2,6-dimethylmorpholine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames-Test: negative (OECD 471)
Chromosomal Aberration Test: negative (OECD 473)
HPRT-Test: negative (OECD 476)

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

no E.coli strain tested

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S9-mix

Test concentrations with justification for top dose:

20, 100, 500, 2500, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: test substance is soluble in water

Untreated negative controls:

yes

Remarks:

sterility control

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoantracene, N-methyl-N'-nitro-N-nitroso-guanidine, 4-nitro-phenylenediamine, 9-aminoacridine hydrochloride

Remarks:

positive control substances depending on metabolic activation conditions and tester strain

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

Evaluation criteria:

In general, a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results

Statistics:

no data

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test substance was completely soluble.
- Precipitation: No precipitation was noted at any concentration.

Standart plate test:

Dose (µg/plate)	TA1535		TA100		TA1537		TA98
	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9
0	17±3	16±3	114±4	132±11	9±2	10±2	27±4	43±8
20	17±3	16±4	104±7	123±13	9±3	10±1	27±3	37±6
100	18±1	15±2	113±7	122±11	7±2	8±2	27±3	30±4
500	18±3	13±3	108±4	128±8	9±2	9±3	31±4	31±6
2500	19±1	14±3	113±9	129±9	8±1	10±3	28±6	26±2
5000	23±6	16±2	131±9	132±6	8±2	8±2	24±4	32±5
2-AA	-	106±8	-	1177±93	-	128±24	-	827±104
MNNG	1623±240	-	1687±95	-	-	-	-	-
AAC	-	-	-	-	571±36	-	-	-
NPD	-	-	-	-	-	-	961±131	-

 

 

Preincubation-test:

Dose (µg/plate)	TA1535		TA100		TA1537		TA98
	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9
0	19±1	17±4	102±10	105±23	12±1	13±5	30±4	38±2
20	22±4	21±2	114±8	122±14	9±2	14±2	33±8	34±9
100	19±3	19±3	131±7	122±18	13±3	10±3	29±6	43±8
500	23±3	17±4	131±11	131±15	17±4	12±5	26±2	44±4
2500	27±3	24±3	143±6	124±7	13±1	9±4	28±11	44±8
5000	28±6	28±3	98±11	123±7	14±4	12±4	25±3	48±6
2AA	-	88±9	-	559±17	-	88±7	-	341±9
MNNG	899±7	-	698±22	-	-	-	-	-
AAC	-	-	-	-	653±88	-	-	-
NPD	-	-	-	-	-	-	756±32	-

 

2-AA: 2-aminoanthracene;

MNNG; N-methyl-N-nitro-N-nitrosoguanidine

NPD: 4-nitro-o-phenylendiamine

AAC: 9-aminoacridine chloride monohydrate

 

 

Conclusions:

No increase in the number of revertants was observed with any strain under any test conditions. No bacteriotoxicity (indicated by reduced number of revertants) was observed. The positive controls led to the expected increase of revertants.



According to the results of the present study, the test substance is not mutagenic in the Ames test under the experimental conditions chosen here.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006-11-20 to 2007-04-03

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)

Version / remarks:

(1997-07-21)

Qualifier:

according to guideline

Guideline:

other: EC Directive 2000/32, B.17 (19 May 2000)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

(Aug 1998)

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

X-linked hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary (CHO) cells

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Additional strain / cell type characteristics:

other: substrain K1

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9-mix

Test concentrations with justification for top dose:

1st Experiment (4-hour exposure period)
without S9-mix: 0, 75, 150, 300, 600, 1200 μg/mL
with S9-mix: 0, 75, 150, 300, 600, 1200 μg/mL

2nd Experiment (4-hour exposure period)
without S9-mix: 0; 400, 600, 800, 1000, 1200 μg/mL
with S9-mix: 0; 400, 600, 800, 1000, 1200 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: culture medium (Ham's F12)
- Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in water, culture medium (Ham's F12) was selected as vehicle.

All media were supplemented with 1% (v/v) penicillin/streptomycin (stock solution: 10 000 μg/mL) and 1% (v/v) amphotericine B (stock solution: 250 μg/mL)
Treatment medium: Ham's F12 medium containing L-glutamine source and hypoxanthine
Culture medium: Ham's F12 medium containing L-glutamine source and hypoxanthine supplemented with 10% (v/v) fetal calf serum (FCS).
Pretreatment medium ("HAT" medium): Ham's F12 medium supplemented with: hypoxanthine, aminopterin, thymidine, fetal calf serum (FCS)
Selection medium ("TG" medium): Hypoxanthine-free Ham's F12 medium supplemented with: 6-thioguanine, L-glutamine, fetal calf serum (FCS)

Untreated negative controls:

yes

Remarks:

historical

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: ethyl methanesulfonate (EMS) without metabolic activation, methylcholanthrene (MCA) with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 20 - 24 hours
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 3 times to first passage, second passage after 6 - 8 days total expression period
- Selection time (if incubation with a selection agent): 6 - 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 16 days

NUMBER OF REPLICATIONS: 6

Statistics:

Due to the clearly negative findings, a statistical evaluation was not carried out.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
Pretests were carried out with concentrations of 1, 5, 10, 50, 100, 500, 1000, 2500, and 5000 µg/mL, 4-hour exposure with and without S-9, 2 replicates per dose.

Table: Summary of results

 

Exp.	Test groups	S9 mix	Prec.^*	Genotoxicity** MFcorr. [per 106 cells]	Cytotoxicity***
					CE1 [%]	CE2 [%]
1	Negative control 75μg/mL 150μg/mL 300μg/mL 600μg/mL 1200μg/mL Positive control^1	- - - - - -	- - - - - -	2.24 3.82 1.96 0.00 0.00 2.01 68.09	100.0 88.2 79.7 93.7 79.5 93.7 90.4	100.0 93.8 89.1 83.7 86.4 93.4 71.3
1	Negative control 75μg/mL 150μg/mL 300μg/mL 600μg/mL 1200μg/mL Positive control^2	+ + + + + +	- - - - - -	1.57 0.92 0.33 1.73 1.04 3.10 115.20	100.0 100.7 111.4 89.4 93.0 85.1 94.4	100.0 99.5 103.4 97.8 91.7 86.7 90.4
2	Negative control 400μg/mL 600μg/mL 800μg/mL 1000μg/mL 1200μg/mL Positive control^1	- - - - - -	- - - - - -	0.63 0.29 0.00 0.00 0.63 0.79 87.69	100.0 93.0 95.5 92.3 104.8 99.1 90.9	100.0 84.8 81.0 83.5 85.2 78.2 60.7
2	Negative control 400μg/mL 600μg/mL 800μg/mL 1000μg/mL 1200μg/mL Positive control^2	+ + + + + +	- - - - - -	0.74 0.00 1.31 0.00 1.95 0.00 72.92	100.0 92.3 112.5 86.4 114.2 123.0 91.9	100.0 93.5 112.9 89.0 84.5 91.6 103.1

* Precipitation in culture medium at the end of exposure period

**Mutant frequency MF_corr : number of mutant colonies per 10⁶cells corrected with the CE₂ value

*** Cloning efficiency related to the respective negative control

¹ : EMS 300 μg/mL         

² : MCA 10 μg/mL

 

 

The negative controls gave mutant frequencies within the range expected for the CHO cell line. Both of the positive control substances, i.e.EMS and MCA, led to the expected increase in the frequencies of forward mutations.

Conclusions:

On the basis of the results of the present study, the test substance did not cause any increase in the mutant frequencies either without S9 mix or after adding a metabolizing system in two experiments performed independently of each other.



Thus, under the experimental conditions of this study, the test substance is considered not to be a mutagenic substance under in vitro conditions in the HPRT locus assay using CHO cells in vitro in the CHO/HPRT forward mutation assay in the absence and the presence of metabolic activation.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006-12-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

(21 Jul 1997)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

(19 May 2000)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

(Aug 1998)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S-9

Test concentrations with justification for top dose:

1st Experiment
4-hour exposure, 18-hour sampling time, without S9 mix: 0; 150; 300; 600; 1200 μg/mL
4-hour exposure, 18-hour sampling time, with S9 mix: 0; 150; 300; 600; 1200 μg/mL

2nd Experiment
18-hour exposure, 18-hour sampling time, without S9 mix: 0; 37.5; 75; 150; 300; 600 μg/mL
18-hour exposure, 28-hour sampling time, without S9 mix: 0; 75; 150; 300; 600 μg/mL
4-hour exposure, 28-hour sampling time, with S9 mix: 0; 150; 300; 600; 1200 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: culture medium
MEM (minimal essential medium with Earle's salts) containing a L-glutamine source supplemented with 10% (v/v) fetal calf serum (FCS), 1% (v/v) penicillin/streptomycin (10 000 IU / 10 000 μg/mL), 1% (v/v) amphotericin B (250 μg/mL).
During exposure to the test substance (only 4-hour treatment), MEM medium was used without FCS supplementation.
- Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in water, the culture medium (MEM: Minimal Essential Medium) was selected as vehicle.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: ethylmethanesulphonate, cycloposphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4, 18 hours
- Sampling time: 18, 28 hours

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 metaphases per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (scoring of 1000 cells/culture)

OTHER EXAMINATIONS:
- Determination of polyploidy: polyploidy index (scoring of 1000 metaphases/culture)

OTHER:
determination of: pH value, osmolarity, solubility

Evaluation criteria:

Acceptance criteria
The V79 in vitro cytogenetic assay is considered valid if the following criteria are met:
- The quality of the slides must allow the identification and evaluation of a sufficient number of analyzable metaphases.
- The numbers of cells with structural/numerical aberrations in the negative control has to be within the range of the historical negative control data.
- The positive control substances both with and without S9 mix have to induce a distinct increase of structural chromosome aberrations.

Assessment criteria
The test substance is assessed as “positive” if the following criteria are met:
- A statistically significant, dose-related and reproducible increase in the number of cells with structural chromosome aberrations (excl. gaps).
- The number of aberrant cells (excl. gaps) exceeds both the concurrent negative/vehicle control value and the historical negative control data range.
A test substance generally is considered as “negative” if the following criteria are met:
- The number of cells with structural aberrations (excl. gaps) in the dose groups is not statistically significant increased above the concurrent negative/vehicle control value and is within the historical negative control data range.

Statistics:

The statistical evaluation of the data was carried out using the MUCHAN program system (internal system).
The proportion of metaphases with structural aberrations was calculated for each group. A comparison of each dose group with the negative control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test was Bonferroni-Holm corrected versus the dose groups separately for each time and was performed one-sided. Statistical significance: * p < 0.05, ** p < 0.01.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Remarks:

not clastogenic at any concentration, but aneugenic at high concentration

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no effect
- Effects of osmolarity: no effect
- Water solubility: yes
- Precipitation: no

RANGE-FINDING/SCREENING STUDIES:
Following the requirements of the current guidelines a test substance should be tested up to a maximum concentration of 5 mg/mL, 5 μL/mL or 10 mM, which is the lowest. In case of toxicity, the top dose should reduce at least one of the toxicity parameters more than 50% of the respective control. For relatively insoluble test substances at least one concentration should be scored showing no precipitation in culture medium at the end of exposure period.
In the pretest the parameter osmolarity was not relevantly influenced by the addition of the test substance preparation to the culture medium at the concentrations measured. However, a pH shift was observed at the highest applied concentration prior to testing. Therefore, the pH of the stock solution was adjusted to a physiological value prior to application using small amounts of 16% (w/v) HCl.
In addition, no test substance precipitation in culture medium was observed up to the highest required concentration of 1200 μg/mL.
After 4 hours treatment in the absence and presence of S9 mix no cytotoxicity indicated by reduced cell numbers/mitotic indices of about or below 50% of control was observed.
In contrary, in the pretest with 18 hours continuous treatment in the absence of S9 mix, the mitotic indices were clearly reduced after treatment with 600 μg/mL.

Clastogenic mode of action

First experiment:

After a treatment time of 4 hours no increase in the number of chromosomally damaged cells was observed either without S9 mix or after the addition of a metabolizing system.

 

Second experiment:

Both with and without S9 mix, no increase in the number of structurally damaged metaphases was observed either after a treatment time of 4 hours or after a continuous treatment of 18 hours at both sampling times, i.e. 18 hours and 28 hours.

 

Aneugenic mode of action

First experiment:

No relevant increase in the number of cells with changes in the number of chromosomes was demonstrated either without S9 mix or after the addition of a metabolizing system.

 

Second experiment:

No increase in the number of cells with changes in the number of chromosomes was demonstrated after a continuous treatment of 18 hours at 18-hour sampling time without S9 mix and after a treatment time of 4 hours after the addition of a metabolizing system.

In contrary, in the absence of S9 mix after 18 hours treatment at 28-hour sampling time the frequency of polyploid cells was clearly increased at 600 μg/mL (20.05% polyploid cells) compared with the respective negative control group (3.00% polyploid cells). At this concentration the cell morphology and cell attachment was influenced by the test substance exposure. In addition, the cell number was clearly reduced (54.2% of control).

 

Mitotic Index

No relevant suppression of the mitotic activity was observed under all experimental conditions at the test groups scorable for cytogenetic damage.

 

Cell counts

Clear growth inhibition indicated by cell numbers of about or below 50% of control was observed at 600 μg/mL (54.2% of control) after 18 hours treatment at 28-hour preparation interval in the absence of S9 mix.

 

Cell morphology

Cell attachment was slightly reduced (i.e. few cells rounded) at several doses and sampling times both with and without S9 mix.

 

Assessment of the slides

In the 2nd Experiment after 18 hours treatment at 18-hour preparation interval in the absence of S9 mix slides non-scorable for cytogenetic damage were obtained at 300 μg/mL and above. This observation is similar to that of the pretest.

 

Treatment conditions

Osmolarity was not influenced by test substance treatment. The pH of the stock solutions was determined and adjusted to a physiological value using small amounts of 16% (w/v) HCl. No test substance precipitation in culture medium occurred within this study.

 

Conclusion: 

The negative controls gave frequencies of aberrations within the range expected for the V79 cell line. Both of the positive control substances, i.e.and cyclophosphamide, led to the expected increase in the number of cells containing structural chromosomal aberrations.

 

 

Conclusions:

On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of structurally aberrant metaphases incl. and excl. gaps at both sampling times either without S9 mix and/or after adding a metabolizing system in two experiments performed independently of each other.

The frequency of cells containing numerical chromosome aberrations was strongly increased in the 2nd experiment after 18 hours exposure at 28 hours-sampling time in the absence of S9 mix. The rate of polyploid cells after treatment with 600 μg/mL (20.05%) clearly exceeded the value of the respective negative control group (3.0%). This observation was corroborated by an increased sample of evaluation of 1000 cells per culture scoring 2 cultures per test group.


Thus, under the experimental conditions of this assay, the test substance is considered not to possess a potent chromosome-damaging (clastogenic) effect under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.

However, based on the observation of a strongly increased frequency of polyploid cells after 18 hours treatment and 10 hours recovery in the absence of metabolic activation it has to be considered that the test substance has an aneugenic potential under the experimental conditions described under in vitro conditions in V79 cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Mouse Micronucleus Test: negative (OECD 474)

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04. Dec 2006 - 05. Mar 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

(Aug 1998)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EC Directive 2000/32, B. 12 (19 May 2000)

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Germany GmbH
- Age at study initiation: 5 – 8 weeks
- Weight at study initiation: ca. 28 g
- Assigned to test groups randomly: yes, according to a randomization plan prepared with an appropriate computer program
- Fasting period before study: no
- Housing: individually in Makrolon cages, type MI
- Diet (ad libitum): Standardized pelleted feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water (ad libitum): drinking water from bottles
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2006-11-28 (arrival at testing facility); 2006-12-04 (animals in study) To: 2006-12-06 or 2006-12-07 (sacrifices)

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in water, purified water was used as vehicle.
- Concentration of test material in vehicle: 20, 100, 200 mg/ml bw for the low, mid and high dose group, respectively
- Amount of vehicle (if gavage or dermal): 10 ml/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The substance to be administered per kg body weight was dissolved in purified water. To achieve a solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly. All test substance formulations were prepared immediately before administration.
The stability of the test substance at room temperature in the vehicle purified water over a period of 4 hours was verified analytically.

Duration of treatment / exposure:

single dose

Frequency of treatment:

single dose

Post exposure period:

sacrifice at 24 and 48 hours post dose

Dose / conc.:

500 other: mg/kg bw

Remarks:

analytical concentration

Dose / conc.:

1 000 other: mg/kg bw

Remarks:

analytical concentration

Dose / conc.:

2 000 other: mg/kg bw

Remarks:

analytical concentration

No. of animals per sex per dose:

5 per group (low dose, mid dose, vehicle control, positive control)
8 per group (high dose)

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): The stability of cyclophosphamide is well-defined under the selected conditions, since the positive control article is a well-established reference clastogen
- Route of administration: oral
- Doses / concentrations: 20 mg/kg bw

vincristine
- Justification for choice of positive control(s): The stability of vincristine is well-defined under the selected conditions, since the positive control article is a well-established reference aneugen
- Route of administration: i.p.
- Doses / concentrations: 0.15 mg/kg bw

Tissues and cell types examined:

femoral bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In a pretest for the determination of the acute oral toxicity, at 2000 mg/kg body weight recommended as the highest dose according to the OECD Guideline all animals (male and female) survived. The clinical signs observed were piloerection, squatting posture, poor general state, irregular respiration and eyelid closure. However, there were no distinct differences in the symptoms between males and females. Thus, only male animals were used for the cytogenetic investigations.
Therefore, 2 000 mg/kg body weight was selected as the highest dose in the main study. 1000 mg/kg bw and 500 mg/kg body weight were administered as further doses.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The animals were treated once orally and samples of bone marrow were taken 24 hours and 48 hours after the administration.

DETAILS OF SLIDE PREPARATION:
The bone marrow was prepared according to the method described by SCHMID W (1976 and 1077) and SALAMONE M. et al (1980).
- The two femora of the animals sacrificed by cervical dislocation were prepared by dissection and removing all soft tissues.
- After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with fetal calf serum (FCS) which was at 37°C (about 2 mL/femur).
- The suspension was mixed thoroughly with a pipette, centrifuged at 300 x g for 5 minutes, the supernatant was removed and the precipitate was resuspended in about 50 μL fresh FCS.
- One drop of this suspension was dropped onto clean microscopic slides, using a Pasteur pipette. Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained.

Staining of the slides
- The slides were stained in eosin and methylene blue (modified May-Grünwald solution or Wrights solution) for about 5 minutes.
- After having briefly been rinsed in purified water, the preparations were soaked in purified water for about 2 - 3 minutes.
- Subsequently, the slides were stained in Giemsa solution (15 mL Giemsa, 185 mL purified water) for about 15 minutes.
- After having been rinsed twice in purified water and clarified in xylene, the preparations were mounted in Corbit-Balsam

METHOD OF ANALYSIS:
Microscopic evaluation
In general, 2000 polychromatic erythrocytes (PCEs) from each of the male animals of every test group were evaluated and investigated for micronuclei (MN). The normochromatic erythrocytes (NCEs) which occured were also scored. The following parameters were recorded:
- Number of polychromatic erythrocytes
- Number of polychromatic erythrocytes containing micronuclei (indicative of clastogenic and/or aneugenic effects)
- Number of normochromatic erythrocytes
- Number of normochromatic erythrocytes containing micronuclei
- Ratio of polychromatic to normochromatic erythrocytes
- Number of small micronuclei (d < D/4) and of large micronuclei (d > D/4) (d = diameter of micronucleus, D = cell diameter) (may indicate the possible mode of action, i.e. a clastogenic or a spindle poison effect)
Slides were coded before microscopic analysis.

Evaluation criteria:

Acceptance criteria
The mouse micronucleus test is considered valid if the following criteria are met:
- The quality of the slides must allow the identification and evaluation of a sufficient number of analyzable cells, i.e. ≥ 2000 PCEs and a clear differentiation between PCEs and NCEs.
- The ratio of PCEs/NCEs in the untreated animals (vehicle control) has to be within the range of the historical negative control data for the animal strain selected.
- The number of cells containing micronuclei in negative control animals has to be within the range of the historical negative control data both for PCEs and for NCEs.
- The positive control substance has to induce a significant increase in the number of PCEs containing micronuclei within the range of the historical positive control data or above.

Assessment criteria
A finding is considered positive if the following criteria are met:
- Statistically significant and dose-related increase in the number of PCEs containing micronuclei.
- The number of PCEs containing micronuclei has to exceed both the concurrent negative control value and the range of the historical negative control data.
A test substance is considered negative if the following criteria are met:
- The number of cells containing micronuclei in the dose groups is not statistically significant above the concurrent negative control value and is within the historical negative control data range.

Statistics:

The statistical evaluation of the data was carried out using the program system MUKERN (internal system).
The asymptotic U test according to MANN-WHITNEY (modified rank test according to WILCOXON) was carried out to clarify the question whether there were significant differences between the control group and dose groups with regard to the micronucleus rate in polychromatic erythrocytes. The relative frequencies of cells containing micronuclei of each animal were used as a criterion for the rank determination for the U test. Significances were identified as follows
* p ≤ 0.05
** p ≤ 0.01

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

2000 mg/kg bw

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY:
In a pretest for the determination of the acute oral toxicity, at 2000 mg/kg bw body weight recommended as the highest dose according to the OECD Guideline all animals (male and female) survived. The clinical signs observed were piloerection, squatting posture, poor general state, irregular respiration and eyelid closure. However, there were no distinct differences in the symptoms between males and females.

RESULTS OF DEFINITIVE STUDY:
1. Microscopic evaluation
The single oral administration of purified water in a volume of 10 mL/kg body weight led to 1.6 ‰ polychromatic erythrocytes containing micronuclei after the 24-hour sacrifice interval or to 1.0 ‰ after the 48-hour sacrifice interval.
After the single administration of the highest dose of 2000 mg/kg bw body weight, 1.1 ‰ polychromatic erythrocytes containing micronuclei were found after 24 hours and 1.4 ‰ after 48 hours.
In the two lower dose groups, rates of micronuclei of about 1.1 ‰ (1000 mg/kg bw group) and 1.0 ‰ (500 mg/kg bw group) were detected at 24 hour sacrifice interval in each case.
With 17.9 ‰ the positive control substance cyclophosphamide for clastogenicity led to an expected statistically significant increase in the number of polychromatic erythrocytes containing mainly small micronuclei.
Vincristine, a spindle poison substance, produced a statistically significant increase of 49.6 ‰ micronuclei in polychromatic erythrocytes. A significant portion increase, 10.2 ‰ was attributable to large micronuclei.
The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the negative control or in the various dose groups at any of the sacrifice intervals.
A distinct inhibition of erythropoiesis induced by the treatment of mice with the test substance was detected at the highest dose of 2000 mg/kg body weight at 48-hour interval.

2. Clinical examinations
The administration of the test substance doses 1000 mg/kg bw and 2000 mg/kg bw led to distinct clinical signs of toxicity (piloerection, hunched posture, reduced general condition, irregular respiration, eyelid closure).
Although, in the pretest all animals survived for at least 2 days after administration of the top dose of 2000 mg/kg bw, in the main experiment animals died at 24-hour sacrifice interval as well as at 48-hour sacrifice interval. Therefore, three additional animals of both dose groups were treated one day later to have the required animal numbers available for scoring. In each of these two high dose groups, totally 3/8 mice died within the first day after dosing.
Neither the single administration of the positive control substance cyclophosphamide in a dose of 20 mg/kg body weight nor that of vincristine in a dose of 0.15 mg/kg body weight caused any evident signs of toxicity.

Conclusions:

According to the results of the present study, the single oral administration of the test substance did not lead to any increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was always within the same range as that of the concurrent negative control in all dose groups and at all sacrifice intervals and within the range of the historical negative control data. An inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected in the dose of 2000 mg/kg body weight (48-hour sacrifice interval).



As a negative control, male mice were administered merely the vehicle, purified water, by the same route and in the same volume as the animals of the dose groups, which gave frequencies of micronucleated polychromatic erythrocytes within the historical negative control range.

Both of the positive control substances, i.e. cyclophosphamide for clastogenicity and vincristine for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei.



Thus, under the experimental conditions chosen here, the test substance does not have any chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

 

In vitro studies:

In an Ames test performed according to OECD Guideline 471 (Bacterial Reverse Mutation Assay) with Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100, test substance concentrations of 20, 100, 500, 2500 and 5000 µg/plate were employed in the presence and absence of Aroclor 1254-induced rat liver S9 mix. At non-toxic test substance concentrations, a significant increase in mutant frequency was not observed (BASF AG, 1995; Val. 2). In a similar AMES test according to OECD Guideline 471 and OECD 472 (Bacterial and E. coli, Reverse Mutation Assay) an isomer mixture of the test substance showed also negative results in the absence and present of Aroclor 1254-induced rat liver S9 mix (BASF AG, 1998; Val. 2).

In two further Ames tests according to a standard protocol approved by the National Toxicology Program, the test substance was tested in four Salmonella typhimurium strains (TA98, TAl00, TAl535 and TAl537) in the presence and absence of Aroclor-induced rat or hamster liver S9 (NTP 1980, 1981; Val. 2). In one test a slight increase in revertants was noted in the Salmonella typhimurium strain TA 1535. No noteworthy increase in the number of revertants was observed for all other strains with and without metabolic activation and also in replicate no increase in revertants in the strain TA 1535 was observed.

 

In an in vitro cytogenetics assay using chinese hamster lung fibroblasts (V79), the test substance was tested both in the absence and presence of metabolic activation (S9 mix), according to the OECD Guideline 473 (BASF AG, 2007; Val.1). The test substance did not cause any biologically relevant increase in the number of structurally aberrant metaphases incl. and excl. gaps at both sampling times either without S9 mix and/or after adding a metabolizing system in two experiments performed independently of each other. The frequency of cells containing numerical chromosome aberrations was strongly increased in the 2nd Experiment after 18 hours exposure at 28 hours-sampling time in the absence of S9 mix. The rate of polyploid cells after treatment with 600 μg/mL (20.05%) clearly exceeded the value of the respective negative control group (3.0%). This observation was corroborated by an increased sample of evaluation of 1000 cells per culture scoring 2 cultures per test group. The test substance is considered not to have a potent chromosome-damaging (clastogenic) effect under in vitro conditions in V79 cells in the absence and the presence of metabolic activation. However, based on the observation of a strongly increased frequency of polyploid cells after 18 hours treatment and 10 hours recovery in the absence of metabolic activation it has to be considered that the test substance has an aneugenic potential under the experimental conditions described under in vitro conditions in V79 cells.

 

In an in vitro gene mutation test in CHO cells according to OECD Guideline 476 (HPRT forward mutation assay), the test substance did not cause any increase in the mutant frequencies either without S9 mix or after adding a metabolizing system in two experiments performed independently of each other (BASF AG, 2007; Val. 1)

 

In vivo studies:

In a micronucleus assay with NMRI mice according to OECD TG 474, 5 male/female animals per group were orally administered single doses of 500, 1000, 2000 mg/kg bw test substance (BASF AG, 2007; Val.1). The single oral administration did not lead to any increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was always within the same range as that of the concurrent negative control in all dose groups and at all sacrifice intervals and within the range of the historical negative control data. An inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected in the dose of 2000 mg/kg body weight (48-hour sacrifice interval). The test substance did not have any chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.

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 1272/2008. Several valid Ames-tests (OECD 471), with or without metabolic activation through S9-Mix, a valid HPRT forward mutation assay (OECD Guideline 476), a valid chromosomal aberration assay (OECD Guideline 473), and a valid in vivo Mammalian Erytrocyte Micronucleus Test (OECD Guideline 474) gave no evidence for a genotoxic effect of the test substance. The observed increased frequency of polyploid cells (aneugenic potential) in the vitro chromosomal aberration assay in V79 cells (OECD 473) could not be confirmed in the in vivo Mammalian Erytrocyte Micronucleus Test (OECD Guideline 474). As a result, the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008, as amended for the seventeenth time in Regulation (EC) No. 2021/849