Reaction mass of hydrogen [1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1) and hydrogen bis[1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1) and hydrogen bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Under the conditions of the study the test material was found to be mutagenic both with and without metabolic activation.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8 December 1992 to 15 January 1993

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

GLP compliance:

yes

Type of assay:

bacterial forward mutation assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
The concentration, homogeneity and stability of the test material preparations were not determined by analysis.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test material was accurately weighed and dissolved in DMSO and appropriate dilutions made on the day of each experiment.

Target gene:

Salmonella typhimurium: Histidine

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Details on mammalian cell type (if applicable):

CELLS USED
- Suitability of cells: The strains used in this assay were all mutants derived from Salmonella typhimurium LT2 and were those recommended for general screening. Prior to being used, characterisation checks were carried out to determine the amino-acid requirement, presence of rfa and R factors and the spontaneous reversion rate.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Overnight sub-cultures of the master slopes were prepared in nutrient broth (Oxoid Limited).
Top agar was prepared using 0.6 % Difco Bacto agar and 0.5 % sodium chloride. 5 mL of 1.0 mM histidine/1.0 mM biotin solution was added to each 100 mL of top agar.
Preliminary Toxicity Study: Sterile plates of Vogel Bonner agar (minimal agar ~30 mL/ plate).
- Properly maintained: Yes, stored at -196 °C in a Statebourne liquid N2 freezer.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Preliminary Toxicity Test
0, 312.5, 625, 1 250, 2 500 and 5 000 μg/plate

Mutation Test: Experiment 1
0, 8.0, 40, 200, 1 000 and 5 000 μg/plate

Mutation Test: Experiment 1
0, 312.5, 625, 1 250, 2 500 and 5 000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (dimethyl sulphoxide).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 4-Nitro-o-phenylenediamine and 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: In agar (plate incorporation).

DURATION: 48 hours expression time

SELECTION AGENT: Histidine

NUMBER OF REPLICATIONS: Experiment 1 and 2: Triplicate

DETERMINATION OF CYTOTOXICITY
- Method: In order to select appropriate dose levels for use in the main study, a preliminary test was carried out to determine the toxicity of the test material to the tester organisms. 0.1 mL of bacterial suspension (TA 100), 2 mL of molten, trace histidine supplemented media (histidine/biotin & top agar), 0.1 mL of test solution and 0.5 mL phosphate buffer were over-layed onto sterile plates of Vogel Bonner agar (minimal agar ~30 mL/ plate). Five doses of the test compound and a solvent control (dimethyl sulphoxide) were tested in duplicate. After approximately 48 hours incubation at 37 °C the plates were scored for revertant colonies and examined for a thinning of the background lawn.

TEST MATERIAL AND NEGATIVE CONTROLS
A 0.1 mL aliquot of one of the bacterial suspensions was placed in sets of sterile test tubes followed by 2.0 mL of molten, trace histidine supplemented, top agar at 45 °C. These sets comprised two test tubes for each bacterial tester strain. 0.1 mL of the appropriately diluted test material or negative control was also added to each of the two test tubes. Into one of the test tubes was placed 0.5 mL of the S9 liver microsome mix; in the other tube 0.5 mL of pH 7.4 buffer was added. This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material.
The second experiment was performed using fresh bacterial cultures, test material and control solutions in triplicate.

POSITIVE CONTROLS WITHOUT ACTIVATION
0.1 mL of one of the positive control solutions (ENNG, 9AA, 4NQO or 4NOPD) was added to a test tube containing 2.0 mL of molten, trace histidine supplemented, top agar and 0.1 mL of the appropriate bacterial suspension. Finally 0.5 mL of pH 7.4 buffer was added to the test tube. This procedure was then repeated, in triplicate, for each of the positive controls.

POSITIVE CONTROLS WITH ACTIVATION
0.1 mL of 2AA or BP solution was added to a test tube containing 2.0 mL of molten trace histidine supplemented top agar and 0.1 mL of one of the test bacterial suspensions. Finally 0.5 mL of S9 mix was added to the test tube. The procedure was then repeated, in triplicate, for each tester strain.
The contents of each test tube were equally distributed onto the surface of Vogel-Bonner agar plates (one tube per plate). These plates were incubated at 37 °C for approximately 48 hours and the number of revertant colonies counted.

Evaluation criteria:

For the test material to be considered positive in this test system, it should have induced a dose-related and statistically significant increase in mutation rate in one or more strains of bacteria in the presence and/or absence of the S9 microsomal enzymes in both experiments at sub-toxic dose levels. If the two experiments give conflicting results or equivocal results are obtained then a third experiment may be used to confirm the correct response. All data are statistically analysed using the methods recommended by the UKEMS. To be considered negative the number of induced revertants compared to spontaneous revertants should be less than twofold at each dose level employed, the intervals of which should be between 2 and 5 fold and extend to the limits imposed by toxicity, solubility or up to the maximum recommended dose of 5 000 μg/plate. In this case the limiting factor was the maximum recommended dose.

Statistics:

All data are statistically analysed using the methods recommended by the UKEMS (Statistical Evaluation of Mutagenicity Test Data).

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON RESULTS
The results for the checks for characteristics, viability and spontaneous reversion rate for each tester strain were all found to be satisfactory.
A significant, dose-related, reproducible increase in the numbers of revertant colonies of bacteria were recorded for all of the strains of Salmonella used, with doses of the test material beginning at 8 μg/plate. A response was observed both with and without metabolic activation.
The positive control substances all produced marked increases in the number of revertant colonies and the activity of the S9 fraction was found to be satisfactory.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In the preliminary toxicity study, precipitation was observed at 5 000 µg/plate in the strain used (TA 100). In Experiments 1 and 2, precipitate was also observed at 5 000 µg/plate. This did not interfere with the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES
The dose range of the test material used in the preliminary toxicity study was 0, 312.5, 625, 1 250, 2 500 and 5 000 μg/plate. The test material was non-toxic in the strain of Salmonella used (TA100).

ADDITIONAL INFORMATION ON CYTOTOXICITY
No toxicity was observed to any of the strains of Salmonella used.

Summary of Experiment 1.

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	TA1538	TA98	TA1537
-	0 8.0 40 200 1 000 5 000	130.3 137.0 142.7 172.7 266.3 278.7	16.7 16.3 26.3 32.3 76.7 43.7	20.7 112.7 210.7 338.0 594.7 637.0	17.7 136.3 222.0 349.3 595.3 805.3	14.7 15.0 15.0 16.3 15.7 15.3
+	0 8.0 40 200 1 000 5 000	161.7 163.7 179.7 212.7 269.7 415.0	14.0 16.0 27.7 22.7 58.0 64.0	22.3 109.7 188.3 436.3 651.0 1237.7	30.0 138.0 251.7 564.7 833.7 1619.7	16.7 14.7 20.3 26.0 26.3 29.7
Positive Controls
-	Name	EENG	EENG	4NOPD	4NQO	9AA
	Concentration (µg/plate)	3.0	5.0	5.0	0.2	50
	Mean no. colonies/plate	475.3	167.7	307.3	185.7	195.3
+	Name	BP	2AA	BP	BP	BP
	Concentration (µg/plate)	5.0	2.0	5.0	5.0	5.0
	Mean no. colonies/plate	407.7	299.3	110.3	192.0	103.0

EENG = N-Ethyl-N'-nitro-N-nitrosoguanidine

4NOPD = 4-Nitroquinoline-1-oxide

9AA = 9-aminoacridine

BP = Benzo(a)pyrene

Summary of Experiment 2.

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	TA1538	TA98	TA1537
-	0 312.5 625 1250 2 500 5 000	105.7 123.7 151.0 222.7 256.0 282.0	12.3 21.3 31.7 53.7 65.7 58.7	12.0 259.0 372.0 473.0 761.3 870.0	15.7 295.7 422.0 491.7 667.3 831.0	12.3 13.3 13.0 13.3 11.0 13.0
+	0 312.5 625 1250 2 500 5 000	110.0 226.3 263.3 260.0 367.7 411.0	13.3 24.3 30.7 47.3 55.0 63.3	15.7 353.0 456.3 588.7 755.3 1166.0	22.3 501.3 594.0 846.0 960.3 1352.7	14.0 13.3 14.7 32.0 30.7 26.7
Positive Controls
-	Name	EENG	EENG	4NOPD	4NQO	9AA
	Concentration (µg/plate)	3.0	5.0	5.0	0.2	50
	Mean no. colonies/plate	472.3	176.7	200.0	169.7	330.3
+	Name	BP	2AA	BP	BP	BP
	Concentration (µg/plate)	5.0	2.0	5.0	5.0	5.0
	Mean no. colonies/plate	426.0	142.7	141.0	160.7	112.7

EENG = N-Ethyl-N'-nitro-N-nitrosoguanidine

4NOPD = 4-Nitroquinoline-1-oxide

9AA = 9-aminoacridine

BP = Benzo(a)pyrene

Conclusions:

Under the conditions of the study the test material was found to be mutagenic both with and without metabolic activation.

Executive summary:

The potential of the test material to cause mutagenic effects in bacteria was assessed in accordance with the standardised guidelines OECD 471 and EU Method B.13/14 under GLP conditions.

Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were treated with the test material through the plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system at 10 % in standard co-factors. The dose range was determined in a preliminary toxicity assay and was 8 to 5 000 μg/plate in the first experiment. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh chemical solutions. In this case the dose range of test material was 312.5 to 5 000 μg/plate.

The test material caused no reduction in the growth of the bacterial lawn at any of the dose levels employed in all of the strains of Salmonella used. The test material was, therefore, tested up to the maximum recommended dose of 5 000 μg/plate. A precipitate was observed at 5 000 μg/plate, this did not interfere with the scoring of revertant colonies.

The solvent (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range and all positive control chemicals produced marked increases in the number of revertant colonies, both with and without the metabolising system. The test was therefore considered to be valid.

A significant, dose-related, reproducible increase in the numbers of revertant colonies was recorded for all of the bacterial strains with doses of test material beginning at 8 μg/plate. A response was observed both with and without metabolic activation.

In conclusion, the test material was found to be mutagenic under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Under the conditions of the test, the test material is not mutagenic.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 January 2005 to 21 March 2005

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

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

NMRI

Remarks:

NMRI BR

Details on species / strain selection:

The NMRI BR mouse is used as test system because it is a readily available rodent species, which is commonly used for this purpose, with documented susceptibility to a wide range of toxic substances. These mice are recommended by international guidelines (e.g. OECD, EEC).

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: Young adult animals were selected (6-8 weeks old).
- Weight at study initiation: Mean body weight immediately prior to dosing with test material ranges from 33.2 ± 1.6 to 35.2 ± 3.6. The body weights of the mice at the start of the treatment were within 20 % of the sex mean.
- Assigned to test groups randomly: Yes, as they came to hand from the delivery boxes.
- Fasting period before study: No
- Housing: Group housing of 5 animals per sex per cage in labelled polycarbonate cages containing Woody Clean bedding. Paper bedding was provided as nest material.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: At least 5 days before start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): Approximately 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours dark per day.

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: Corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material was suspended in corn oil. Test material concentrations were blended and treated with ultra-sonic waves to obtain a homogeneous suspension. Test material concentrations were dosed within 2.5 h after preparation.

TREATMENT
The mice received an intraperitoneal injection of a maximum tolerated (high), an intermediate and a low dose of test material. The route of administration was chosen to maximize the chance of the test material reaching the target tissue.
The dosing volume was 10 mL/kg body weight. The route and frequency of administration and the volume administered of the negative and the positive control was the same as those of the test material.

Duration of treatment / exposure:

The test material was administered as a single intraperitoneal injection.

Frequency of treatment:

All test groups received a single intraperitoneal injection.

Post exposure period:

Bone marrow of the groups treated with the test material was sampled 24 or 48 hours after dosing.
Bone marrow of the negative control group was isolated 24 hours after dosing and bone marrow of the positive control group was isolated 48 hours after dosing.

Dose / conc.:

62.5 mg/kg bw (total dose)

Dose / conc.:

125 mg/kg bw (total dose)

Dose / conc.:

250 mg/kg bw (total dose)

No. of animals per sex per dose:

5 animals per sex per dose group. There were two test groups for the animals dosed at 250 mg/kg body weight.

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control: Cyclophosphamide dissolved in physiological saline was used to treat the positive control group.
- Route of administration: A single intraperitoneal injection.
- Doses / concentrations: 50 mg/kg body weight.

Tissues and cell types examined:

Bone marrow erythrocytes.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Selection of an adequate dose range for the micronucleus main test was based on a dose range finding study. Five dose groups, three comprising 1 male and 1 female, and two groups comprising 3 males and 3 females received a single dose of the test material. The study duration per dosing was two to three days. During this period mortality and physical condition were recorded at least once daily.

DETAILS OF SLIDE PREPARATION:
The animals were sacrificed by cervical dislocation. Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone was flushed with approximately 2 mL of foetal calf serum. The cell suspension was collected and centrifuged at 1 000 rpm (approximately 100 g) for 5 min.
The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed with the serum by aspiration with the remaining serum. A drop of the cell suspension was placed on the end of a slide, which was previously cleaned (24 h immersed in a 1:1 mixture of 96 % (v/v) ethanol/ether and cleaned with a tissue) and marked (with the study identification number and the animal number). The drop was spread by moving a clean slide with round-whetted sides at an angle of approximately 45 ° over the slide with the drop of bone marrow suspension. The preparations were air-dried, fixed for 5 min in 100 % methanol and air-dried overnight. Two slides were prepared per animal.
The slides were automatically stained using the "Wright-stain-procedure" in an "Ames" HEMAtek slide stainer. The dry slides were dipped in xylene before they were embedded in MicroMount and mounted with a coverslip.

METHOD OF ANALYSIS:
All slides were randomly coded before examination. An adhesive label with study identification number and code was stuck over the marked slide. At first the slides were screened at a magnification of 100x for regions of suitable technical quality, i.e. where the cells were well spread, undamaged and well stained. Slides were scored at a magnification of 1 000 x. The number of micronucleated polychromatic erythrocytes was counted in 2 000 polychromatic erythrocytes. The ratio polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1 000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated.

Evaluation criteria:

A micronucleus test is considered acceptable if it meets the following criteria:
a) The positive control substance induced a statistically significant (Wilcoxon Rank Sum Test, two-sided test at P < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes.
b) The incidence of micronucleated polychromatic erythrocytes in the control animals should reasonably be within the laboratory historical control data range (mean ± three times the standard deviation):
Males: 1 .4 ‰ ± 4.1 ‰ indicated are means for n = 231.
Females: 1.5 ‰ ± 3.7 ‰ indicated are means for n = 160.
Equivocal results should be clarified by further testing using modification of experimental conditions.

Statistics:

A test material is considered positive in the micronucleus test if:
- It induces a statistically significant (Wilcoxon Rank Sum Test; two sided test at P < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) in the combined data for both sexes or in the data for male or female groups separately.

A test material is considered negative in the micronucleus test if:
- None of the tested concentrations or sampling times showed a statistically significant (P < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes neither in the combined data for both sexes nor in the data for male or female groups separately.

The preceding criteria are not absolute and other modifying factors may enter into the final evaluation decision.

Key result

Sex:

female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 250, 500, 750, 1 000 and 2 000 mg/kg.
- Solubility: The test material was suspended in corn oil and treated with ultrasonic waves to obtain a homogenous suspension.
- Clinical signs of toxicity in test animals: There were deaths and clinical signs including lethargy, hunched posture, rough coat and hairless body parts dark coloured.


RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity in test animals: The animals of the negative and positive control groups showed no abnormalities. During the first two hours after dosing two male animals and one female animal of the groups treated with 250 mg/kg body weight had a hunched posture. One of these male animals also had a rough coat. In the groups treated with 125 mg/kg body weight two female animals had a hunched posture and a rough coat. In the groups treated with 62.5 mg/kg body weight the animals showed no reaction to treatment.
Within 20 hours after dosing all animals treated with the test material had dark coloured hairless body parts. Two male animals treated with 250 mg/kg body weight also had a hunched posture and one of these animals also had a rough coat.
Within 43 hours after dosing all animals of the groups treated with 250 mg/kg body weight had a hunched posture, a rough coat and their hairless body parts were dark coloured. Two male animals were also lethargic.

- Induction of micronuclei: The mean number of micronucleated polychromatic erythrocytes scored in test material treated groups were compared with the corresponding solvent control group.
No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of test material treated animals compared to the vehicle treated animals.
The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historical solvent control data range.
Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes in both sexes. Hence, the acceptability criteria of the test were met.

- Ratio of PCE/NCE: The animals of the groups which were treated with the test material showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which reflects a lack of toxic effects of this compound on the erythropoiesis. The animals of the groups treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes, demonstrating toxic effects on erythropoiesis.

Conclusions:

Under the conditions of the test, the test material is not mutagenic.

Executive summary:

The test material was assessed in the Micronucleus Test in mice, to evaluate its genotoxic effect on erythrocytes in bone marrow according to OECD Test Guideline 474 and EU Method B.12.

The test material was a black powder with a purity of >85.5 %, and was suspended in corn oil.

Five male and five female animals were used in each of the six treatment groups, including negative and positive controls. All groups received a single intraperitoneal injection. The negative and positive control groups were treated with vehicle and 50 mg/kg body weight of cyclophosphamide (CP), respectively. Animals were dosed with the test material at 250 (two groups), 125 (one group), and 62.5 (one group) mg/kg body weight. After dosing the animals of the dose level of 250 mg/kg body weight showed the following toxic signs: Lethargy (2 male animals), hunched posture (7 male and 6 female animals), rough coat (5 male and 5 female animals) and dark coloured hairless body parts (all animals). All animals of the dose level of 125 and 62.5 mg/kg body weight had dark coloured hairless body parts after dosing.

Two female animals dosed with 125 mg/kg body weight had a rough coat and a hunched posture.

Bone marrow of the groups treated with the test material was sampled 24 or 48 (highest dose only) hours after dosing. Bone marrow of the negative and positive control groups was harvested 24 and 48 hours after dosing, respectively.

No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with the test material.

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historical solvent control data range. Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes in both sexes. Hence, both criteria for an acceptable assay were met.

The groups that were treated with the test material showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, which reflects a lack of toxic effects of this compound on the erythropoiesis. The groups that were treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, demonstrating toxic effects on erythropoiesis.

It is concluded that the test material is not mutagenic in the micronucleus test under the experimental conditions described.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Ames Test

The potential of the test material to cause mutagenic effects in bacteria was assessed in accordance with the standardised guidelines OECD 471 and EU Method B.13/14 under GLP conditions. The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were treated with the test material through the plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system at 10 % in standard co-factors. The dose range was determined in a preliminary toxicity assay and was 8 to 5 000 μg/plate in the first experiment. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh chemical solutions. In this case the dose range of test material was 312.5 to 5 000 μg/plate.

The test material caused no reduction in the growth of the bacterial lawn at any of the dose levels employed in all of the strains of Salmonella used. The test material was, therefore, tested up to the maximum recommended dose of 5 000 μg/plate. A precipitate was observed at 5 000 μg/plate, this did not interfere with the scoring of revertant colonies.

The solvent (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range and all positive control chemicals produced marked increases in the number of revertant colonies, both with and without the metabolising system. The test was therefore considered to be valid.

A significant, dose-related, reproducible increase in the numbers of revertant colonies was recorded for all of the bacterial strains with doses of test material beginning at 8 μg/plate. A response was observed both with and without metabolic activation.

In conclusion, the test material was found to be mutagenic under the conditions of the test.

 

In Vivo Mouse Micronucleus

The test material was assessed in the Micronucleus Test in mice, to evaluate its genotoxic effect on erythrocytes in bone marrow according to OECD Test Guideline 474 and EU Method B.12. The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

The test material was a black powder with a purity of >85.5 %, and was suspended in corn oil.

Five male and five female animals were used in each of the six treatment groups, including negative and positive controls. All groups received a single intraperitoneal injection. The negative and positive control groups were treated with vehicle and 50 mg/kg body weight of cyclophosphamide (CP), respectively. Animals were dosed with the test material at 250 (two groups), 125 (one group), and 62.5 (one group) mg/kg body weight. After dosing the animals of the dose level of 250 mg/kg body weight showed the following toxic signs: Lethargy (2 male animals), hunched posture (7 male and 6 female animals), rough coat (5 male and 5 female animals) and dark coloured hairless body parts (all animals). All animals of the dose level of 125 and 62.5 mg/kg body weight had dark coloured hairless body parts after dosing.

Two female animals dosed with 125 mg/kg body weight had a rough coat and a hunched posture.

Bone marrow of the groups treated with the test material was sampled 24 or 48 (highest dose only) hours after dosing. Bone marrow of the negative and positive control groups was harvested 24 and 48 hours after dosing, respectively.

No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with the test material.

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historical solvent control data range. Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes in both sexes. Hence, both criteria for an acceptable assay were met.

The groups that were treated with the test material showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, which reflects a lack of toxic effects of this compound on the erythropoiesis. The groups that were treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, demonstrating toxic effects on erythropoiesis.

It is concluded that the test material is not mutagenic in the micronucleus test under the experimental conditions described.

Justification for classification or non-classification

In accordance with the criteria for classificaton as defined in Annex I, Regulation (EC) No. 1272/2008, the test material does not require classification for genetic toxicity.