Registration Dossier

The test substance did not induce mutations (base-pair substitution and frame-shift type) in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 as well as in E. coli WP2 uvr A both in presence and absence of metabolic activation (Harlan 2013).

The test substance was tested in L5178Y cells in presence and absence of metabolic activation in a study performed according to OECD 476. In both experiments the test substance did not induce an increase in mutant frequency at the TK +/- locus and is therefore considered to be non-mutagenic (Harlan 2015).

In a chromosome aberration test in CHO-WBL cells, structural (with metabolic activation) and numerical (without metabolic activation) aberrations were reported after 3-hour treatment at concentrations up to cytotoxicity. Prolonged testing without metabolic activation did not induce any effects. Therefore it cannot be excluded that the substance exhibits clastogenic effects.

Reference

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:: 22 Sep 2014 to 25 Feb 2015
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: other: Guidelines study under GLP, positive and vehicle controls do not fall clearly within historical control ranges in part of the experiments
Qualifier:: according to guideline
Guideline:: OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:: yes
Type of assay:: in vitro mammalian chromosome aberration test
Species / strain / cell type:: Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):: - Source: Genetic Toxicology Laboratory at Pfizer, Inc., Groton, CT, USA
- Type and identity of media: McCoy’s complete media
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes, 12-14 hour cycling time, average 21 chromosomes

Each culture was prepared by seeding with 1.4 × 10E6 CHO cells per 75 cm2 flask in McCoy’s complete media and incubated at 37 ± 1°C in vented flasks in a humidified atmosphere of 5 ± 1% CO2 for approximately 24 hours prior to the initiation of treatment.
Metabolic activation:: with and without
Metabolic activation system:: Aroclor™ 1254-induced rat liver S9 fraction
Test concentrations with justification for top dose:: Concentrations are based on the results of 3 pre-tests

Exp 1 (3 h without metabolic activation) : stock solution of 150 mg/mL further diluted with DMSO/ethanol(1/1) to:
6.25, 12.5, 25.0, 50.0, 75.0, 100, 125, 150, 225, 300, and 500 µg/mL
Exp 2 (20 h without metabolic activation) : stock solution of 50 mg/mL further diluted with DMSO/ethanol(1/1) to:
6.25, 12.5, 25.0, 50.0, 75.0, 100, 125, 150, 225, 300, and 500 µg/mL
Exp 3 (3 h with metabolic activation) : stock solution of 50 mg/L further diluted with DMSO/ethanol(1/1) to:
6.25, 12.5, 25.0, 50.0, 75.0, 100, 125, 150, 225, 300, and 500 µg/mL
Vehicle / solvent:: - Vehicle(s)/solvent(s) used: DMSO/Ethanol (1/1)
- Justification for choice of solvent/vehicle: pre-test at 500 mg/L
Negative solvent / vehicle controls:: yes
Remarks:: DMSO/Ethanol (1/1)
Positive controls:: yes
Positive control substance:: mitomycin C
Remarks:: tests without metabolic activation
Negative solvent / vehicle controls:: yes
Remarks:: DMSO/Ethanol (1/1)
Positive controls:: yes
Positive control substance:: cyclophosphamide
Remarks:: test with metabolic activation
Details on test system and experimental conditions:: METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: exp 1 3 hours without metabolic activation, exp 2 20 hours without metabolic activaton, exp3 3 hours with metabolic activation
- Expression time: until 20 hours

SPINDLE INHIBITOR: Colcemid (0.1 µg/mL) 2 hours before harvesting
FIXATIVE: 3:1 methanol:glacial acetic acid

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 metaphases per replicate (+ 15 aberrant cells per replicate)

DETERMINATION OF CYTOTOXICITY
- Method: relative population doubling (with Coulter counter) + mitotic index (1000 cells)+ visible signs of cytotoxicity

OTHER EXAMINATIONS:
- Determination of polyploidy: 200 cells/replicate
- Determination of endoreplication: 200 cells/replicate
Evaluation criteria:: POSITIVE RESPONSE:
The test item would be considered positive for inducing chromosomal aberrations if a significant increase (p ≤0.01) in the mean percent of cells with chromosomal aberrations is observed at one or more dose levels, or if a significant increase (p ≤0.05) in the mean percent of cells with chromosomal aberrations is observed at two or more dose levels. If a significant increase is seen at one or more dose levels, a dose-response should be observed.
NEGATIVE RESPONSE
The test item would be considered negative for inducing chromosomal aberrations if no statistically significant increase (p ≤0.05) is observed in the mean percent of cells with chromosomal aberrations at any of the test concentrations.
Statistics:: One-tailed Fisher’s Exact test on cell numbers with aberrations
A response would be considered statistically significant for dose-response trend in the Cochran-Armitage test if p ≤0.05.
Species / strain:: Chinese hamster Ovary (CHO)
Metabolic activation:: without
Genotoxicity:: ambiguous
Remarks:: no dose response
Cytotoxicity / choice of top concentrations:: cytotoxicity
Remarks:: at 150 ug/mL and above
Vehicle controls validity:: other: within historical control ranges
Positive controls validity:: other: MMC (0.75 ug/mL) within historica control values
Species / strain:: Chinese hamster Ovary (CHO)
Metabolic activation:: without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: cytotoxicity
Remarks:: at 225 ug/mL and above; precipitate at 125 ug/mL and above
Vehicle controls validity:: other: outside historical control ranges
Positive controls validity:: other: MMC (0.10 ug/mL) within historical control ranges
Species / strain:: Chinese hamster Ovary (CHO)
Metabolic activation:: with
Genotoxicity:: positive
Remarks:: dose response
Cytotoxicity / choice of top concentrations:: cytotoxicity
Remarks:: at 75 ug/mL and above
Vehicle controls validity:: other: at the upper range of historical control values
Positive controls validity:: other: CP (0.75 ug/mL) at the lower range of historical control values
Additional information on results:: TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not reported
- Effects of osmolality: not reported
- Precipitation: see above

RANGE-FINDING/SCREENING STUDIES: performed, but only cytotoxicity part reported

COMPARISON WITH HISTORICAL CONTROL DATA: see above
Remarks on result:: other: other: 3 hours exposure
Remarks:: Migrated from field 'Test system'.
Conclusions:: Interpretation of results (migrated information):
ambiguous

The substance was considered positive for inducing structural aberrations in CHO-WBL cells with metabolic activation and equivocal for inducing structural aberrations in CHO-WBL cells in the short-treatment without metabolic activation under the conditions of this test system. A significant test item-related increase in numerical aberrations (endoreduplication) was observed in the short-treatment without metabolic activation
Executive summary:: In a chromosome aberration test in CHO-WBL cells, structural (with metabolic activation) and numerical (without metabolic activation) aberrations were reported after 3-hour treament at concentrations upto cytotoxicity. Prolonged testing without metabolic activation did not induce any effects. Therefore it cannot be excluded that the substance exhibits clastogenic effects.

Endpoint conclusion:

adverse effect observed (positive)

In a study according to OECD 474 5 male mice/groupwere dosed twice via oral gavage with vehicle (corn oil), the substance at 2000, 1000 and 500 mg /kg bw or with 40 mg/kg cyclophosphamide (dosed only once). One animal dosed at 1000 mg/kg bw died and one at 500 mg/kg bw showed lethargy and a rough coat after the second dosing but recovered within 23 hours. No treatment related clinical signs or mortality were noted in the other animals. Blood samples collected at 0.5, 1, 2, 4, 6 and 8 hours after dosing from three satellite animals (dosed at 2000 mg/kg bw) per time point treated with 2000 mg showed concentrations of the substance above the upper limit of quantification (> 10000 ng/L), which confirms exposure.

Bone marrow was sampled 48 hours after the first dosing. No biological relevant increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with the substance compared to the vehicle treated animals. No toxicity on erythropoiesis of the substance was observed (expressed as the ratio of polychromatic to normochromatic erythrocytes).

The substance is not clastogenic in the bone marrow micronucleus test of mice up to a dose of 2000 mg/kg.

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 April 2015 to 28 September 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This testing was performed for the purpose of chemical notification in China. Therefore no testing proposal was included.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2014

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

other: in vivo micronucleus teste

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

recommended by international guidelines

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 7 weeks
- Weight at study initiation: 31-40 g
- Assigned to test groups randomly: yes
- Fasting period: withheld 3 - 4 hours prior to dosing until 3 - 4 hours after administration
- Housing:
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH,
Soest, Germany) ad libitum)
- Water : tap water ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.5 - 22.6°C
- Humidity (%): 38-73%
- Air changes (per hr): ca 10/h
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: solubility/stability of the substance
- Amount of vehicle (if gavage or dermal): 10 mL

Details on exposure:

dosing solutions prepared on day of treatment (doses based on the outcome of a pre-test in 3 males and 3 females at 2000 mg/kg bw)

Duration of treatment / exposure:

2 days

Frequency of treatment:

once at 0 and 24 h

Post exposure period:

24 hour after last dose

Dose / conc.:

500 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

Dose / conc.:

2 000 mg/kg bw/day

No. of animals per sex per dose:

5 males/ dose + additional 18 animals at 2000 mg/kg for blood sampling at 0.5, 1, 2, 4, 6 and 8 hours after the second dosing

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide;
- Justification for choice of positive control(s): according to the guideline
- Route of administration: oral gavage
- Doses / concentrations: 40 mg/kg bw

Tissues and cell types examined:

bone marrow for normo- and poly-chromated erithocytes

Details of tissue and slide preparation:

Bone marrow was sampled 48 hours after the first dosing from both femurs, flushed withetal calf serum and the cell suspension was centrifuged at 216 g for 5 min. Smears were prepared after removal of the supernatant (one drop left behind) and suspended on a clean slide that was coloured by Giemsa based staining (Wright-stain-procedure)

Evaluation criteria:

A test substance is considered positive in the micronucleus test if:
It induces a biologically as well as a statistically significant (Dunnett’s test, one-sided, p < 0.05)
increase in the frequency of micronucleated polychromatic erythrocytes and the number of
micronucleated polychromatic erythrocytes in the animals should be above the historical control
data range.

A test substance is considered negative in the micronucleus test if:
None of the tested concentrations show a statistically significant (Dunnett’s test, one-sided,
p < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes and the number
of micronucleated polychromatic erythrocytes in the animals should be within the historical control
data range.

Statistics:

ToxRat Professional version 3.0

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

One animal dosed with 1000 mg/kg test substance died immediately after the second dosing due to a technical error while dosing and one animal dosed with 500 mg/kg showed lethargy and a rough coat after the second dosing but recovered within 23 hours after second dosing. No treatment-related clinical signs or mortality were noted in the other animals.

Analyzed blood concentrations of the substance (from the 2000 mg/kg bw additional mice) were above the upper limit of quantification as validated in the analytical method (> 10000 ng/L).

GroupTreatment	Dose (mg/kg bw		Number of micronucleatedpolychromatic erythrocytes (mean ± S.D.)^(1,2	Ratio polychromatic/ normochromatic erythrocytes (mean ± S.D.)^(1,2
Vehicle control		0	3.6 ±0.9	0.95 ±0.07
Test substance		2000	4.0 ±1.0	0.90 ±0.08
Test substance		1000	4.5 ±1.7	0.94 ±0.05
Test substance		500	3.4 ±1.1	0.91 ±0.06
CP		40	38.2 ± 6.8⁽⁴⁾	0.54 ±0.12

Vehicle control = Corn oil

CP = Cyclophosphamide.

(1) Four to five animals per treatment group.

(2) At least 4000 polychromatic erythrocytes were evaluated with a maximum deviation of 5%.

(3) The ratio was determined from at least the first 1000 erythrocytes counted.

(4) Significantly different from corresponding control group (Studentsttest, P = 0.01).

Conclusions:

The substance is not clastogenic in the bone marrow micronucleus test of mice up to a dose of 2000 mg/kg

Executive summary:

In a study according to OECD 474 5 male mice/groupwere dosed twice via oral gavage with vehicle (corn oil), the substance at 2000, 1000 and 500 mg /kg bw or with 40 mg/kg cyclophosphamide (dosed only once). One animal dosed at 1000 mg/kg bw died and one at 500 mg/kg bw showed lethargy and a rough coat after the second dosing but recovered within 23 hours. No treatment related clinical signs or mortality were noted in the other animals. Blood samples collected at 0.5, 1, 2, 4, 6 and 8 hours after dosing from three satellite animals (dosed at 2000 mg/kg bw) per time point treated with 2000 mg showed concentrations of the substance above the upper limit of quantification (> 10000 ng/L), which confirms exposure.

Bone marrow was sampled 48 hours after the first dosing. No biological relevant increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with the substance compared to the vehicle treated animals. No toxicity on erythropoiesis of the substance was observed (expressed as the ratio of polychromatic to normochromatic erythrocytes).

The substance is not clastogenic in the bone marrow micronucleus test of mice up to a dose of 2000 mg/kg.

Endpoint conclusion:

no adverse effect observed (negative)

The potential clastogenic effects seen in the in vitro chromosome aberration test were further investigated in an in vivo micronucleus test, where blood test substance concentrations (above the upper limit of quantification as validated in the analytical method (> 10000 ng/L)) were confirmed. No increase of the frequency of micronucleated polychromatic erythrocytes was reported at any of the doses tested.

Therefore it can be concluded that the substance is not clastogenic and does not need to be classified.

The available in vitro data together with the in vivo micronucleus test are not indicative for mutagenic or clastogenic effects of the substance and therefore the criteria for classification according to Regulation (EC) 1272/2008 are not met.

Treatment	% Mitotic Reduction	% Cytotoxicity RPD	% Cells w/Abs	% Cells w/>1 Abs	% Endo Cells	% Polyploid Cells
Vehicle (1%)	0.00	0.00	0.0	0.0	0.0	4.8
MMC 0.75 µg/mL	35.39	10.69	66.7*	46.7*	0.0	4.5
37.5 µg/mL	0.00	0.33	2.5**	0.0	0.0	2.8
75.0 µg/mL	0.00	0.67	2.5**	0.0	0.3	3.3
150 µg/mL^a	0.00	7.17	2.5**	0.0	15.0*	3.3

Treatment	% Cytotoxicity RPD	% Cells w/Abs	% Cells w/>1 Abs	% Endo Cells	% Polyploid Cells
Vehicle (1%)	0.00	4.0	0.0	0.0	5.0
MMC 0.10 µg/mL	15.21	50.8*	27.1*	0.0	4.8
75.0 µg/mL	13.14	1.0	0.0	0.0	5.3
100 µg/mL	23.02	2.0	0.0	0.0	6.5
125µg/mL^a	33.15	3.5	0.0	0.0	3.3

Treatment	% Cytotoxicity RPD	% Cells w/Abs	% Cells w/>1 Abs	% Endo Cells	% Polyploid Cells
Vehicle (1%)	0.00	2.0	0.0	8.5	4.8
CP 7.5 µg/mL	19.40	15.5*	3.5*	5.3	4.3
12.5 µg/mL	6.71	1.5	0.0	9.8	4.5
25.0 µg/mL	30.33	6.0**	1.0	9.8	5.0
75.0 µg/mL^a	54.04	12.5*	3.0**	9.0	3.0