromatic to normochromatic erythrocytes
Statistical evaluation (see below)

Evaluation criteria:

95% level of significance for comparisons

Statistics:

Wilcoxon (paired, one-sided, increase) for number of cells and Wilcoxon (paired, two-sided) for the ratio

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

red coloured faeces but free of clinical signs of toxicity after 48 hours

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2500 mg/kg body weight
- Clinical signs of toxicity in test animals: red coloured faeces
- Other: lethality: 0 out of 3 males and 0 out of 3 females

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no statistically significant increase of micronucleated polychromatic erythrocytes in dosed animals
- Ratio of PCE/NCE (for Micronucleus assay): the ratio remained essentially unaffected by the test compound
- Statistical evaluation: see above

Mean mutation indices in polychromatic erythrocytes:

Vehicle control group -- dose group -- positive control group

24 hours male: 1.0 -- 1.8 -- 23.5

24 hours female: 1.0 -- 1.3 -- 21.1

48 hours male: 1.0 -- 0.7 -- not done

48 hours female: 1.0 -- 0.3 -- not done

72 hours male: 1.0 -- 1.7 -- not done

72 hours female: 1.0 -- 1.0 -- not done

Conclusions:

Interpretation of results: negative
Oral administration of the test item (Pigment Red 122) did not lead to a substantial increase of micronucleated polychromatic erythocytes and was not mutagenic in the in vivo mouse micronucleus test

Executive summary:

The test item was tested in the micronucleus test. The test compound was suspended in sesame oil and dosed once oral at 2500 mg per kg bodyweight to male and female mice, upon the results of the previously conducted dose range finding assay. According to the test procedure the animals were killed 24, 48 or 72 hours after administration.

Endoxan^R was used as positive control substance and was administered orally at a dose of 50 mg per kg bodyweight.

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 unaffectedby the treatment with the test item and was statistically not different from the control values.

Endoxan^R induced in both males and females a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity af the system.

The results indicate that, under the conditions of the present study, the test item is not mutagenic in the micronucleus test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

not applicable

Justification for classification or non-classification

No classification

The test material did not caus muatagenic effects in bacteria, mammalian cell as well in vivo in mice.