[2-[[4-[(2-chloro-4-nitrophenyl)azo]phenyl]ethylamino]ethyl](2-hydroxypropyl)dimethylammonium chloride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genotoxicity of the target substance was assessed in two in vitro tests (OECD 471 and OECD 490). The test substance was mutagenic in the bacterial reverse mutation test, but tested negative in the in vitro mammalian cell gene mutation test.

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

Study period:

1991-02-06 to 1991-07-08

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:

only 4 strains tested (instead of five)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine locus

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Type and identity of media:
The test followed the directions of Ames et al. (1973, 1975) and Maron and Ames (1983): 0.1 mL compound, 0.1 mL bacteria, 0.5 mL S9 mix or buffer, 2.0 mL soft agar, 45 °C in water bath, max. 30 sec, transfer to petri dish with solid agar

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

First test: 5000, 1000, 200, 40 and 8 µg/plate;
Repeat tests: 400, 200, 100, 50 and 25 µg/plate

Vehicle / solvent:

The solvent was deionized water and for the positive controls DMSO.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: nitrofurantoin

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-nitro-1,2-phenylene diamine

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-1,2-phenylene diamine

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 0.1 mL TS + 0.1 mL bacteria + 0.5 mL S9 + 2 mL soft agar: 30 sec at 45 °C
- Exposure duration: 48 hours at 37°C

NUMBER OF REPLICATIONS: 4 plates/strain/concentration

DETERMINATION OF CYTOTOXICITY
- Method: - background growth
- marked and dose-dependent reduction in mutant count compared to negative controls
- titer determination

- OTHER:
Acceptance criteria:
a) The negative controls had to be within the expected range, as defined by published data (i.e. Maron and Ames, 1983) and the laboratory's own historical data
b) The positive controls had to show sufficient effects, as defined by the laboratory's experience
c) Titer determinations had to demonstrate sufficient bacterial density in the suspension.

An assay which did not comply with at least one of the above criteria was not used for assessment. Furthermore, the data generated in this assay needed to be confirmed by two additional independent experiments. Even if the criteria for points (a), (b) and (c) were not met, an assay was accepted if it showed mutagenic activity of the test compound.

Evaluation criteria:

A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100 and TA 98 this increase should be about twice the amount of negative controls, whereas for TA 1537, at least a threefold increase should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by scientific judgement.

Statistics:

N.A.

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

There was no indication of bacteriotoxic effects of the test item at doses of up to and including 100 µg per plate. The total bacteria counts consistently produced results comparable to the negative controls, or differed only insignificantly. Nor was any inhibition of growth noted. Higher doses had a strong, strain-specific bacteriotoxic effect. Therefore they could only be used up to 400 µg per plate for assessment purposes. Three of the four strains concerned revealed a dose-related increase in mutant counts to well over double those of negative controls. Strains TA 100, TA 1537 and TA were affected. The findings were confirmed by a repeat test. The lowest dose at which this finding was reproducible was approximately 200 µg per plate for Salmonella typhimurium TA 100 and TA 1537, and approximately 25 µg per plate for TA 98. Positive findings were obtained with and without S9 mix, the effects being strengthened by S9 mix.

 Table 1: Summary of mean values with S9-mix from the first test

µg/plate		Strain
		TA 1535		TA 100		TA 1537		TA 98
30% S9-mix
0		12		107		11		32
8		12		170		12		49
40		14		185		21		101
200		10		250		77		467
1000		11		10		0		--
5000		0		0		0		0
2-AA		140		498		31		182

 

Table 2: Summary of mean values with S9-mix from repeat test

µg/plate		Strain
		TA 1535		TA 100		TA 1537		TA 98
30% S9-mix
0		13		165		12		44
25		16		226		26		397
50		12		213		24		185
100		14		223		58		757
200		14		245		82		802
400		9		174		25		486
2-AA		127		662		70		307

 Table 3: Summary of mean values without S9-mix from the first test

µg/plate		Strain
		TA 1535		TA 100		TA 1537		TA 98
0		13		91		11		24
8		9		115		10		58
40		11		141		24		196
200		10		37		--		208
1000		--		0		0		0
5000		0		0		0		0
Na-azid		641
NF				289
4-NPDA						40		57

 

Table 4: Summary of mean values without S9-mix from the repeat test

µg/plate		Strain
		TA 1535		TA 100		TA 1537		TA 98
0		12		116		11		25
25		12		201		27		157
50		10		140		22		389
100		10		113		16		303
200		7		28		0		198
400		6		13		0		--
Na-azid		824
NF				361
4-NPDA						60		58

Conclusions:

A dose dependent increase in the mutant count at non-bacteriotoxic concentrations in comparison to the negative controls was observed after treatment with the test item in the presence and absence of metabolic activation. Therefore, the test item is considered to be mutagenic with and without S9 mix in this Salmonella/microsome test.

Executive summary:

In a reverse gene mutation assay in bacteria (equivalent to OECD 471), strains TA 1535, TA 100, TA 1537 and TA 98 of S. typhimurium were exposed to the test item (95.6 % purity) in deionized water at concentrations of 8, 40, 200, 1000 and 5000 µg/plate (first/pre-test) and at concentrations of 25, 50, 100, 200 and 400 µg/plate (repeat test) in the presence and absence of mammalian metabolic activation. 

The test item was tested up to the limit concentration (5000 µg/plate). Based on bacteriotoxic effects, dose groups above 400 µg per plate were not used for assessment purposes. The positive controls induced the appropriate responses in the corresponding strains. In Salmonella typhimurium strains TA 100, TA 1537 and TA 98 a mutagenic effect was observed at non-cytotoxic doses. In the Salmonella typhimurium strain TA 1535 no mutagenic effect was observed. Based on the result, it can be stated that the test item can be considered as a bacterial mutagen in this assay.

This study is classified as acceptable. The study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2015-12-16 to 2016-05-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

The mutation assay method used in this study is based on the identification of L5178Y colonies which have become resistant to a toxic thymidine analogue trifluorothymidine (TFT). This analogue can be metabolised by the enzyme thymidine kinase (TK) into nucleosides, which are used in nucleic acid synthesis resulting in the death of TK-competent cells. TK-deficient cells, which are presumed to arise through mutations in the TK gene, cannot metabolise trifluorothymidine and thus survive and grow in its presence. In the L5178Y mouse lymphoma cells, the gene which codes for the TK enzyme is located on chromosome 11. Cells which are heterozygous at the TK locus (TK+/-) may undergo a single step forward mutation to the TK-/- genotype in which little or no TK activity remains. The mouse lymphoma assay often produces a bimodal size distribution of TFT resistant colonies designated as small or large. It has been evaluated that point mutations and deletions within the active allele (intragenic event) produce large colonies. Small colonies result in part from lesions that affect not only the active TK allele but also a flanking gene whose expression modulates the growth rate of cells.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium supplemented with 10% Foetal Calf Serum (RPMI
complete)
- Properly maintained: yes; permanent stock of mouse lymphoma L5178Y cells are stored in liquid nitrogen and subcultures are prepared from the frozen stocks for experimental use.
- Periodically checked for Mycoplasma contamination: yes
- The generation time, plating efficiency and mutation rates (spontaneous and induced) have been checked in this laboratory.
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 tissue fraction: Species: Rat, Strain: Sprague Dawley, Tissue: Liver, Inducing Agents: Phenobarbital – 5,6-Benzoflavone, Producer: MOLTOX, Molecular Toxicology, Inc. Batch Number 3512

Test concentrations with justification for top dose:

A preliminary cytotoxicity assay was performed at the following dose levels: 1000, 500, 250, 125, 62.5, 31.3, 15.6, 7.81 and 3.91 µg/mL. Based on the results obtained in the preliminary cytotoxicity assay, two independent assays for mutation at the TK locus were performed using the following dose levels:
Main Assay I (-S9; treatment time 3 hours): 60.2, 43.0, 30.7, 22.0, 15.7 and 11.2 μg/mL
Main Assay I (+S9; treatment time 3 hours): 100, 50.0, 25.0, 12.5, 6.25 and 3.13 μg/mL
Main Assay II (-S9; treatment time 24 hours): 35.0, 25.0, 17.9, 12.8, 9.11 and 6.51 μg/mL

Vehicle / solvent:

Test item solutions were prepared using dimethylsulfoxide (DMSO)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

methylmethanesulfonate

Details on test system and experimental conditions:

A preliminary cytotoxicity test was performed in order to select appropriate dose levels for the mutation assays. In this test a wide range of dose levels of the test item was used and the survival of the cells was subsequently determined. Treatments were performed in the absence and presence of S9 metabolic activation for 3 hours and for 24 hours only in the absence of S9 metabolic activation. A single culture was used at each test point. The mutation assays were performed including vehicle and positive controls, in the absence and presence of S9 metabolising system. Duplicate cultures were prepared at each test point, with the exception of the positive controls which were prepared in a single culture. In the first experiment, the cells were exposed to the test item for a short treatment time (3 hours). Since negative results were obtained without metabolic activation, the second experiment in the absence of S9 metabolism was performed, using a longer treatment time (24 hours). After washing in Phosphate Buffered Saline (PBS), cells were resuspended in fresh complete medium (10%) and incubated to allow expression of the mutant phenotype. At the end of the expression period cells were plated for the evaluation of 5-trifluorothymidine resistance and for viability.

Evaluation criteria:

For a test item to be considered mutagenic in this assay, it is required that:
1. The induced mutant frequency (IMF) is higher than the global evaluation factor (GEF)
suggested for the microwell method (126x10^-6) at one or more doses.
2. There is a significant dose-relationship as indicated by the linear trend analysis.
Results which only partially satisfy the above criteria will be dealt with on a case-by-case basis. Similarly, positive responses seen only at high levels of cytotoxicity will require careful interpretation when assessing their biological significance. Any increase in mutant frequency should lie outside the historical control range to have biological relevance.

Statistics:

Statistical analysis was performed according to UKEMS guidelines (Robinson W.D., 1990).

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Survival after treatment:
In the first experiment, in the absence of S9 metabolic activation, no cell survived after treatment at the highest dose level, moderate toxicity reducing relative total growth (RTG) to 14% of the concurrent negative control was noted at 43.0 µg/mL, slight to mild toxicity was observed between 15.7 and 30.7 µg/mL, while no relevant toxicity was noted at the lowest concentration tested. In the presence of S9 metabolism, treatment with the test item at 100 µg/mL yielded moderate toxicity reducing RTG to 24% of the concurrent negative control value, slight toxicity was noted at the next lower concentration, while no relevant toxicity was observed over the remaining concentrations tested. In the second experiment, in the absence of S9 metabolic activation using a long treatment time, the highest dose level selected (35 µg/mL) yielded marked toxicity reducing RTG to 8% of the concurrent negative control value. The next two lower dose levels of 25.0 and 17.9 µg/mL yielded moderate toxicity reducing RTG to 15 and 26%, respectively. Dose-related toxicity was seen over the remaining dose levels tested.

Mutation results:
In Main Assay I, statistically significant increases in mutant frequency were observed at the lowest and at two intermediate concentrations, in the absence of S9 metabolism, and at the highest dose level in its presence. A linear trend was indicated, both in the absence and presence of S9 metabolism. However, the observed increases were lower than the Global Evaluation Factor both in the absence and presence of S9 metabolism, thus they were considered of no biological relevance. In Main Assay II, no increases in mutant frequency were observed at any concentration tested.

Conclusions:

Under the experimental conditions reported, the test item is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay using mouse lymphoma L5178Y cells.

Executive summary:

In a mammalian cell gene mutation assay conducted according to OECD 490, mouse lymphoma L5178Y cells cultured in vitro were exposed to the test item (87.9% purity) in dimethylsulfoxide at concentrations of 11.2, 15.7, 22.0, 30.7, 43.0 and 60.3 µg/mL (main assay I, without S9 mix) and at 3.13, 6.25, 12.5, 25.0, 50.0 and 100 µg/mL (main assay I, with S9 mix) and in main assay II, without S9 mix at concentrations of 6.51, 9.11, 12.8, 17.9, 25.0 and 35.0 µg/mL. The test item was tested in the main assay I up to 60.3 µg/mL (without metabolic activation) and up to 100 µg/mL (with metabolic activation) and in main assay II up to 35.0 µg/mL (without metabolic activation) based on data obtained from the preliminary cytotoxicity test. The positive controls did induce the appropriate response. There was no evidence of induced mutant colonies over background. 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 490.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Genotoxicity of the target substance was assessed in an in vivo micronucleus test (OECD 474). The test substance was not gentoxic.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1992-03-17 to 1993-01-11

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

GLP compliance:

yes

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: F. Winkelmann, Borchen, Germany
- Age at study initiation: 8 - 12 weeks of age
- Weight at study initiation: 28 - 43 g
- Housing: females: in groups of a maximum of three mice; males: individually in Macrolon type I cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 1.5
- Humidity (%): 40 -70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: physiological saline

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was dissolved in physiological saline solution, stirred with a magnetic mixer during administration and injected intraperitoneally.

Duration of treatment / exposure:

test item dose groups: 16, 24 and 48 hours,
negative/positive control: 24 hours

Frequency of treatment:

once

Post exposure period:

no

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

three test item related experimental dose groups: 16, 24 and 48 hours

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control used: cyclophosphamide
- Route of administration: intraperitoneal
- Dosis: 20 mg/kg bw

Tissues and cell types examined:

bone marrow derived erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The selection of the dose was based on a pilot test, in which groups of five animals, including both males and females, were i.p. administered 10 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 75 mg/kg and 100 mg/kg bw. For the results please box "Additional information on results".

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
see Table 1 in box "Any other information on materials and methods incl. tables".

DETAILS OF SLIDE PREPARATION:
Bone marrow was flushed into a tube containing fetal bovine serum and centrifuged (5 min, 1000 rpm)
Air dried smears were automatically stained with an Ames HemaTek Slide Stainer and then destained with methanol, rinsed with deionized water and dried. After drying, the slides were covered with xylene and a cover glass.

METHOD OF ANALYSIS:
Coded slides were evaluated using a light microscope at a magnification of about 1000. Micronuclei appear as stained chromatin particles in the anucleated erythrocytes. Normally, 1000 polychromatic erythrocytes were counted per animal. The incidence of cells with micronuclei was established by scanning the slides in a meandering pattern. Moreover, the ratio of polychromatic to normochromatic erythrocytes was determined (number of normochromatic erythrocytes per 1000 polychromatic ones). Additionally, the number of normochromatic erythrocytes showing micronuclei was also established.

Evaluation criteria:

A test was considered positive if at any of the intervals, there was a relevant and significant increase in the number of polychromatic erythrocytes showing micronuclei in comparison to the negative control. A test was considered negative if there was no relevant or significant increase in the rate of micronucleated polychromatic erythrocytes at any time. A test was also considered negative if there was a significant increase in that rate which, according to the laboratory’s experience, was within the range of negative controls. A test was considered equivocal if there was an increase of micronucleated polychromatic erythrocytes above the range of attached historical negative controls, provided the increase was not significant and the result of the negative control was not closely related to the data of the respective treatment group.

Statistics:

The test item group(s) with the highest mean and the positive control were checked by Wilcoxon’s non parametric rank sum test with respect to the number of polychromatic erythrocytes having micronuclei and the number of normochromatic erythrocytes. A variation was considered statistically significant if its error probability was below 5% and the treatment group figure was higher than that of the negative control.
The rate of normochromatic erythrocytes containing micronuclei was examined if the micronuclear rate for polychromatic erythrocytes was already relevantly increased. In this case, the group with the highest mean was compared with the negative control using the one-sided chi²-test. A variation was considered statistically significant, if the error probability was below 5% and the treatment group figure was higher than that of the negative control. In addition, standard deviations (1s ranges) were calculated for all the means.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
The selection of the dose was based on a pilot test, in which groups of five animals, including both males and females, were i.p. administered 10 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 75 mg/kg and 100 mg/kg bw. The following symptoms were recorded for up to 48 hours, starting at 10 mg/kg bw: apathy, roughened fur, red discoloration of hairless parts of skin, staggering gait, spasm, leaping coloured urine. In addition, 3 of 5 animals died in the 75 mg/kg group and all animals died in the 100 mg/kg bw group. Based on the results, 50 mg/kg bw was considered as 1 MTD for the main test.

RESULTS OF DEFINITIVE STUDY
- Clinical signs: After application the animals showed the following signs of toxicity: apathy, roughened fur, staggering gait, spasm, twitching, difficulty in breathing and orange discoloured urine. No symptoms were recorded for the control groups.
- Mortality: One of forty treated animals died during the test period due to acute toxicity. No animals died in the control group.
- Induction of micronuclei (for Micronucleus assay): no clastogenic effect, see Table 2
- Ratio of PCE/NCE (for Micronucleus assay): slightly changed, see Table 2
- Statistical evaluation: see Table 2

Table 2: Summary of results of micronucleus test with the test item
experimental groups		Number of evaluated poly-chromatic erythrocytes (PCE)		Number of normo-chromatic erythrocytes per 1000 PCE		micronucleated cells per 1000
						normo-chromatic erythrocytes		poly-chromatic erythrocytes
Negative control		10000		811 +/- 208		1.2 +/- 1.6		1.5 +/- 1.1
Test item_16 hours		10000		1321* +/- 319		1.3 +/- 1.0		1.6 +/- 1.0
Test item_24 hours		10000		1088 +/- 549		1.3 +/- 1.6		1.5 +/- 1.0
Test item_48 hours		10000		775 +/- 199		0.4 +/- 0.7		1.3 +/- 1.2
Positive control		10000		557 +/- 231		0.4 +/- 1.3		12.6* +/- 6.9

Concentration: test item: 50 mg/kg bw; positive control: 20 mg/kg bw

* p< 0.01 (tested by non-parametric Wilcoxon ranking test)

Conclusions:

The test item did not induce structural and/or numerical chromosomal damage in the immature erythrocytes of the mouse. Therefore, the test item is considered to be non-mutagenic with respect to clastogenicity and aneugenicity in the mammalian erythrocyte micronucleus test.

Executive summary:

In a NMRI mouse bone marrow micronucleus test conducted in accordance with OECD Guideline 474, 5 mice/sex/treatment group were treated intraperitoneally once with the test item (95.6% purity) at doses of 0 and 50 mg/kg bw. The vehicle used was physiological saline. The animals were sacrificed and bone marrow cells were harvested after 16 hours (test item), 24 hours (negative/positive control and test item) and after 48 hours (test item).

After application the animals showed the following signs of toxicity: apathy, roughened fur, staggering gait, spasm, twitching, difficulty in breathing and orange discoloured urine. The test item was tested at an adequate dose based on a preliminary dose range finding test. The positive control induced the appropriate response. There was not a significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time (16, 24 and 48 hours).

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 474 for in vivo cytogenicity.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The target substance was tested positive in a bacterial reverse mutation test (OECD 471), but was tested negative in the higher tier studies conducted in accordance with OECD 474 (in vivo micronucleus test) and 490 (in vitro mammalian cell gene mutation).

Justification for classification or non-classification

The target substance was genotoxic in the bacterial reverse mutation test, but was tested negative in higher tier studies (OECD TG 474 and OECD TG 490). Therefore, it can be concluded based on a weight-of-evidence approach of the available data that no classification of the target substance is warranted.