4H-Pyrazino[2,1-a]isoquinolin-4-one, 2-acetyl-1,2,3,6,7,11b-hexahydro-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An Ames Test according to OECD TG 471 was negative with and without S9 (reference 7.6.1 -1).

A gene mutation assay in mammalian cells (MLA Assay) according to OECD TG 476 was negative with S9 and weakly positive without S9 (reference 7.6.1 -2).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004-12-21 to 2005-05-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S-9 mix.

Test concentrations with justification for top dose:

Concentration range in the 1st main test (with and without metabolic activation): 5.00, 15.8, 50.0, 158, 500, 1580 and 5000 µg/plate
Concentration range in the 2nd main test (with and without metabolic activation): 50.0, 158, 500, 1580 and 5000 µg/plate

Vehicle / solvent:

Solvent: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

benzo(a)pyrene

cumene hydroperoxide

other: Daunomycin and N-ethyl-N'-nitro-N-nitrosoguanidine in the absence of S9 mix. 2-Aminoanthracene with S9 mix.

Rationale for test conditions:

According to the guideline

Evaluation criteria:

A test material is defined as non-mutagenic in this assay if:
- "no" or "weak increases" occur in the first and second series of the main experiment. ("Weak increases" randomly occur due to experimental variation.)

A test material is defined as mutagenic in this assay if:
- a dose-related (over at least two test material concentrations) increase in the number of revertants is induced, the maximal effect is a "clear increase", and the effects are reproduced at similar concentration levels in the same test system;
- "clear increases" occur at least at one test material concentration, higher concentrations show strong precipitation or cytotoxicity, and the effects are reproduced at the same concentration level in the same test system.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Table 1 Summary of mean revertant numbers, run 1

Test Material	Concentration [µg/plate]	+/- S9 Mix	Mean revertant colonies /
			TA 98	TA 100	TA 102
Solvent control	0	-	17	139	237
Test item	5	-	20	127	237
	15.8	-	23	127	221
	50	-	19	130	211
	158	-	20	133	266
	500	-	25	124	220
	1580	-	21	136	197
	5000	-	18	110	150
Solvent control	0	+	32	162	310
Test item	5	+	28	157	305
	15.8	+	32	159	307
	50	+	23	145	313
	158	+	28	149	318
	500	+	31	162	281
	1580	+	31	153	146
	5000	+	23	140	211
Positive controls	Name	-	DAUN	ENNG	CUM
	Conc [µg/plate]		4	5	200
	Revert. Colonies/plate		271	548	1126
	Name	+	2-AA	2-AA	B(a)P
	Conc [µg/plate]		2	2	10
	Revert. Colonies/plate		401	614	1659

Test Material	Concentration [µg/plate]	+/- S9 Mix	Mean revertant colonies /
			1535	1537	E. coli WP2 uvrA
Solvent control	0	-	18	6	43
Test item	5	-	24	3	41
	15.8	-	27	6	46
	50	-	27	5	44
	158	-	20	3	44
	500	-	17	4	46
	1580	-	24	7	43
	5000	-	19	11	23
Solvent control	0	+	24	8	47
Test item	5	+	25	7	55
	15.8	+	25	8	53
	50	+	23	8	44
	158	+	22	6	46
	500	+	21	6	49
	1580	+	23	7	43
	5000	+	16	2	28
Positive controls	Name	-	ENNG	9-AA	ENNG
	Conc [µg/plate]		10	50	5
	Revert. Colonies/plate		312	347	1027
	Name	+	2-AA	2-AA	2-AA
	Conc [µg/plate]		2	5	10
	Revert. Colonies/plate		215	265	407

Table 2 Summary of mean revertant numbers, run 2

Test Material	Concentration [µg/plate]	+/- S9 Mix	Mean revertant colonies /
			TA 98	TA 100	TA 102
Solvent control	0	-	17	132	211
Test item	50	-	20	130	222
	158	-	16	123	241
	500	-	16	125	221
	1580	-	16	126	187
	5000	-	11	105	154
Solvent control	0	+	27	189	207
Test item	50	+	27	184	194
	158	+	31	197	193
	500	+	23	191	189
	1580	+	21	182	184
	5000	+	21	161	170
Positive controls	Name	-	DAUN	ENNG	CUM
	Conc [µg/plate]		4	5	200
	Revert. Colonies/plate		522	597	1077
	Name	+	2-AA	2-AA	B(a)P
	Conc [µg/plate]		2	2	10
	Revert. Colonies/plate		113	180	568

Test Material	Concentration [µg/plate]	+/- S9 Mix	Mean revertant colonies /
			1535	1537	E. coli WP2 uvrA
Solvent control	0	-	19	7	47
Test item	50	-	20	5	38
	158	-	16	8	44
	500	-	18	9	37
	1580	-	21	6	37
	5000	-	15	12	31
Solvent control	0	+	23	10	50
Test item	50	+	23	11	53
	158	+	28	12	49
	500	+	23	9	41
	1580	+	23	7	42
	5000	+	16	7	32
Positive controls	Name	-	ENNG	9-AA	ENNG
	Conc [µg/plate]		10	50	5
	Revert. Colonies/plate		436	391	910
	Name	+	2-AA	2-AA	2-AA
	Conc [µg/plate]		2	10	10
	Revert. Colonies/plate		120	209	152

DAUN: Daunomycin

ENNG: N-Ethyl-N'-nitro-N-nitroso-guanidine

CUM: Cumene hydroperoxide

2-AA: 2-Aminoanthracene

B(a)p: Benzo(a)pyrene

9-AA: 9-Aminoacridine

Conclusions:

Under the conditions of this assay the test item was not mutagenic to bacterial cells.

Executive summary:

The investigations for the mutagenic potential of the test material were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA according to OECD TG 471. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed. In the two series with S9 mix, 10 or 30 % S9 in the S9 mix were used in the 1st and 2nd series, respectively. The test item was dissolved in dimethyl sulfoxide (DMSO) and tested at concentrations ranging from 5.00 to 5000 µg/plate. Precipitation of the test material on the agar plates did not occur. Toxicity to the bacteria was not observed. Daunomycin, N-ethyl-N'-nitro-N-nitrosoguanidine, 9-aminoacridine and cumene hydroperoxide served as strain specific positive control test materials in the absence of S9 mix. 2-Aminoanthracene and benzo[a]pyrene were used for testing the bacteria and the activity of the S9 mix. Each treatment with the test materials used as positive controls led to a clear increase in revertant colonies, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used. With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005-01-31 to 2005-06-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

thymidine kinase (TK +/-)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 with heat-inactivated horse serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

- S9 mix: 15.8 , 50.0, 158, 500, 889 and 1580 µg per mL medium
+ S9 mix: 15.8 , 50.0, 158, 500, and 1580 µg per mL medium

Vehicle / solvent:

Dimethyl sulfoxide (DMSO), routinely used, fits purpose

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

7,12-dimethylbenzanthracene

Details on test system and experimental conditions:

The test material was assayed for its ability to induce mutations at the TK locus (5-trifluorothymidine resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of two independent experimental series, each conducted in the absence and presence of an exogenous metabolizing system (S9 mix from livers of rats pretreated with Aroclor 1254). The exposure time in the first experimental series was 3 hours in the presence and 24 hours in the absence of S9 mix. In the analysis, colony sizing was performed in order to discriminate between large and small colonies. Molecular analysis has indicated that the large colonies tend to represent events within the gene (base-pair substitutions and deletions) whereas small colony mutants often involve large genetic changes frequently visible as chromosome aberrations (Applegate et al., 1990; Moore et al., 1985; Thacker, 1985). Thus, in this system, gene mutations within the TK gene (11-13 kilobases) and chromosomal events involving the gene may be detected.

Rationale for test conditions:

According to the guideline

Evaluation criteria:

The effects of the test material upon the mutation frequency are defined as
- "No effect" or "no increase" in the mutation frequency if the mean frequency of the parallel incubations of a given test material concentration is less than 2.0-fold above the mean of the actual negative controls or the mean mutation falls within the historical range of the negative controls.
- "Clear effect" or "clear increase" in the mutation frequency if the test material induces at least a 3.0-fold increase above the mean of the actual negative controls and the mean mutation frequency for a given test material concentration is at least 1.5-fold above the highest value of the historical negative controls.

All other results are defined as a "weak effect" or a "weak increase" of the mutation frequency.

Test materials are assessed as negative or non-mutagenic in this test system if:
- the assay is considered valid and
- no effect (no increase in the mutation frequency) occurs in the two experimental series performed or
- a weak effect (weak increase) occurs in one series and no effect (no increase) in the other series of experiments.

Test materials are assessed as positive or mutagenic in this test system if:
- the assay is considered valid and
- a clear effect (clear increase in the mutation frequency) occurs at similar concentrations of the test material in the two experimental series performed, or
- a clear effect (clear increase) occurs in one series and a weak effect (weak increase) in the other series of experiments at identical concentrations, or
- weak effects (weak increases) occur dose-dependently (over at least two test material concentrations) and reproducibly at identical concentrations in the two experimental series performed.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

in the cytotoxic dose range

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(>= 500 µg/mL)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(>= 500 µg/mL)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no effects
- Effects of osmolality: no effects
- Evaporation from medium: no
- Water solubility: soluble
- Precipitation: not observed
- Other confounding effects: no

RANGE-FINDING/SCREENING STUDIES:

COMPARISON WITH HISTORICAL CONTROL DATA: negative controls are within the historical range

ADDITIONAL INFORMATION ON CYTOTOXICITY: Toxicity to the cells was observed at concentrations >=500 µg/mL

Conclusions:

From the results of this study it is concluded that the test item is weakly mutagenic in this test system in the cytotoxic dose range without external metabolic activation from S9-mix.

Executive summary:

The test material was assayed for its ability to induce mutations at the TK locus (5-trifluorothymidine resistance) in mouse lymphoma cells using a fluctuation protocol according to OECD TG 476. The study consisted of two independent experimental series, each conducted in the absence and presence of an exogenous metabolizing system (S9 mix from livers of rats pretreated with Aroclor 1254). The exposure time in the first experimental series was 3 hours in the presence and 24 hours in the absence of S9 mix.
In the analysis, colony sizing was performed in order to discriminate between large and small colonies. Molecular analysis has indicated that the large colonies tend to represent events within the gene (base-pair substitutions and deletions) whereas small colony mutants often involve large genetic changes frequently visible as chromosome aberrations (Applegate et al., 1990; Moore et al., 1985; Thacker, 1985). Thus, in this system, gene mutations within the TK gene (11-13 kilobases) and chromosomal events involving the gene may be detected.
The following concentrations have been investigated: without S9 mix: 15.8 , 50.0, 158, 500, 889 and 1580 µg per mL medium with S9 mix: 15.8 , 50.0, 158, 500, and 1580 µg per mL medium. Precipitation of the test material in the incubation medium was not observed. Clear cytotoxic effects, i.e. a relevant decrease in either the % relative survival or % total growth of the test cells, occurred at concentrations 500 µg/mL, in the absence of S9 mix. The doses tested were selected to determine viability and mutagenicity (5 -trifluorothymidine (TFT) resistance) 2 days after treatment. Negative (solvent) and positive control treatments were included in each mutation experiment in the absence and presence of S9 mix. Mutant frequencies in negative control cultures fell within normal ranges, and clear increases in mutation were induced by the positive control chemicals 4 -nitroquinoline N-oxide (without S9 mix) and 7,12 -dimethylbenz[a]anthracene (with S9 mix). Therefore, the study was accepted as valid. The test item increased the mutant frequency in the two experimental series performed in the absence of S9 mix at concentrations of 889 µg/mL and 1180 µg/mL. At this concentration level, less than 29 % viability (% relative total growth) of the test cells was determined. In addition, the frequency of small to large colony counts was reproducibly increased in this dose range. No increased the mutant frequency was seen in the presence of S9 mix.
From the results of this study it is concluded that the test item is weakly mutagenic in this test system in the cytotoxic dose range without external metabolic activation from S9 -mix.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

A micronucleus test in vivo according to OECD 475 was negative (reference 7.6.2 -1).

Link to relevant study records

Reference

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005-11-14 to 2006-07-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH Tripartite Harmonised Guideline on Genotoxicity: Specific Aspects of Regulatory Tests

Version / remarks:

1995

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian bone marrow chromosome aberration test

Species:

mouse

Strain:

CD-1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: young adult; 5-7 weeks
- Weight at study initiation: 26-32 g (DRF), 25-33 g (main study); group weights differed from the overall mean by no more than 5%
- Assigned to test groups randomly: yes, under following basis: system of random numbers
- Fasting period before study: no
- Housing: in cages that conform to the Code of Practice for the housing and care of animals used in scientific procedures
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25°C
- Humidity: 40-70%.
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: methyl cellulose
- Justification for choice of solvent/vehicle: not toxic to mice, good application profile
- Concentration of test material in vehicle: 12.5 - 75 mg/mL, depending on dose level
- Constant application volume of 5 mL/kg bw

Details on exposure:

In the dose-range finding study the test article was administered on a single occasion to groups of three male mice at doses of 500 and 750 mg/kg.
In the main study, the test article was administered on a single occasion to groups of six male mice at doses of 125, 250 and 500 mg/kg.

Duration of treatment / exposure:

single administration

Frequency of treatment:

once

Post exposure period:

24 and 48 h

Dose / conc.:

125 mg/kg bw/day (actual dose received)

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

Male: 125 mg/kg; No. of animals: 6; Sacrifice time: 24 hours
Male: 250 mg/kg; No. of animals: 6; Sacrifice time: 24 hours
Male: 500 mg/kg; No. of animals: 6; Sacrifice time: 24 hours
Male: 500 mg/kg; No. of animals: 6; Sacrifice time: 48 hours

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamid (CPS), 40 mg/kg, dissolved in saline at 2 mg/mL.

Tissues and cell types examined:

bone marow smeares from both femurs

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: pre-test

DETAILS OF SLIDE PREPARATION:
Both femurs from each animal were exposed, removed, cleaned of adherent tissue and the ends removed from the shanks. Using a syringe and needle, bone marrows were flushed from the marrow cavity with 1 mL foetal calf serum into appropriately labelled centrifuge tubes (one per animal).

A further 2 mL of foetal calf serum was added and the tubes centrifuged and the serum was aspirated to leave one or two drops and the cell pellet. The pellet was mixed into this small volume of serum in each tube and from each tube a small volume of suspension was placed on the end of each of two slides labelled with the appropriate study number, sampling time, sex, date of preparation and animal number. The latter served as a code so analysis could be conducted "blind". A smear was made from the drop by drawing the end of a clean slide along the labelled slide.

Slides were allowed to air-dry and were fixed for 5 minutes in absolute methanol, followed by rinsing several times in water. One slide from each set of two was then taken, the other was kept in reserve. After a second fixing/rinsing procedure, slides were stained for 10 minutes in filtered Giemsa stain diluted 1:6 (v/v) in distilled water. Stained slides were rinsed, and allowed to dry thoroughly before clearing in xylene for 3 minutes. When dry, the slides were mounted with coverslips.

METHOD OF ANALYSIS:
Slides from the CPA-treated mice were initially checked to ensure the system was operating satisfactorily. The slides from all control and dose groups were arranged in numerical order by sampling time and analysed by a person not connected with the dosing phase of the study.

Initially the relative proportions of polychromatic erythrocytes (PCE), seen as pale blue or blue/grey enucleate cells, and normochromatic erythrocytes (NCE), seen as smaller yellow/orange-stained enucleate cells, were determined until a total of at least 1000 cells (PCE plus NCE) had been analysed.

Counting continued (but of PCE only) until at least 2000 PCE had been observed. All PCE containing micronuclei observed during these two phases of counting were recorded. The vernier coordinates of all cells containing micronuclei were recorded to a maximum of six per 2000 cells scored.

After completion of microscopic analysis and decoding of the data, the ratio of PCE to NCE (expressed as %PCE) for each animal and the mean for each group was calculated. The individual and group mean frequency of micronucleated PCE ± standard deviation (%MNPCE) were also determined.

%PCE values were examined to see if there was any decrease in groups of treated animals that could be taken as evidence of bone marrow toxicity.

The frequencies of micronucleated PCE in vehicle control animals were compared with the historical negative control data to determine whether or not the assay was acceptable. For each group, inter-individual variation in the numbers of micronucleated PCE was estimated by means of a heterogeneity Chi2 test.

The numbers of micronucleated PCE in each treated group were then compared with the numbers in vehicle control groups by using a 2 x 2 contingency table to determine Chi2. Probability values of P<=0.05 were to be accepted as significant. A further statistical test (for linear trend) was used to evaluate possible dose-response relationships.
If the heterogeneity Chi2 test provided evidence of significant (p <= 0.05) variability between animals within at least one group, non-parametric analysis was more appropriate and provision made to use the Wilcoxon rank sum test.

Additional statistical analysis was not required in this study.

Evaluation criteria:

The data were evaluated as to whether exposure to the test article was associated with:
1. a statistically significant increase in the frequency of micronucleated PCE occurring at one or more dose levels.

2. an incidence and distribution of micronucleated PCE at such a point that exceeded the laboratory's historical vehicle control data.

3. a dose-response trend in the proportion of micronucleated PCE (where more than two dose levels were analysed).

The test article was to be considered positive in this assay if all of the above criteria were met.

The test article was to be considered negative in this assay if none of the above criteria were met.

Data that do not fall into either of the above categories were to be judged on a case by case basis. Evidence of a dose-response is considered useful but not essential in the evaluation of a positive result. Biological relevance is taken into account, for example consistency of response within and between dose levels and any confirmatory experiments.

Statistics:

The frequencies of micronucleated PCE in vehicle control animals were compared with the historical negative control data to determine whether or not the assay was acceptable. For each group, inter-individual variation in the numbers of micronucleated PCE was estimated by means of a heterogeneity Chi2 test.

The numbers of micronucleated PCE in each treated group were then compared with the numbers in vehicle control groups by using a 2 x 2 contingency table to determine Chi2. Probability values of P<=0.05 were to be accepted as significant. A further statistical test (for linear trend) was used to evaluate possible dose-response relationships.
If the heterogeneity Chi2 test provided evidence of significant (p <= 0.05) variability between animals within at least one group, non-parametric analysis was more appropriate and provision made to use the Wilcoxon rank sum test.

Additional statistical analysis was not required in this study.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Doses producing toxicity: Clinical signs at 500 mg/kg.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Clinical signs including lethargy, palpebral closure, bradypnoea and transient unconsciousness were observed in all animals receiving 500 mg/kg indicating that the animals were systemically exposed to substantial amounts of the test material and that 500 mg/kg was a suitable estimate of the maximum tolerated dose.

Negative (vehicle) control mice exhibited group mean frequencies of polychromatic erythrocytes (PCE) to normochromatic erythrocytes (NCE); ratio expressed as %PCE that were within (48-hour data), or slightly below (24 hour data) historical vehicle control (normal) values. Individual frequencies of micronucleated PCE (MN PCE) in the vehicle control groups were consistent with historical vehicle distribution data.

Mice treated with the test material exhibited group mean %PCE values that were similar to the concurrent vehicle control and which were within (or close to) the historical negative control (normal) range for all dose groups. These data did not represent evidence of bone marrow toxicity (as may be evidenced by a decrease in %PCE compared to concurrent control values).

Group mean frequencies of micronucleated PCE were similar to, and not statistically different from those seen in the concurrent vehicle control group for all dose groups (24 + 48 hour data). Individual frequencies of MN PCE for all treated animals were consistent with historical vehicle control distribution data.

Conclusions:

It is concluded that the test item did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of male mice treated up to 500 mg/kg (considered a suitable estimate of the maximum tolerated dose in the mouse under the experimental conditions employed). Thus, the test item was not genotoxic in vivo in the test system described.

Executive summary:

The test material was assayed in vivo in a mouse bone marrow micronucleus test at three dose levels according to OECD TG 475. The choice of dose levels was based on an initial toxicity range-finding study in which the test material, formulated in 0.5% (wlv) methyl cellulose (0.5% MC) was administered to mice via intraperitoneal injection. The test article was administered on a single occasion to groups of three male mice at doses of 500 and 750 mg/kg. Observations were made over a 2 day period following administration and signs of toxicity recorded. The negative (vehicle) control in the study was 0.5% MC also administered via intraperitoneal injection on a single occasion. Two groups of six male mice treated with this were killed and sampled 24 or 48 hours after administration. Cyclophosphamide (CPA), the positive control, was dissolved in saline and administered via intraperitoneal injection as a single dose of 40 mg/kg to a group of six male mice which were killed after 24 hours. In the main study, the test item was formulated as described and administered at 125, 250 and 500 mg/kg to groups of six male mice killed 24 or 48 hours after dose administration. Clinical signs including lethargy, palpebral closure, bradypnoea and transient unconsciousness were observed in all animals at a dose level of  500 mg/kg indicating that the animals were systemically exposed to substantial amounts and that 500 mg/kg was a suitable estimate of the maximum tolerated dose.
Negative (vehicle) control mice exhibited group mean frequencies of polychromatic erythrocytes (PCE) to normochromatic erythrocytes (NCE); ratio expressed as %PCE that were within (48-hour data), or slightly below (24 hour data) historical vehicle control (normal) values. Individual frequencies of micronucleated PCE (MN PCE) in the vehicle control groups were consistent with historical vehicle distribution data.
Positive control animals exhibited increased numbers of micronucleated PCE such that the frequency in the positive control group was significantly greater than in the concurrent controls with all animals exhibiting marked increases in numbers of MN PCE.
The assay system was therefore considered as both sensitive and valid. Mice treated with the test material exhibited group mean %PCE values that were similar to the concurrent vehicle control and which were within (or close to) the historical negative control (normal) range for all dose groups. Group mean frequencies of micronucleated PCE were similar to, and not statistically different from those seen in the concurrent vehicle control group for all dose groups (24 + 48 hour data). Individual frequencies of MN PCE for all test item treated animals were consistent with historical vehicle control distribution data.

It is concluded that the test material did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of male mice treated up to 500 mg/kg (considered a suitable estimate of the maximum tolerated dose in the mouse under the experimental conditions employed). Thus, the test item was not genotoxic in vivo in the test system described.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Amest Test

The investigations for the mutagenic potential of the test material were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA according to OECD TG 471. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed. In the two series with S9 mix, 10 or 30 % S9 in the S9 mix were used in the 1st and 2nd series, respectively. The test item was dissolved in dimethyl sulfoxide (DMSO) and tested at concentrations ranging from 5.00 to 5000 µg/plate. Precipitation of the test material on the agar plates did not occur. Toxicity to the bacteria was not observed. Daunomycin, N-ethyl-N'-nitro-N-nitrosoguanidine, 9-aminoacridine and cumene hydroperoxide served as strain specific positive control test materials in the absence of S9 mix. 2-Aminoanthracene and benzo[a]pyrene were used for testing the bacteria and the activity of the S9 mix. Each treatment with the test materials used as positive controls led to a clear increase in revertant colonies, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used. With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.

MLA

The test material was assayed for its ability to induce mutations at the TK locus (5-trifluorothymidine resistance) in mouse lymphoma cells using a fluctuation protocol according to OECD TG 476. The study consisted of two independent experimental series, each conducted in the absence and presence of an exogenous metabolizing system (S9 mix from livers of rats pretreated with Aroclor 1254). The exposure time in the first experimental series was 3 hours in the presence and 24 hours in the absence of S9 mix.
In the analysis, colony sizing was performed in order to discriminate between large and small colonies. Molecular analysis has indicated that the large colonies tend to represent events within the gene (base-pair substitutions and deletions) whereas small colony mutants often involve large genetic changes frequently visible as chromosome aberrations (Applegate et al., 1990; Moore et al., 1985; Thacker, 1985). Thus, in this system, gene mutations within the TK gene (11-13 kilobases) and chromosomal events involving the gene may be detected.
The following concentrations have been investigated: without S9 mix: 15.8 , 50.0, 158, 500, 889 and 1580 µg per mL medium with S9 mix: 15.8 , 50.0, 158, 500, and 1580 µg per mL medium. Precipitation of the test material in the incubation medium was not observed. Clear cytotoxic effects, i.e. a relevant decrease in either the % relative survival or % total growth of the test cells, occurred at concentrations 500 µg/mL, in the absence of S9 mix. The doses tested were selected to determine viability and mutagenicity (5 -trifluorothymidine (TFT) resistance) 2 days after treatment. Negative (solvent) and positive control treatments were included in each mutation experiment in the absence and presence of S9 mix. Mutant frequencies in negative control cultures fell within normal ranges, and clear increases in mutation were induced by the positive control chemicals 4 -nitroquinoline N-oxide (without S9 mix) and 7,12 -dimethylbenz[a]anthracene (with S9 mix). Therefore, the study was accepted as valid. The test item increased the mutant frequency in the two experimental series performed in the absence of S9 mix at concentrations of 889 µg/mL and 1180 µg/mL. At this concentration level, less than 29 % viability (% relative total growth) of the test cells was determined. In addition, the frequency of small to large colony counts was reproducibly increased in this dose range. No increased the mutant frequency was seen in the presence of S9 mix. From the results of this study it is concluded that the test item is weakly mutagenic in this test system in the cytotoxic dose range without external metabolic activation from S9 -mix.

MNT, in vivo

The test material was assayed in vivo in a mouse bone marrow micronucleus test at three dose levels according to OECD TG 475. The choice of dose levels was based on an initial toxicity range-finding study in which the test material, formulated in 0.5% (wlv) methyl cellulose (0.5% MC) was administered to mice via intraperitoneal injection. The test article was administered on a single occasion to groups of three male mice at doses of 500 and 750 mg/kg. Observations were made over a 2 day period following administration and signs of toxicity recorded. The negative (vehicle) control in the study was 0.5% MC also administered via intraperitoneal injection on a single occasion. Two groups of six male mice treated with this were killed and sampled 24 or 48 hours after administration. Cyclophosphamide (CPA), the positive control, was dissolved in saline and administered via intraperitoneal injection as a single dose of 40 mg/kg to a group of six male mice which were killed after 24 hours. In the main study, the test item was formulated as described and administered at 125, 250 and 500 mg/kg to groups of six male mice killed 24 or 48 hours after dose administration. Clinical signs including lethargy, palpebral closure, bradypnoea and transient unconsciousness were observed in all animals at a dose level of  500 mg/kg indicating that the animals were systemically exposed to substantial amounts and that 500 mg/kg was a suitable estimate of the maximum tolerated dose.
Negative (vehicle) control mice exhibited group mean frequencies of polychromatic erythrocytes (PCE) to normochromatic erythrocytes (NCE); ratio expressed as %PCE that were within (48-hour data), or slightly below (24 hour data) historical vehicle control (normal) values. Individual frequencies of micronucleated PCE (MN PCE) in the vehicle control groups were consistent with historical vehicle distribution data.
Positive control animals exhibited increased numbers of micronucleated PCE such that the frequency in the positive control group was significantly greater than in the concurrent controls with all animals exhibiting marked increases in numbers of MN PCE.
The assay system was therefore considered as both sensitive and valid. Mice treated with the test material exhibited group mean %PCE values that were similar to the concurrent vehicle control and which were within (or close to) the historical negative control (normal) range for all dose groups. Group mean frequencies of micronucleated PCE were similar to, and not statistically different from those seen in the concurrent vehicle control group for all dose groups (24 + 48 hour data). Individual frequencies of MN PCE for all test item treated animals were consistent with historical vehicle control distribution data.

It is concluded that the test material did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of male mice treated up to 500 mg/kg (considered a suitable estimate of the maximum tolerated dose in the mouse under the experimental conditions employed). Thus, the test item was not genotoxic in vivo in the test system described.

Overall conclusion

The test item revealed a weakly positive (mutagenic) result in the MLA study. However, the positive findings were only observed at cytotoxic concentrations and thus have to be treated with caution. Moreover, a micronucleus test in vivo confirmed that there is no concern in regards to clastogenicity with the test item.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genotoxicity, the test item does not require classification as mutagenic according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation (EU) 2019/521.