Acetophenone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- in vitro gene mutation study in bacteria (OECD Guideline 471, GLP): negative with and without metabolic activation

- In Vitro Mammalian Chromosome Aberration Test (OECD Guideline 473, GLP, V9 CHL): negative without S9-mix, positive with S9-mix

- In Vitro Mammalian Cell Gene Mutation Test (OECD Guideline 476, GLP, L5178Y cells) negative with and without S9-mix

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 04-AUG-2006 to 25-MAY-2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

GLP compliance:

yes (incl. QA statement)

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:

phenobarbital/ß-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

- S9: 32.3 - 517.5 µg/mL
+ S9: 64.7 - 1100 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: solubility

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: without S9: ethylmethane sulfonate; with S9: cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: without S9: 4, 18, 28 hrs; with S9: 4 hrs
- Fixation time (start of exposure up to fixation or harvest of cells): 18 or 28 hrs

SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicates, verification by repeat assays

NUMBER OF CELLS EVALUATED: 200 metaphases per test concentration

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Evaluation criteria:

Clastogenicity: positive test result if structural chromosome aberrations excluding gaps are higher than the range of the historical control of 0.0 - 4.0 %, and either a concentration related or significant increase (p<0.05) of structural chromosome aberrations is observed
Aneugenicity: positive test result if numerical chromosome aberrations are higher than the range of the historical control of 0.0 - 5.6 %

Statistics:

Fisher's exact est

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

induction of structural chromosomal aberrations

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not affected
- Effects of osmolality: not affected
- Precipitation: at >= 513 µg/mL both without and with S9

RANGE-FINDING/SCREENING STUDIES:
no cytotoxicity up to 1035 µg/mL after 4 hr treatment; clear cytotoxicity at >= 517.5 µg/mL after 24 hr treatment

COMPARISON WITH HISTORICAL CONTROL DATA:
without S9: Experiment IA: single signifcant increase of aberrant cells after 4 hr-treatment and 18 hr-preparation interval at 32.2 µg/mL, however, the aberration rate of 2.0 % was clearly within the historical control range; therefore regarded to be of no biological relevance
with S9: increases of aberrant cells exceeding the laboratory's control range were observed in experiment IA, IIA and IIB (see Table)

RANGE-FINDING/SCREENING STUDIES:
precipitation of test substance at >= 513 µg/mL both without and with S9; no cytotoxicity up to 1035 µg/mL after 4 hr treatment; clear cytotoxicity at >= 517.5 µg/mL after 24 hr treatment

COMPARISON WITH HISTORICAL CONTROL DATA:
without S9: single signifcant increase after 4 hr-treatment and 18 hr-preparation interval at 32.2 µg/mL, however, the aberration rate of 2.0 % was clearly within the historical control range; therefore regarded to be of no biological significance

ADDITIONAL INFORMATION ON CYTOTOXICITY:
without S9 after 4 hr treatment at 129.4 µg/mL, after 18 hr treatment at 517.5 µg/mL, after 28 hr treatment at 258.8 µg/mL; with S9 after 4 hr treatment at >= 900 µg/mL

Table: Significant changes of structural chromosomal aberrations after treatment with acetophenone in the presence of metabolic activation

Experiment	Test concentration (µg/mL)	% aberrant cells excluding gaps	Significance level p-value	% aberrant cells with exchanges
Experiment IA	Solvent control	1.5	-	1.0
	64.7	4.0	0.070	1.5
	129.4	5.0 *	0.027	1.5
	258.8	4.5 *	0.044	0.5
Experiment IB (Repeat)	Solvent control	2.5	-	1.0
	100.0	3.0	0.386	0.5
	200.0	3.0	0.386	0.0
	300.0	3.5	0.288	0.5
Experiment IIA	Solvent control	1.5	-	1.0
	258.8	3.0	0.169	0.0
	517.5	2.5	0.252	0.0
	1035.0	9.5 *	< 0.001	9.5 ^§
Experiment IIB (Repeat)	Solvent control	0.5	-	0.0
	900	10.0 *	< 0.001	5.5 ^§
	1000	38.0 *	< 0.001	14.0 ^§
	1100	20.0 *	< 0.001	7.5 ^§

* effect of statistical significance and exceeding the historical control range

^§ effect exceeding the historical control range

As the significant effect of experiment IA was not repeatable in experiment IB , it was regarded as biologically not relevant.

The significant increase of structural aberrations in experiment IIA at 1035 µg/mL was confirmed at doses of 900 to 1100 µg/mL in experiment IIB. Distinct increases in the number of cells with exchanges provided additional evidence for a clastogenic potential.

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation
positive with metabolic activation

A clastogenic potential of acetophenone is indicated in the presence of metabolic activation by S9 mix by the induction of structural chromosomal aberrations which is confirmed by increased frequencies of exchanges.

Executive summary:

In a chromosomal aberration test with V79 cells according to OECD Guideline 473, a clastogenic potential of acetophenone is indicated in the presence of metabolic activation by S9 mix by the repeatable induction of structural chromosomal aberrations (test concentrations of 900 µg/mL and higher), which is confirmed by increased frequencies of exchanges. There was no biologically relevant increase of structural aberrations in the absence of metabolic activation nor were there any increased frequencies of polyploid cells.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August - October 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital/ß-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Experiment 1: 3, 10, 33, 100, 333, 1000, 2500, 5000 µg/plate
Experiment 2: 33, 100, 333, 1000, 2500, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: solubility

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: sodium azide for TA1535, TA100; 4-nitro-o-phenylenediamine for TA1537, TA98; methylmethanesulfonate for TA102

Details on test system and experimental conditions:

METHOD OF APPLICATION: experiment 1 plate incorporation assay; experiment 2 preincubation

DURATION
- Preincubation period: 60 min
- Exposure duration: atmleast 48 hrs

NUMBER OF REPLICATIONS: triplicates, 2 experiments

Evaluation criteria:

mutagenic response if revertant rate is twice the solvent control for strain TA98, TA100, TA102 and thrice the solvent control for strain TA1535 and TA1537

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA: all assays with the test substance and negative and positive controls in the range of historical control data

Conclusions:

Test OECD 471: negative.
There was no indication of genotoxic activity in the Salmonella reverse mutation assay with S. typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 both without and with metabolic activation.

Executive summary:

There was no indication of genotoxic activity in the Salmonella reverse mutation assay with S. typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 both without and with metabolic activation when acetophenone was tested according to OECD Guideline 471 at concentrations up to 5000 µg/plate .

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 29-AUG-2006 to 19-FEB-2007

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640, RPMI 1640 plus HT, RPMI 1640 plus HAT
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from phenobarbital/ß-naphthoflavone induced rat liver

Test concentrations with justification for top dose:

Experiment 1: - S9: 0, 150, 300, 600, 900, 1200 µg/mL (ca. 10 mM); +S9: 0, 75, 150, 300, 600, 900 µg/mL
Experiment 2: - S9: 0, 150, 300, 600, 900, 1200 µg/mL (ca. 10 mM); +S9: 0, 600, 800, 900, 1000, 1100 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: solubility

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension

DURATION
- Exposure duration: 4 hrs in experiment 1; 24 hrs without S9 and 4 hrs with S9 in experiment 2
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): 10-.15 d
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays): TFT

NUMBER OF REPLICATIONS: 2 parallel cultures in two separate experiments

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

Number of colonies with evaluation of colony size distribution by (a) absolute size of the colony: large colony > 1/3 of the well; (b) optical density of small colony > large colony
Mutation frequency: positive test if the induced mutation frequency reproducibly exceeds a threshold of > 126 colonies per 1,000,000 cells above the solvent control

Statistics:

dose dependence by linear regression analysis, significant if p<0.05

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

No substantial and reproducible dose-dependent increase in mutant colony numbers; an isolated increase in one culture at 900 µg/mL was considered as biologically irrelevant fluctuation

Conclusions:

Interpretation of results (migrated information): negative

Acetophenone showed no mutagenic activity in the mouse lymphoma TK assay both in the absence and presence of metabolic activation.

Executive summary:

Acetophenone was tested for genotoxic activity in the mouse lymphoma thymidine kinase assay both in the presence and absence of S9 mix up to cytotoxic concentrations (dose range 75 -1200 µg/mL) in two separate experiments with two replicates each. Exposure duration was 4 or 24 hrs without S9 and 4 hrs with S9. The test was performed according to OECD Guideline 476 (GLP study). There was no indication of a mutagenic activity at the thymidine kinase locus in mouse lymphoma L5178Y cells.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In vivo micronucleus assay (OECD 474, GLP, mouse, i.p.): negative

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:

February to June 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Arald Winkelmann
- Age at study initiation: minimum 7 weeks (6-12 weeks at start of acclimation)
- Weight at study initiation:
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing: groups of 5
- Diet (e.g. ad libitum)
- Water (e.g. ad libitum
- Acclimation period: 1 week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 55 +- 10
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: cotton seed oil
- Justification for choice of solvent/vehicle: solubility and relative non-toxicity
- Concentration of test material in vehicle: 10.3-51.5 mg/mL
- Amount of vehicle : 10 mL/kg bw

Frequency of treatment:

single

Post exposure period:

44 hrs; additionally 68 hrs fur a further negative control and high-dose group

Dose / conc.:

515 mg/kg bw (total dose)

Dose / conc.:

257.5 mg/kg bw (total dose)

Dose / conc.:

103 mg/kg bw (total dose)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: i.p.
- Doses / concentrations: 40 mg/kg bw

Tissues and cell types examined:

peripheral blood erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Dose selection based on preceeding study on toxicity for determination of MTD: MTD = 515 mg/kg bw/d after intraperitoneal application; doses correspond to 0.2-, 0.5- and 1-fold MTD

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
44 and 68 hrs after application

METHOD OF ANALYSIS:
at least 10,000 cells scored per animal

Evaluation criteria:

Relative PCE = proportion of immature polychromatic erythrocytes among total number of erythrocytes
Criteria for positive results:
- dose-related increase of micronucleated erythrocytes
- biologically relevant increase of micronucleated erythrocytes for at least one of the dose groups
- according to OECD Guideline 422 a statistitically significant increase additionally to the biologically relevant increase

Statistics:

Non-parametric Mann-Whitney test

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Clinical signs of toxicity in test animals: at the MTD all treated animals showed reduction of spontaneous activity, prone position, apathy, palpebral closure, uncoordinated movements
- Evidence of cytotoxicity in tissue analyzed: yes, relative changes of PCE

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no increase for test substance
- Appropriateness of dose levels and route: yes, testing up to MTD
- Statistical evaluation: no significant changes

Conclusions:

Interpretation of results: negative
There were no increased incidences of micronuclei in mouse peripheral blood erythrocytes.

Executive summary:

No increased incidences of micronuclei were observed in mouse peripheral blood erythrocytes after single intraperitoneal application of dosages of 103, 257.5, 515 mg/kg bw corresponding to 0.2, 0.5 and 1.0 of the MTD. The test was performed according to OECD Guideline 474 with sampling times of 44 and 68 hrs.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro, acetophenone was found not to induce gene mutations in a Salmonella typhimurium reverse mutation assay (Key study/guideline study: Sokolowski, 2006) and in a mouse lymphoma cell mutagenicity assay at the thymidine kinase locus (Key study/guideline study: Wollny et al., 2007) when tested up to cytotoxic concentrations. The absence of a mutagenic activity in bacteria is supported by further data from Salmonella typhimurium reverse mutation studies (Commoner, 1976; Elliger et al., 1984; Florin et al., 1980), from a mutagenicity assay with Escherichia coli strains being deficient and proficient in DNA repair (DNA polymerase activity) (Fluck et al., 1976), and from a DNA repair assay in Salmonella typhimurium TA 1535/pSK1002 (umu test) (Ono et al., 1991).

 

A clastogenic potential was indicated in an in vitro chromosome aberration test in V79 cells, only in the presence of metabolic activation and at test concentrations of at least 900 µg/mL (Key study/guideline study: Höpker, 2007). In contrast, there was no indication of a clastogenic activity in an in vivo micronucleus assay in peripheral blood erythrocytes after application up to the MTD (515 mg/kg bw by intraperitoneal injection) (Key study/guideline study: Hofman-Hüther, 2008).

 

There exists some weight of evidence from in vitro studies with artificial test conditions that acetophenone principally has the capacity to be activated in the presence of UV radiation (photosensitising effect) or oxidative agents to induce modifications in DNA or nucleotides or in repair deficient strains of E. coli (Demidov et al., 1991; Epe et al., 1993; Adam et al., 2001, 2002; Mennigmann, 1972.; Fix and Bockrath, 1983; Midorikawa et al., 2004). All data come from test systems that are not validated for assessment of a mutagenic potential under biologically relevant conditions. Up to now, there are no data to assess if mechanisms of photosensitization and photooxidation play any role in mammalian cells in vitro or in vivo.

Justification for classification or non-classification

According to CLP and EC regulation 1272/2008 no classification of acetophenone for germ cell mutagenicity results. Based on the available key studies, there is no mutagenic potential in vitro. There is no evidence of a clastogenic effect in somatic cells in vivo in a guideline study, so that the indication of a possible clastogenic effect from an vitro study in the presence of metabolic activation is not supported in the in vivo condition.