Reaction product of Chloro[29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]aluminium, sulphuric acid and caustic soda

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the weight of evidence of a number of different studies, it is concluded that the substance does not display genotoxic toxicity in vitro.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102 is not included in the test system

GLP compliance:

no

Remarks:

Study pre-dates GLP regulations

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name as used in study report: FAT 60 149/A
- Batch: 124-4+5

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced liver S-9

Test concentrations with justification for top dose:

25, 75, 225, 675 and 2025 μg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

cyclophosphamide

other: daunoblastin (TA98 without S9), N-methyl-N'-nitro-N-nitrosoguanidine (TA1535 without S9), 9(5)aminoacridine hydrochloride (TA1537 without S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

Evaluation criteria:

The test substance was considered to be non-mutagenic if the colony count in relation to the negative control was not doubled at any concentration.

Statistics:

When the colonies had been counted, the arithmetic mean was calculated.

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The values of 6 to 7 back-mutant colonies at the concentrations of 25, 75 and 225 μg/plate without microsomal activation on Strain TA1537 (controls: 3 back-mutant colonies) are attributed to a higher rate of spontaneous revertant colonies.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Only tested up to 1000 µg/plate

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Only tested up to 1000 µg/plate

GLP compliance:

no

Remarks:

Study pre-dates GLP regulations

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name as used in the study report: FAT 60 149/B
- Batch No.: Partie 1

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced S9 Mix

Test concentrations with justification for top dose:

1, 5, 10, 50, 100, 500, 1000 μg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

cyclophosphamide

other: Daunorubicin-HCl (TA98 without S9), N-methyl-N'-nitro-N-nitrosoguanidine (TA1535 and E.Coli without S9), 9(5)aminoacridine hydrochloride (TA1537 without S9), 2-aminoanthracene (TA1538 with S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48-55 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: In order to estimate a growth-inhibiting effect of the test substance on the bacteria, a plate test without microsomal activation was performed on Strain TA 100 with the following concentrations: 0.0001, 0.001, 0.01, 0.1, 1, 10, 50, 100, 500, 1000 and 2000 μg/plate.

Evaluation criteria:

A test substance is generally considered to be nonmutagenic if the colony count in relation to the negative control is not doubled at any concentration.

Statistics:

When the colonies had been counted, the arithmetic mean was calculated.

Species / strain:

other: S. typhimurium TA98, TA100, TA1535, TA1537, TA1538, E. coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: Slight increase in the number of back-mutant colonies observed in the experiment with microsomal activation on Strain TA 1537 is attributed to a higher incidence of spontaneously occurring back-mutants

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Tested without metabolic activation, only two dose levels tested and no positive controls tested

Qualifier:

no guideline followed

Principles of method if other than guideline:

The test substance was applied to mouse lymphoma cells (L5178Y) in vitro at concentrations of 296 and 372 µg/mL for either 4 or 18 hours.

GLP compliance:

no

Remarks:

Study pre-dates GLP regulations

Type of assay:

other: mouse lymphoma assay

Specific details on test material used for the study:

- Name as used in study report: FAT 60 149/B
- Batch No.: 4204

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Test concentrations with justification for top dose:

A toxicity test was first performed in vitro to determine the concentrations to be used in the mutagenicity assay. The concentrations, causing an 80% cell-kill. The investigations were performed with the concentrations of 296 and 372 μg/ml.

Vehicle / solvent:

Fischer's medium containing 10% horse serum

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

no

Details on test system and experimental conditions:

TOXICITY TEST
A toxicity test was first performed in vitro to determine the concentrations to be used in the mutagenicity assay. The concentrations, best suited for mutagenicity test, are those causing an 80% cell-kill. The L5178Y cells used in this assay were taken from cultures in an exponential phase of growth. The substance was prepared in Fischer's medium containing 10% horse serum in various doses ranging from 10 to 10000 yg/ml, and the incubation was continued for 4 or 18 h in a 5% CO2 atmosphere in 5-ml Falcon flasks. After the removal of the test substance, samples were taken for determination of viability. For each concentration, eight tubes each containing 100 cells in semisolid agar were prepared and incubated for 10 days at 37°C in a CO2 atmosphere. At the end of the incubation period a clone count was carried out and the number of clones in the control was set at 100%. As a rule, from the results obtained, the concentration required to produce an 80% cell-kill is calculated. For the 4 h incubation as well as for the 18 h incubation an 80% cell-kill was obtained. Thus, in the mutagenicity experiments the concentrations of 372 μg/ml and of 296 μg/ml respectively were employed.

MUTAGENICITY TEST
The mutagenicity test was carried out by treating L5178Y cells with the selected concentration at a cell density of 10E6 /ml in Falcon flasks. The exposure time was 4 h and 18 h respectively. After removal of the substance, the cells were incubated for three days, the cell density being adjusted daily to 2E5 cells/ml. At the end of the expression period, the cells were set up at a density of 4xE5 cells/5 ml in a semi-solid agar containing antimetabolites in culture tubes. The antimetabolites added were methotrexate (LEDERLE), thymidine (FLUKA) and cytosine arabinoside (SERVA). Parallel with these cultures a cell-viability control was carried out. For this purpose 100 cells per 5 ml were seeded in agar of the same quality but without antimetabolites. The incubation time of the mutagenicity test cultures was 14 days, that for cell-viability control 10 days. The values obtained from the viability control are used to normalize the results received from the mutagenicity test, i.e. to preclude a 100%-viability of the cells seeded in cultures of the mutagenicity test.

Evaluation criteria:

The calculated mutant frequency corresponds to the number of clones per 100,000 cells. The mutation factor is calculated by dividing the mutant frequency of the treated cells by the mutant frequency of the control cells. The test substance is considered to be nonmutagenic if the mutation factor is not greater than 2.5

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

not examined

Additional information on results:

The cells were incubated together with the substance in a concentration of 372 μg/ml for four hours and of 296 μg/ml for 18 hours. These concentrations did produce the required 80% cell-kill. In the mutagenicity experiments performed with FAT 60 149/B in the above-mentioned concentration, there was no increase in the number of mutant colonies found in comparison with the control, neither after 4 h treatment nor after 18 h treatment.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

no

Remarks:

Study pre-dates GLP regulations

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name as used in Study report: Tinolux 4204

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S-9 was purchased from Kikkoman Co., Ltd

Test concentrations with justification for top dose:

0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 500, 1000 and 5000 μg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48-55 h

NUMBER OF REPLICATIONS: 2

Evaluation criteria:

A test substance is generally considered to be nonmutagenic if the colony count in relation to the negative control is not doubled at any concentration.

Statistics:

When the colonies had been counted, the arithmetic mean was calculated.

Species / strain:

other: S. typhimurium TA98, TA100, TA1535, TA1537, TA1538 and E. coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Based on the weight of evidence of three different studies, it is concluded that the substance does not show genotoxic toxicity in vivo.

Link to relevant study records

Reference

Reference 1

Endpoint:

genetic toxicity in vivo, other

Remarks:

Dominant lethal test

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)

Deviations:

yes

Remarks:

No positive controls used; 3 separate studies performed

GLP compliance:

no

Remarks:

Study pre-dates GLP regulations

Type of assay:

rodent dominant lethal assay

Specific details on test material used for the study:

- Name as used in the study report: FAT 60 149/B

Species:

mouse

Strain:

Tif:MAGf

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: closed SPF breeding colony
- Age at study initiation: 2-3 months
- Weight at study initiation: 28-53 g
- Diet: standard laboratory diet (NAFAG No.890)
- Water: ad libitum:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26
- Humidity (%): 34-78
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

Polyethylene glycol 400

Details on exposure:

The substance was administered orally by intubation in single doses of 666 and 2000 mg/kg to each 20 males. The control group was treated with the vehicle only. The females remained untreated.

Duration of treatment / exposure:

Singe doses

Frequency of treatment:

Once

Post exposure period:

6 hours

Dose / conc.:

666 mg/kg bw (total dose)

Dose / conc.:

2 000 mg/kg bw (total dose)

No. of animals per sex per dose:

20 males

Control animals:

yes, concurrent vehicle

Positive control(s):

no

Details of tissue and slide preparation:

The number of live embryos and embryonic deaths were listed. In addition, the uteri were placed in a solution of ammonium sulphide in order to detect sites of early embryonic resorptions.

Statistics:

To compare the totals of the number of implantations indicating possible pre-implantation losses - the t-test or Mann-Whitney's U-test was used. The totals of the numbers of mated.and pregnant dams or embryonic deaths were compared with the aid of the Χ2-test or - Fisher's exact test.

Sex:

male

Genotoxicity:

negative

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Additional information on results:

In the first study, the date on the mating ratio and the number of implantations were comparable in all groups. The data on embryonic deaths showed a statistically significant increase in the high-dose group in the second (14.5%) and the third (16.3%) mating periods in comparison to the respective controls (7.5% and 6.9%) (p<0.01). In the second study, the findings made in first study were not reproduced. In this study, in the second mating period, there was a statistically significant increase in the number of embryonic deaths in the low-dose group in comparison with the control (10.8%; control 4.9%) (p<0.01). In addition, a statistically significantly lower rate of pregnancies was found in the control group (69.2%) during the third period, in comparison with the respective treatment groups (>90%). In the third study, the statistically significant finding made in both, the 1 and the 2 study were not reproduced. In this study, only in the low-dose group during the first period was a lower number of implantations (mean value 9.84) found than in the controls (mean value 11.1).

Since the deviations observed in the three studies were each only reproducible on the individual investigations and not in one or both of the others, they can be regarded as purely fortuitous and the results in their entirety therefore cannot be interpreted as evidence of dominant lethal effects in the progeny of male mice treated with the test substance.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

GLP compliance:

no

Remarks:

Study pre-date GLP regulations

Type of assay:

other: Chromosome Studies in Somatic Cells, Bone Marrow

Specific details on test material used for the study:

- Name as used in the study report: FAT 60 149/B
- Batch No.: P.1

Species:

hamster, Chinese

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 20 - 29 g
- Housing: individual cages
- Diet: Standard diet: NAFAG No.924.
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22
- Humidity (%): 29-40
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: unspecified

Vehicle:

0.1% aqueous solution of sodium-carboxymethylcellulose

Details on exposure:

1250, 2500 and 5000 mg/kg in 20 ml/kg 0.1% aqueous solution of sodium-carboxymethylcellulose

Duration of treatment / exposure:

Treatment consisted of one daily dose on 2 consecutive days.

Dose / conc.:

1 250 mg/kg bw/day (nominal)

Dose / conc.:

2 500 mg/kg bw/day (nominal)

Dose / conc.:

5 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

4

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide: 64 mg/kg in 20 ml/kg sodium-carboxymethylcellulose

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

Bone marrow harvested from the shafts of both femurs was suspended in 1% sodium citrate solution to induce hypotonicity, kept in a waterbath at 4 to 6 °C for 45 min and then centrifuged for 10 min at 200 x g. The pellets were then fixed in methanol-acetic acid 3:1 for a period of 30 min at room temperature. After resuspension of the pellet and centrifugation for 5 min at 150 x g, the fixative was changed and the specimen stored overnight at 4°C. Finally the pellet was again resuspended, centrifuged for 5 min at 150 x g and resuspended in some 0.5 ml fixative in order to obtain a more concentrated cell suspension. These specimens were pipetted onto wet slides and stained with acetic-orcein.

Four animals (two females and two males) from each group were evaluated. 100 metaphase plates from each animal were analysed by reference to the following criteria: a) Chromatid-type aberrations. These changes include "breaks" and "exchanges". All discontinuations exceeding the diameter of a chromatid were designated as breaks. b) Chromosome-type aberrations. This category comprises "acentric fragments", "minutes" (very small chromosome fragments), "ring chromosomes" and "dicentrics". c) Chromatid gaps. All interruptions of the chromatid continuity which were smaller than the diameter were designated as gaps. d) Chromosome pulverizations. Completely pulverized metaphase chromosomes were distinguished from incomplete pulverizations with recognizable chromosome fragments.

Statistics:

The significance of difference was assessed by chi-square test.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: In the intermediate-dose group one female animal died after the second application

Additional information on results:

In the control group, in the intermediate and in the high dosage group no aberrations were detected. In the low-dose group one metaphase with a chromatid exchange was found in altogether 400 metaphases. In contrast to the test substance, cyclophosphamide, 64 mg/kg, used as positive control caused a marked increase of chromatid-type aberrations (26.25%) and chromosome-type aberrations (0.5%). In addition, pulverizations were recorded (1.0%). The difference is highly significant from the concurrent negative control. The number of gaps (i.e. achromatid regions in the intact chromatid) was not related to the treatment with the test subtsance. In all dosage groups the percentage did not exceed the control value.

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

no

Remarks:

Study pre-dates GLP regulations

Type of assay:

other: Mammalian Erythrocyte Micronucleus Test

Specific details on test material used for the study:

- Name as used in the study report: FAT 60 149/B
- Batch: P.1

Species:

hamster, Chinese

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 21 - 31 g
- Housing: individual cages
- Diet: Standard diet: NAFAG No.924.
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23-24
- Humidity (%): 53-67
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

Polyethylene Glycol 400

Details on exposure:

Treatment consisted of daily one application on 2 consecutive days.

Frequency of treatment:

One application on 2 consecutive days

Post exposure period:

24 h

Dose / conc.:

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

Dose / conc.:

2 500 mg/kg bw/day (actual dose received)

Dose / conc.:

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

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide: 128 mg/kg in 20 ml/kg PEG 400

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

Bone marrow was harvested from the shafts of both femurs. In a siliconized pipette filled with approx. 0.5 vil rat serum the bone marrow was drawn up. In order to receive a homogeneous suspension the content of pipette was aspirated gently about three times. Small drops of the mixture were transferred on the end of a slide, spread out by pulling it behind a polished cover glass and the preparations were air-dried. Three hours later, the slides were stained in undiluted May-Grunwald solution for 2 min then in May-Grunwald solution/water 1/1 for 2 min and then in Giemsa's, 40% for 20 min. After being rinsed in methanol 55% for 5-8 sec and washed off twice in water, they were left immersed in water for approx. 2 min. After rinsing with distilled water and air-drying, the slides were cleared in Xylol and mounted in Eukitt.

METHOD OF ANALYSIS: The slides of three female and three male animals each of the negative control group, the positive control group and of the groups treated with various doses of the substance were examined. 1000 bone marrow cells each were scored per animal and the following anomalies were registered: a) Single Jolly bodies, b) fragments of nuclei in erythrocytes, c) micronuclei in erythroblasts, d) micronuclei in leucopoietic cells, e) polyploid cells.

Statistics:

The significance of difference was assessed by chi-square test.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

In the high-dose group one female animal and in the intermediate dose group one male animal died after the second and the first application, respectively. In all dosage groups the percentage of cells displaying anomalies of nuclei did not differ significantly from the negative control. By contrast, the positive control (cyclophosphamide, 128 mg/kg) yielded a marked increase of the percentage of cells with anomalies. Here the mean percentage of anomalies was 16.0, whereas the negative control yielded a percentage of 0.1. The difference is highly significant (p<0.05).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro: Ames

Three reliable Ames tests, all with and without microsomal activation, and two mouse lymphoma assays are available. The first Ames test, with S. typhimurium strains TA98, TA100, TA1535, TA1537, did not contain a strain that may detect certain oxidising mutagens, cross-linking agents and hydrazines (CIBA-GEIGY, 1979). Exposure up to 2025 μg/plate was tested. While there were slightly increased back-mutant colonies in one strain, these were not dose-related and considered to be attributable to a higher incidence of spontaneously occurring back-mutants. In the second Ames test, next to the strains included in the first test, also TA1538 and E. coli WP2 uvrA were included (CIBA-GEIGY, 1980). Exposure up to 1000 μg/plate was tested. Again, there was a slight increase in back-mutant colonies in one strain, which was not dose related and considered to be caused by a higher incidence of spontaneously occurring back-mutants. In the third Ames test, the same strains were included as in the second test and exposure was up to 5000 μg/plate (Ishizu, 1981). In this test, comparison of the number of back-mutant colonies in the controls and the cultures treated with the various concentrations revealed no marked deviations.

In vitro: Mutagenicity

One mouse lymphoma assay, similar to OECD guideline 476, studied mutagenic effects on mouse lymphoma cells (L5178Y) in vitro in concentrations of 296 and 372 μg/mL in the absence of metabolic activation (CIBA-GEIGY, 1980). The concentrations produced a 80% cell-kill in the 4- and 18 hour toxicity test. No positive controls were tested. Comparison of the number of forward mutant colonies of the treated and control cells after exposure to the antimetabolites revealed no differences attributable to treatment with the substance.

In vivo: Clastogenicity

A micronucleus test was performed in Chinese hamsters, using one daily dose, given by oral gavage, of 1250, 2500 or 5000 mg/kg on two consecutive days (CIBA-GEIGY, 1981). The bone marrow smears from animals treated with various doses showed no significant difference from the control. The incidence of bone marrow cells with anomalies of nuclei corresponds to the frequency observed in the control group. By contrast, a "positive control" experiment with cyclophosphamide (128 mg/kg) yielded 16.0% cells with anomalies of nuclei. This is significantly different from the controls treated with the vehicle (PEG 400) alone.

A chromosome aberration study, similar to OECD guideline 475, was performed in Chinese hamsters (CIBA-GEIGY, 1982). The substance was administered by oral gavage. Treatment consisted of one daily dose of 1250, 2500 or 5000 mg/kg on each of 2 consecutive days. The chromosome displays from the animals of the negative control group, of the intermediate-dose group and of the high-dose group showed no aberrations. In the animals of the low-dose group one metaphase per 400 cells with a chromatid-type aberration in the form of an exchange was detected. By contrast, treatment of the animals with cyclophosphamide 64 mg/kg (positive control) yielded 26.3% of cells with chromatid-type aberrations and 0.5% cells with chromosome-type aberrations. 1.0% of the cells showed pulverizations.

A dominant lethal study in mice was performed by administering orally in single doses to male albino mice (Tif: MAG f [SPF]), which were then mated with untreated females of the same strain. The experiments were carried out over six mating periods in two studies and over three mating periods in one study. At the end of each mating period the females were replaced by new ones. Doses of 666 and 2000 mg/kg were given. Three separate studies were performed in this way. In the first study, the females mated with males which had been treated did not differ significantly from the females mated with control males, either in the mating ratio or in the number of implantations. The data on embryonic deaths, however, showed a statistically significant increase in the highdose group in the second and third mating periods. To ascertain whether this finding was due to an effect of the substance, the study was repeated for the first three mating periods. On this second occasion, the findings of the first study were not reproduced. In this study, a statistically significant increase in the number of embryonic deaths was observed in the second mating period in the low-dose group. In addition, in the third period, the number of pregnant females in the control group was significantly lower than in the two treatment groups. Owing to the not unequivocal results, a third study was carried out under identical conditions to the first. On this occasion, the statistically significant findings made in both the first and the second study were not reproduced. Since the deviations observed in the three studies were each only reproducible on the individual investigations and not in one or both of the others, they can be regarded as purely fortuitous and the results in their entirety therefore cannot be interpreted as evidence of dominant lethal effects in the progeny of male mice treated with the test substance.

In vivo: Mutagenicity

A second mouse lymphoma assay was performed in a host-mediated assay (CIBA GEIGY, 1980). The investigations were performed with a dose 6000 mg/kg, which was the highest dose tested in a single administration toxicity test. This dose did not lead to a decrease in the number of cells. The mutagenicity test was performed in mice. L5178Y-cells were inoculated intraperitoneally (1E6 cells/animal). Three days after inoculation of the target cells the substance was given orally to four animals in the stated dose (6000 mg/kg). Three days after the administration of the substance, the cells were removed from the peritoneal cavity under aseptic precautions. These cells were used for the test with the antimetabolites. In the mutagenicity test the mutant frequency in the cultures of treated cells was not increased, thus a mutation factor of 2.5 or more was not obtained.

Justification for classification or non-classification

Based on the weight of evidence from both in vitro and in vivo tests, it is concluded that the criteria for classification for genetic toxicity of the CLP Regulation (EC) No 1272/2008 are not met. Results from the dominant lethal test are not unequivocal, but all other tests indicate no genetic toxicity.