1,4-Diazabicyclo[2.2.2]octane-2-methanol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471): negative with S. typhimurium TA 1535, TA 1537, TA 98 and TA 100, and E. coli WP2 uvrA, with and without metabolic activation
Chromosome aberration test (OECD 473): negative in Chinese hamster lung cells with and without metabolic activation
Gene mutation test in mammalian cells (OECD 476): negative in L5178Y mouse lymphoma cells with and without metabolic activation

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:

experimental study

Adequacy of study:

key study

Study period:

12 - 28 Dec 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted Jul 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: “Standards for Mutagenicity Tests using Microorganisms” (Notification No. 77 and 120) and the “Amendment of the Reporting Form of the Results of the Mutagenicity Tests using Microorganisms” (Notification No. 653), Ministry of Labour, Japan

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon (for S. typhimurium strains)
trp operon (for E. coli strain)

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

preliminary study: 1.2, 4.9, 20, 78, 313, 1250 and 5000 μg/plate
main test 1 and 2: 313, 625, 1250, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle used: water
- Justification for choice of solvent/vehicle: Based on the information from the sponsor that the test substance was soluble at 5% and more in water.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: benzo[a]pyrene (B[a]P), 2-aminoanthracene (2AA), 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine x 2HCl (ICR-191), sodium azide (NaN3)

Remarks:

+S9: B[a]P (5 µg/plate, TA100, TA98, TA1537), 2AA (2 µg/plate, TA1535; 10 µg/plate, WP2 uvrA); -S9: AF-2 (0.01 µg/plate, TA100, WP2 uvrA; 0.1 µg/plate, TA98); ICR-191 (1 µg/plate, TA1537); NaN3 (0.5 µg/plate, TA1535)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: growth inhibition

Evaluation criteria:

If the number of revertant colonies on the test plates increased significantly in comparison with that on the control plates (based on twice as many as that of the negative control), and dose-response and reproducibility were also observed, the test substance was to be judged positive.

Statistics:

Mean values and standard deviation were calculated.

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

PRECIPITATION
No precipitation on the plates was observed.

RANGE-FINDING/SCREENING STUDIES:
No growth inhibition by the test substance was observed in any strain with and without metabolic activation. Therefore, as the highest dose level of the test substance in the main tests, the 5000 μg/plate dose was selected for all strains.

COMPARISON WITH HISTORICAL CONTROL DATA:
The revertant colonies of the positive controls and solvent controls were within the limits (mean±3SD) of historical control data.

Conclusions:

Interpretation of results (migrated information):
negative

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 Feb - 10 Mar 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

Jul 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

other: Chinese hamster lung cells (CHL/IU)

Details on mammalian cell type (if applicable):

- Type and identity of media: Eagle MEM culture medium supplemented with sodium bicarbonate and L-glutamin and 10% fetal bovine serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

6 h treatement: 375, 750, 1125 and 1500 µg/mL with and without S9 mix
24 h treatment: 375, 500, 625, 750, 875 and 1000 µg/mL without S9 mix

Vehicle / solvent:

- Vehicle/solvent used: Japanese Pharmacopoeia physiological saline
- Justification for choice of solvent/vehicle: The test subtance is soluble in physiological saline at 50 mg/mL, and in DMSO at 500 mg/mL, with no exothermic reation, foaming, or discoloration. Accordingly physiological saline was selected as the vehicle to prepare the test substance.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

mitomycin C

Remarks:

mitomycin (MMC): 0.1 µg/mL (6h, -S9), 0.05 µg/mL (24 h, -S9); 3,4-benzopyrene (BP): 10 µg/mL (6 h, +S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 6 and 24 h
- Fixation time (start of exposure up to fixation or harvest of cells): 6 h treatment: 24 h; 24 h treatment: 24 h

SPINDLE INHIBITOR (cytogenetic assays): colcemid, 0.2 µg/mL
STAIN (for cytogenetic assays): 3% Giemsa

NUMBER OF REPLICATIONS: 2 plates

NUMBER OF CELLS EVALUATED: 100 metaphase cells per plate (200 metaphase cells per dose)

DETERMINATION OF CYTOTOXICITY
- Method: cell growth

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

Validity criteria for the test:
The validity of the test was verified when the two criteria shown below were met.
a) Both the incidences of cells with structural aberrations and numerical aberrations in the negative control group of all test series are <5%.
b) The incidence of structural aberrations of chromosomes in the positive control group of all test series are >= 10%.
The present results of test series met both of them.

Evaluation of test results:
The results were determined to be negative when both the incidences of cells with structural aberrations and numerical aberrations were < 5%. When either or both of them were >=5% but <10%, a confirmatory test should be conducted, and the results were determined to be equivocal when good reproducibility was obtained. The results were determined to be positive when either or both incidences were >= 10% and good reproducibility or dose-dependent increase in the incidences was observed.

Statistics:

Statistical analysis was not conducted.

Species / strain:

other: Chinese hamster lung cells (CHL/IU)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 750 µg/mL and higher following the 24 h treatment without S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The culture medium in the plate was visually observed for color change at the start and end of treatment with the test substance precipitations. Since no color change was noted, treatment with the test substance preparations was considered to have no effect on the pH of culture medium.
- Precipitation: The culture medium in the plate was macroscopically observed for precipitation of the test substance at the start and end of treatment with the test preparations. No precipitation occurred.


RANGE-FINDING/SCREENING STUDIES:
A cell growth inhibition test was performed in three test series (6 h treatment with and without S9 mix and 24 h treatment without S9 mix) at concentrations of 11.7, 23.4, 46.9, 93.8, 188, 375, 750, 1500 µg/mL. The highest dose is equivalent to approx. 10 mM. Two plates were used at each concentration. IC50 of 24 h continuous treatment was 727.5 µg/mL. Cell growth was not inhibited by more than 50% following short-term treatment with and without S9 mix.

Table 1. Results of chromosomal aberration test*

Test item	Concentration	Growth rate (%)	Aberrant cells in %
	in µg/mL	in %	Structural aberrations	Numerical aberrations
Exposure period 6 h, fixation time 24 h, without S9 mix
Physiological saline		100	0	0
MMC	0.1		35	0
Test substance	375	101.5	n.d.	n.d.
	750	96.5	0	0.5
	1125	75.5	0.5	1
	1500	61.5	0	2
Exposure period 6 h, fixation time 24 h, with S9 mix
Physiological saline		100	0.5	0
BP	10		42	0
Test substance	375	101.0	n.d.	n.d.
	750	105.5	1	0
	1125	102.0	0.5	0
	1500	87.0	0.5	0
Exposure period 24 h, fixation time 24 h, without S9 mix
Physiological saline		100	0	0.5
MMC	0.05		47.5	0.5
Test substance	375	103	n.d.	n.d.
	500	94.5	0	0.5
	625	79.0	0	0
	750	48.5	0.5	1.0
	875	34.5	n.d.	n.d.
	1000	29.0	n.d.	n.d.

MMC: mitomycin C; BP: 3,4-benzopyrene (positive controls)

n.d.: not determined

* data provided by the sponsor in an extra sheet

Conclusions:

Interpretation of results (migrated information):
negative

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 Apr - 17 June 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 21 Jul 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Health Care Inspectorate, Ministry of Health, Welfare and Sport, Den Haag, The Netherlands

Type of assay:

mammalian cell gene mutation assay

Target gene:

TK locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI Hepes buffered medium containing penicillin/streptomycin (50 U/mL and 50 µg/mL, respectively), 1mM sodium pyruvate, 2 mM L-glutamin and heat-inactivated horse serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital (80 mg/kg bw) and beta-naphthoflavone (100 mg/kg bw)

Test concentrations with justification for top dose:

dose-range finding test: 17, 52, 164, 512, 1422 µg/mL

1st mutagenicity test (3 h):
without S9 mix: 5.2, 17, 52, 164, 512, 725, 1000 and 1422 µg/mL
with S9 mix: 0.5, 1.7, 5.2, 17, 52, 164, 512 and 1422 µg/mL

2nd mutagenicity test (24 h):
without S9 mix: 5.4, 17, 52, 164, 280, 512, 630, 800, 1000* and 1260* µg/mL (* not used for mutation frequency measurement, since
these dose levels were too toxic for further testing).

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Remarks:

-S9 mix: methylmethanesulfonate (MMS), in DMSO, 15 and 5 µg/mL for a 3 and 24 h treatment period, respectively; +S9 mix: cyclophosphamide (CP), in Hanks' balanced salt solution without Ca and Mg, 7.5 µg/mL;

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 1st experiment: 3 h exposure with and without S9 mix; 2nd experiment: 24 h without S9 mix
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11-12 days
- Fixation time (start of exposure up to fixation or harvest of cells): 13-15 days

SELECTION AGENT (mutation assays): trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: quintuplicates in 96-well microtiter plates for mutation frequency (MF) determination in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency and relative total growth

Evaluation criteria:

Acceptability of the assay:
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120% in order to have an acceptable number of surviving cells analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 106 survivors and ≤ 170 per 1E+07 survivors.
c) The growth rate (GR) over the 2-day expression period for the negative controls should be between 8 and 32 (3 hours treatment) and between 32-180 (24 hours treatment).
d) The mutation frequency of MMS should not be below 500 per 1E+07 survivors, and for CP not below 700 per 1E+07 survivors.


Evaluation criteria:
The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126 (Moore et al., 2000. Environmental and Molecular Mutagenesis 35: 185-190).

A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.

In addition to the criteria stated above, any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

1st test: at 1422 µg/mL: RTG was reduced by 80% (-S9 mix) and 77% (+S9 mix); 2nd test: at 630 and 800 µg/mL: RTG was reduced by 67% and 92%, respectively compared to RTG of the solvent control

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH of a concentration of 1422 μg/mL was 8.0 (compared to 7.4 in the solvent control).
- Effects of osmolality: The osmolarity of a concentration of 1422 μg/mL was 0.405 Osm/kg (compared to 0.412 Osm/kg in the solvent control).
- Precipitation: The test substance did not precipitate in the exposure medium up to and including the concentration of 1422 μg/mL (= 10 mM).

RANGE-FINDING/SCREENING STUDIES:
In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test substance concentration range of 17 to 1422 µg/mL in the absence of S9 mix with a 3 and 24 h treatment period and in the presence of S9 mix with a 3 h treatment period.
3 h treatment: In the absence of S9 mix, the relative suspension growth was 32% at the highest test substance concentration of 1422 μg/mL compared to the relative suspension growth of the solvent control. In the presence of S9 mix, no toxicity in the relative suspension growth was observed up to test substance concentrations of 1422 μg/mL compared to the solvent control.
24 h treatment: In the absence of S9 mix, the relative suspension growth was 46% at the test substance concentration of 512 μg/mL compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at the test substance concentration of 1422 μg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay. Except the response of one of the solvent control cultures in the first experiment in the absence of S9-mix. However since this response was just below the lower limit of the range and clear negative results were obtained, the validity of the test was considered to be not affected.

Table 1: Experiment 1: Cytotoxic and mutagenic response in the mouse lymphoma L5178Y test system

Dose	RSG	CEday2	RSday2	RTG)	Mutation frequency per 1E+06 survivors
(µg/mL)	(%)	(%)	(%)	(%)	total	(small	large)
Without metabolic activation, 3 h treatment
DMSO 1	100	104	100	100	54	(23	30)
DMSO 2		111			45	(21	23)
5.2	88	115	107	94	51	(26	23)
17	80	123	114	91	55	(24	29)
52	93	107	99	92	49	(27	21)
164	96	94	87	83	63	(26	36)
512	78	97	90	70	75	(37	36)
725	58	105	98	57	84	(38	42)
1000	42	121	113	47	32	(13	18)
1422	19	110	102	20	70	(30	37)
MMS	64	66	62	39	620	(329	216)
With metabolic activation, 3 h treatment
DMSO 1	100	98	100	100	71	(21	48)
DMSO 2		99			62	(19	41)
0.5	102	102	104	106	55	(14	39)
1.7	111	78	79	88	75	(19	54)
5.2	113	89	90	102	52	(18	33)
17	99	90	91	90	50	(19	30)
52	111	83	84	93	47	(18	28)
164	101	75	76	77	61	(14	46)
512	92	88	89	81	46	(14	31)
1422	36	64	65	23	96	(37	47)
CP	25	14	14	4	2773	(1227	1280)

RSG: relative suspension growth; CE: cloning efficiency; RS: relative survival; RTG: relative total growth; MMS: methylmethanesulfonate; CP: cyclophosphamide

 

Table 2: Experiment 2: Cytotoxic and mutagenic response in the mouse lymphoma L5178Y test system

Dose	RSG	CEday2	RSday2	RTG)	Mutation frequency per 1E+06 survivors
(µg/mL)	(%)	(%)	(%)	(%)	total	(small	large)
Without metabolic activation, 24 h treatment
DMSO 1	100	101	100	100	56	(31	23)
DMSO 2		86			62	(41	20)
5.4	103	86	92	95	68	(43	23)
17	95	102	109	104	64	(47	16)
52	97	98	105	102	56	(28	26)
164	100	71	76	76	89	(51	34)
280	84	83	88	74	89	(55	31)
512	69	94	100	70	76	(55	19)
630	38	80	86	33	36	(29	7)
800	12	58	61	8	11	(7	4)
MMS	95	72	77	74	741	(426	224)

RSG: relative suspension growth; CE: cloning efficiency; RS: relative survival; RTG: relative total growth; MMS: methylmethanesulfonate; CP: cyclophosphamide

The numbers of small and large colonies in the test substance treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Conclusions:

Interpretation of results (migrated information):
negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

- Gene mutation in bacteria

A bacterial gene mutation assay with the test substance was performed in compliance with OECD Guideline 471 and GLP (Tosoh Corporation, 2014a). In two independent experiments, the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and the Escherichia coli strain WP2 uvrA were exposed to the test substance dissolved in water using the preincubation method. Since no cytotoxicity and precipitation was noted up to the limit concentration of 500 µg/plate in a preliminary study, test concentrations of 313, 625, 1250, 2500 and 5000 µg/plate were used with and without metabolic activation in the two main studies. No increase in the mean number of revertants per plate was observed in any of the test strains compared to the control. The number of spontaneous mutations of the negative control was found to be within the respective historical control ranges and the positive controls induced the expected increase in the number of reverse mutants. No growth inhibition was noted after exposure of the test strains with any of the test concentrations in both experiments. Under the conditions of this experiment, the test substance did not show mutagenicity in the selected strains of S. typhimurium and E. coli in the presence and absence of metabolic activation.

 

- Chromosome aberrations

The clastogenic activity of the test substance was investigated in an in vitro mammalian chromosome aberration test in Chinese hamster lung cells performed according to OECD Guideline 473 and GLP (Tosoh Corporation, 2014b). In a preliminary cytotoxicity test, cells were exposed to concentrations of the test substance ranging from 11.7 to 1500 µg/mL (equivalent to approx. 10 mM ) for a short-term 6 h-exposure period with and without metabolic activation (S9 mix) and a continuous 24 h-exposure period without S9 mix. In all experiments, a fixation time of 24 h was chosen after incubation of cells for the respective time points. IC50 of 24 h continuous treatment was 727.5 µg/mL. Cell growth was not inhibited by more than 50% following short-term treatment with and without S9 mix.

Based on the results of the preliminary test, cell cultures of the main experiment were exposed to concentrations of 375, 750, 1125 and 1500 µg/mL with and without S9 mix for a short-term 6 h-exposure period and to concentrations of 375, 500, 625, 750, 875 and 1000 µg/mL without S9 mix for a continuous 24 h-exposure period. Concentrations of 750, 1125 and 1500 µg/mL (6 h treatment, with and without S9 mix) and 500, 625 and 750 µg/mL (24 h treatment, without S9 mix) were selected for metaphase analysis. The incidences of structural and numerical aberrations of chromosomes were less than 5% at all doses in all test series compared to controls and thus the test substance was considered not-clastogenic under the conditions of this test. The positive controls included during short-term and continuous exposure showed the expected results, thus confirming the sensitivity of the test method.

- Gene mutation in mammalian cells

The mutagenic activity of the test substance was evaluated in an in vitro mammalian cell gene mutation test according to OECD Guideline 476 and in compliance with GLP (Tosoh Corporation, 2014c). Based on the results of a dose range finding test, L5178Y mouse lymphoma cells were exposed for 3 h to the test substance up to concentrations of 1422 µg/mL (10 mM) in the absence and presence of metabolic activation. The relative total growth of the highest test substance concentration was reduced by 80 and 77% in the absence and presence of S9 mix, respectively compared to the total growth of the solvent controls. No significant increase in the mutation frequency at the TK locus was observed after treatment with the test substance either in the absence or in the presence of S9 mix. In a second independent experiment the test substance was tested up to a concentration of 800 µg/mL in the absence of S9 mix with a modified treatment period of 24 h. The relative total growth of the 800 µg/mL test substance concentration was reduced by 92% compared to the total growth of the solvent controls. No significant increase in the mutation frequency at the TK locus was observed after treatment with the test substance either. In addition, no significant difference in the numbers of small and large colonies in the test substance treated cultures were observed. Mutation frequencies in cultures treated with positive control chemicals were increased by 13-fold for methylmethansulfonate in the absence of S9-mix, and by 42-fold for cyclophosphamide in the presence of S9 mix, demonstrating that the test conditions were appropriate for the detection of a mutagenic response.

Thus, under the experimental conditions the test substance is not mutagenic in the TK mutation test system.

 

In conclusion, the test substance showed no evidence of clastogenic or mutagenic potential with and without metabolic activation in 3 in vitro test systems.

 

Justification for classification or non-classification

The available data on genetic toxicity of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.