1,3,3-trimethyl-2-[[methyl(p-tolyl)hydrazono]methyl]-3H-indolium chloride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on results of the in vitro tests performed following OECD 471 and OECD 476 , no mutagenic effects were observed.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

other: experimental result from similar substance

Adequacy of study:

key study

Study period:

1991-07-01 to 1991-07-09

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study with one minor deviation from current guideline. Only four strains were tested (instead of five).

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

No E. coli strain tested

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. (1973a, 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 degree 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/plateRepeat tests: 800, 400, 200, 100, 50 and 25 µg/plate 70, 60, 50, 40, 30, 20 and 10 µg/plate TA 100 with 10 %, 30 %, 50 % S9

Vehicle / solvent:

The solvent employed for the test item was methanol 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

Remarks:

10 µg per plate, only for strain TA 1535 w/o S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: nitrofurantoin

Remarks:

0.2 µg per plate, only for TA 100 w/o S9

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

Remarks:

10 µg per plate, only for TA 1537 w/o S9

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

Remarks:

0.5 µg per plate, only for strain TA 98 w/o S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

3 µg per plate, all strains with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)DURATION- Preincubation: 0.1 mL TS+0.1 mL bacteria+0.5 mL S9+2 mL soft agar: 30 sec at 45 °C- Incubation period: 48 hours at 37°CNUMBER OF REPLICATIONS: 4 plates/strain/concentrationDETERMINATION OF CYTOTOXICITY - Method: - background growth - marked and dose-dependent reduction in mutant count compared to negative controls - titer determination 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 datab) The positive controls had to show sufficient effects, as defined by the laboratory's experiencec) 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 good scientific judgement.

Statistics:

N.A.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

TA 100: 1.6-fold increase at cytotoxic concentrations

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

There was no indication of a bacteriotoxic effect of the test item at 8 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 to a limited extent up to 1000 µg per plate for assessment purposes.In the first trial, none of the four strains concerned showed a dose related and biologically relevant increase in mutant counts over those of the negative controls. This applied both to the tests with and without S9 mix.The negative findings for Salmonella typhimurium TA 1535, TA 1537 and TA 98 were confirmed by further repeat tests. In the TA 100 strain, a 1.6-fold increase in comparison of the negative controls was found in the second test. The findings of the second trial for Salmonella typhimurium TA 100 were confirmed by further repeat tests using 30 % and 50 % S9 mix. No relevant increase was found using 10 % S9 mix. The lowest dose at which this finding was reproducible was approximately 30 µg per plate for Salmonella typhimurium TA 100. Minimally increased mutation rates - below the threshold for positive effects ( 2 fold) - were obtained only with S9 mix containing 30 % and 50 % S9 fraction in S9 mix.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1: Summary of mean values without S9-mix from the pre-test
Pre-Test: µg/plate		Strain
		TA 1535		TA 100		TA 1537		TA 98
0		8		89		7		20
8		11		84		7		18
40		12		84		6		22
200		10		88		7		22
1000		1		20		4		19
5000		0		0		0		0
Na-azid		631
NF				382
4-NPDA						56		86

Table 2: Summary of mean values with S9-mix from the pre-test
Pre-Test: µg/plate		Strain
		TA 1535		TA 100		TA 1537		TA 98
30% S9-mix
0		17		117		10		34
8		16		137		8		28
40		20		145		10		39
200		8		148		12		40
1000		--		11		3		30
5000		0		0		0		0
2-AA		99		817		134		427

Table 3: Summary of mean values without S9-mix from the repeat tests
Repeat-Test: µg/plate		Strain
		TA 1535		TA 100		TA 1537		TA 98
0		15		130		11		32
25		16		141		12		30
50		16		124		10		39
100		13		131		10		41
200		12		138		14		37
400		13		100		13		29
800		5		42		4		20
Na-azid		793
NF				416
4-NPDA						58		105

Table 4: Summary of mean values with S9-mix from the repeat tests
Repeat-Test: µg/plate		Strain
		TA 1535		TA 100		TA 1537		TA 98
30% S9-mix
0		34		190		17		53
25		27		241		21		71
50		28		303		20		74
100		13		197		20		64
200		6		106		20		50
400		--		12		3		13
800		0		--		--		--
2-AA		242		1297		68		570

Table 5: Summary of mean values with different S9-mix concentrations tested in strain TA 100
µg/plate		Strain TA 100
		30 % S9		10% S9		30% S9		50% S9
0		155		150		149		177
10		204		142		160		233
20		189		169		185		199
30		237		175		201		207
40		253		173		212		243
50		261		174		246		261
60		168		174		256		250
70		162		185		209		271
2-AA		534		1406		942		613

Conclusions:

Interpretation of results (migrated information):negative without metabolic activationnegative with metabolic activationNo biologically relevant and 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 non-mutagenic with and without S9 mix in the Salmonella/microsome test.

Executive summary:

In a reverse gene mutation assay in bacteria, strains TA 1535, TA 100, TA 1537, TA 98 of S. typhimurium were exposed to the test item (98.5% purity) in methanol at concentrations of 8, 40, 200, 1000 and 5000 µg/plate (first/pre-test) and at concentrations of 25, 50, 100, 200, 400, 800 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 only the dose groups up to 1000 µg per plate were used to a limited extent for assessment purposes. The positive controls induced the appropriate responses in the corresponding strains. In the studies with metabolic activation, a slight increase (up to 1.6 fold) in the mutant count was observed. This slight increase was not dose-dependent and did not occur in all experiments. In the studies without metabolic activation no induction of mutant counts was observed. Therefore, the test item is considered to be non-mutagenic with and without S9 mix in the Salmonella/microsome test.

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

Remarks:

Type of genotoxicity: gene mutation

Type of information:

other: experimental result on similar substance

Adequacy of study:

key study

Study period:

2015-07-31 to 2016-02-18

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Compliant with GLP and testing guidelines; coherence between data, results and conclusions.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

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 acidsynthesis 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 TKlocus (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 affectnot 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 andsubcultures 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 Numbers 3453 and 3483

Test concentrations with justification for top dose:

A preliminary cytotoxicity assay was performed at the following dose levels: 4340, 2170, 1090, 543, 271, 136, 67.8, 33.9 and 17.0 μg/mLAn additional cytotoxicity assay was performed using the 24 hour treatment time. The test item was assayed at the following dose levels: 17.0, 8.50, 4.25, 2.13, 1.06, 0.531, 0.266, 0.133 and 0.0664 μg/mL.two independent assaysfor mutation at the TK locus were performed using the dose levels described in the following table:Main Assay I (+/-S9; treatment time 3 hours): 68.0, 54.4, 43.5, 34.0, 17.0, 8.50 and 4.25 μg/mLMain Assay II (-S9; treatment time 24 hours): 6.80, 5.23, 4.02, 2.01, 1.01 and 0.503 μg/mLMain Assay II (+S9; treatment time 3 hours): 68.0, 45.3, 30.2, 20.1 and 13.4 μg/mL

Vehicle / solvent:

Test item solutions were prepared using dimethylsulfoxide (DMSO)

Negative solvent / vehicle controls:

yes

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 metabolicactivation. 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 withoutmetabolic 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 (126 x 10^-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 biologicalsignificance. 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).

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Survival after treatment: In the first experiment, in the absence of S9 metabolic activation, severe toxicity reducing relative total growth (%RTG) to 2-17% was noted at the four highest dose levels, from 68.0 to 17.0 μg/mL. Dose-related toxicity was observed over the remaining dose levels tested. In the presence of S9 metabolic activation, marked toxicity reducing RTG to15% of the concurrent negative control value was observed at the highest dose level tested (68.0 μg/mL).Moderate toxicity reducing RTG in a range of 36-42%, was noted between 54.4 and 34.0 μg/mL. No relevant toxicity was observed over the remainingdose levels tested.In the second experiment, in the absence of S9 metabolic activation using a long treatment time, no cells were recovered after treatment at the highestdose level selected (6.80 μg/mL). The next lower dose level (5.23 μg/mL) yielded marked toxicity reducing RTG to 10% of the concurrent negativecontrol value. Dose-related toxicity was seen over the remaining dose levels tested. In the presence of S9 metabolism, marked toxicity reducing RTG to 14% of the concurrent negative control value was noted at the highest dose level tested (68.0 μg/mL). Dose-related toxicity from moderate to slight was seen over the remaining dose levels tested.Mutation results: The induced mutation frequency (IMF) was lower than the global evaluation factor (GEF) following treatment with the test item at all concentration levels, both in the absence and presence of S9 metabolic activation.In Main Assay I, both in the absence and presence of S9 metabolism, and in Main Assay II, only in the presence of S9, a linear trend was indicated anda statistically significant increase in mutant frequency was observed at the highest dose level tested.However, the IMFs at these concentrations were lower than the GEF and the mutant frequencies observed were within the historical control range observed at RTC. Hence, these increases in mutant frequency were considered to be of no biological relevance.For the negative and positive controls, the number of wells containing small colonies and those containing large colonies were reported. The small and large colony mutant frequencies were estimated and the proportion of small mutant colonies was calculated. An adequaterecovery of small colony mutants was observed following treatment with the positive controls.The pH values and osmolality of the post-treatment media were determined. The addition of the test item solution did not have any obvious effect on the osmolality or pH of the treatment medium.

See Final Report

Conclusions:

Interpretation of results (migrated information):negative with and without metabolic activationIt is concluded that the test item does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro inthe absence or presence of S9 metabolic activation, under the reported experimental conditions.

Executive summary:

The test item (94.3% purity) was examined for mutagenic activity by assaying for the induction of 5-trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the absence and presence of S9 metabolic activation, using a fluctuation method. The test item was dissolved in dimethylsulfoxide (DMSO) at the concentration of 434 mg/mL, corresponding to 4340 μg/mL in the final treatment medium (0.01 M; upper limit indicated in the Study Protocol). A first cytotoxicity assay was performed, both in the absence and presence of S9 metabolic activation, the test item was assayed at a maximum dose level of 4340 μg/mL and at a wide range of lower dose levels: 2170, 1090, 543, 271, 136, 67.8, 33.9 and 17.0 μg/mL. In the absence of S9 metabolic activation, using the 3 hour treatment time, no cells survived treatment of 136 μg/mL or higher. At the next lower dose level of 67.8 μg/mL, severe toxicity was observed, reducing relative survival (RS) to 14% of the concurrent negative control value, while slight toxicity was noted over the remaining levels tested. Using the 24 hour treatment time, no cells survived to treatment at all dose levels tested, with the exception of 17.0 μg/mL were RS was reduced to 1%. Following treatment in the presence of S9 metabolic activation, using the short treatment time (3 hours), no cells survived treatment of 271 μg/mL or higher. Toxicity was seen over the remaining dose levels ranging from 1% to 66% RS of the concurrent negative control value. An additional cytotoxicity assay was performed using the 24 hour treatment time. The test item was assayed at a maximum dose level of 17.0 μg/mL and at a wide range of lower dose levels: 8.50, 4.25, 2.13, 1.06, 0.531, 0.266, 0.133 and 0.0664 μg/mL. Severe toxicity was seen at the two highest dose levels, while at the next lower dose level of 4.25 μg/mL, moderate toxicity was observed, reducing relative survival (RS) to 39% of the concurrent negative control value. No toxicity was observed over the remaining dose levels tested. Based on the results obtained in the preliminary trial, two independent assays for mutation at the TK locus were performed using the dose levels described in the following table:

Main Assay I (+/-S9): 68.0, 54.4, 43.5, 34.0, 17.0, 8.50 and 4.25 μg/mL

Main Assay II (-S9): 6.80, 5.23, 4.02, 2.01, 1.01 and 0.503

Main Assay II (+S9): 68.0, 45.3, 30.2, 20.1 and 13.0 μg/mL

Adequate levels of cytotoxicity, covering a range from the maximum to slight or no toxicity, were observed in all treatment series. No relevant increases in mutant frequencies were observed following treatment with the test item, in the absence or presence of S9 metabolism. Negative and positive control treatments were included in each mutation experiment in the absence and presence of S9 metabolism. The mutant frequencies in the solvent control cultures fell within the normal range. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system. It is concluded that the test item does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Based on the in vivo test result, no mutagenic effect were observed.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

other: experimental result from similar substance

Adequacy of study:

key study

Study period:

1992-03-17 to 1993-01-27

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: Winkelmann, Borchen, Germany- Age at study initiation: 8-12 weeks- Weight at study initiation: 28-43 g- Assigned to test groups randomly: yes- Housing: males: single; females: 3/cage (Macrolon type I cages)- Diet (ad libitum): ad libitum- Water (ad libitum): ad libitum- Acclimation period: at least one weekENVIRONMENTAL CONDITIONS- Temperature (°C): 22.5 to 23 °C- Humidity (%): 39 to 43%- Air changes (per hr): 10- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

intraperitoneal

Vehicle:

physiological saline

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:The test item was dissolved in physiological saline solution using sonication for 15 minutes and injected intraperitoneally at a volume of 10 mL/kg body weight

Duration of treatment / exposure:

Negative control: 24 hTest substance: 16, 24, and 48 hPositive control: 24 h

Frequency of treatment:

Single

Post exposure period:

N.A.

Remarks:

Doses / Concentrations:5 mg/kg body weightBasis:nominal conc.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide- Route of administration: intraperitoneal- Doses / concentrations: 20 mg/kg bw

Tissues and cell types examined:

Femoral bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The selection of the test item dose was based on a pilot test, in which groups of five animals, including both males and females, were intraperitoneally administered 1 mg/kg, 5 mg/kg, 7.5 mg/kg, 10 mg/kg, 50 mg/kg and 100 mg/kg bw of the test item. The following symptoms were recorded for up to 48 hours, starting at 5 mg/kg: apathy, roughened fur, staggering gait, sternal recumbency, spasm and difficulty in breathing. In addition, 3 of 5 animals died in the 7.5 mg/kg group and all animals died in the higher dose groups. Based on these results, 5 mg/kg test item was chosen as 1 MTD for this test.TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):Single administrationSampling time was: Test substance: 16, 24, and 48 hours after administration Negative control: 24 hours after administrationPositive Control: 24 hours after administrationDETAILS 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:1000 polychromatic erythrocytes per animal were scored for incidence of micronuclei. The number of cells with micronuclei was recorded, not the number of individual micronuclei. 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.In addition, 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. In this case, a second test had to be performed at the most sensitive interval.

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.

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 DEFINITIVE STUDYClinical signs: After a single intraperitoneal administration of 5 mg/kg test item, treated animals showed the following compound-related symptoms until sacrifice: apathy, roughened fur, staggering gait, spasm, difficulty in breathing and slitted eyes. Their feeding behavior was normal. One of 40 treated animals died during the test period, due to the acute toxicity of 5 mg/kg test item. No symptoms were recorded for the control groups. No animals died in these groups.Microscopic evaluation: Concerning the assessment of the clastogenic potential of the test item there were no relevant variation in results between males and females.Therefore, they were evaluated jointly.The ratio of polychromatic to normochromatic erythrocytes was altered by the treatment with the test item, being 1000: 811 (1s=208) in the negative control, 1000: 1770 (1s=652) in the 16 hours group, 1000: 1620 (1s=745) in the 24 hours group and 1000: 1210 (1s=892) in the 48 hours group. The results with the test item gave no relevant induction of micronucleated polychromatic erythrocytes after a single intraperitoneal treatment with 5 mg/kg. Similarly, the number of micronucleated normochromatic erythrocytes did not increase relevantly in any of the treatment groups.The positive control caused a clear increase in the number of micronucleated polychromatic erythrocytes.

Table 1: 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		1770* +/- 662		0.8 +/- 0.7		1.9 +/- 1.4
Test item_24 hours		10000		1620 +/- 745		1.1 +/- 0.5		1.9 +/- 1.4
Test item_48 hours		10000		1210 +/- 892		0.9 +/- 1.0		1.9 +/- 1.4
Positive control		10000		557 +/- 231		0.4 +/- 1.3		12.6* +/- 6.9

Concentration: test item: 5 mg/kg bw; positive control: 20 mg/kg bw

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

Conclusions:

Interpretation of results (migrated information): negativeThe 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 clatogenicity and aneugenicity in the mammalian erythrocyte micronucleus test.

Executive summary:

In a NMRI mouse bone marrow micronucleus assay (OECD 474), 5 animals/sex/dose were treated intraperitoneal with the test item (98.5%) at doses of 0 and 5 mg/kg bw. Bone marrow cells were harvested at 16, 24 and 48 hours post-treatment. The vehicle was physiological saline. There were signs of toxicity during the study such as apathy, roughened fur, staggering gait, spasm, difficulty in breathing and slitted eyes. One animal died during the test period. The test item was tested at an adequate dose based on the results from a dose range finding study. The positive control induced the appropriate response. There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time.

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)

Justification for classification or non-classification

Based on the both in vitro and in vivo tests, BY29 could be considered as not mutagen.