Pentane-2,4-dione

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test, Salmonella strains TA1535, 1537, 1538, 98, and 100 (+/- S9-mix): negative.
HGPRT in vitro, CHO cells (+/- S9-mix): negative
SCE, CHO cells (+/- S9-mix): positive

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Conduction and documentation of study acceptable. Study report available.

Principles of method if other than guideline:

2,4-pentanedione concentrations used in the test did not produce excessive cytotoxic inhibition of mitotic CHO cells as derived from preliminary investigations. The highest three doses were selected for the determination of the incidence of chromosomal aberrations. Chromosomes were prepared by standard methods and stained using the Fluorescence plus Giemsa (FPG) technique that is used for visualization of sister chromatid exchanges. CHO cells were exposed to 2,4-pentanedione and appropriate controls for a 6 hour period in the absence of metabolic activation. Indirect genotoxic potential, requiring metabolic activation by liver S9-homogenate, was studied with a 2 hour exposure period to the test substance and the S9 activation system. Following the exposure period, cells were rinsed, fresh medium was added and the cells were then harvested at 14 or 22 hours after the start of exposure for testing performed with or without activation. A total of fifty cells/culture/harvest interval was examined for chromosome damage using duplicate cultures for the test agent and controls. At least 5 dose levels were tested both with and without metabolic activation. Incidence of chromosome damage was determined for the highest 3 doses which did not produce excessive cytotoxic inhibition of cell division (mitosis). The number of chromatid and chromosome type aberrations, the total number of aberrations per 50 cells examined (with and without including gaps in the total) and the level of statistical significance were determined. Cyclophosphamide (1 .5 µg/mL, with S9-mix) and triethylenamine (1.5 µg/mL, without S9-mix) were used as the positive control agents to assure the reliability and sensitivity of the test system for detecting clastogens dependent and independent of metabolic activation.

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):

CHO-K1-BH4 (subclone D1)

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix from livers of Sprague-Dawley rats pretreated with Aroclor 1254

Test concentrations with justification for top dose:

0.04-0.12 mg/mL without S9-mix; 0.06-0.14 mg/mL with S9-mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 100 µg ethylmethanesulfonate/mL without S9-mix and 300 µg dimethylnitrosamine/mL with S9-mix

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In tests performed without S9 activation, all three of the dose levels of 2,4-pentanedione evaluated produced significant increases in the number of CAs. The proportion of cells with chromosome aberrations ranged from 95 % to 100 % in comparison to an incidence of 3% aberrant cells for the negative control. In contrast, cells tested under more realistic physiologica l conditions (namely in the presence of an S9 activation system) did not show increased numbers of CA in comparison to values of control cultures. 2,4-Pentanedione was considered by the study authors to be highly clastogenic (chromosome breaking) to CHO cells in tests performed without metabolic activation but it was not clastogenic when tested in the presence of a metabolic activation under the conditions of this in vitro test system.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Cells were cultured for an additional 10 hours following the normal division time of 12 hours (2,4-pentanedione produced a significant delay in cell division cycle), thus allowing the cells a period of 22 hours to recover and complete DNA synthesis. This extended growth period enabled higher dose levels to be evaluated, and allowed the cells sufficient time to replicate DNA which is necessary for observation of synthesis-dependent types of chromosome damage. Extending the chromosome sampling period to compensate for cell cycle delays increased the sensitivity of the test system, and unequivocally demonstrated that the test substance produced CAs when tested for direct clastogenic potential in CHO cells. 2,4-Pentanedione was considered by the study authors to be highly clastogenic (chromosome braking) to CHO cells in the tests performed without metabolic activation. However there are some peculiarities in the test without metabolic activation which raise doubts about the quality of the experimental performance of the study: It is an extremely unusual finding that a test substance produces in 98 % of the target cells chromosome aberrations at a dose level (0.03 mg/mL) that practically does not increase the cell cycle time (increase by only 5%) and reduces the cell number by only 34% (data from a pre-study, measured 6 h after a 6 h treatment). Although in the present study the preparation interval was 10 h longer (16 h), a dramatic change in cell proliferation in a mass culture would not be expected within this difference of time. This is confirmed by the statement of the authors that the highest three doses which did not produce excessive cytotoxic inhibition of mitotic cells (0.03, 0.10 and 0.12 mg/mL) were scored for incidence of chromosome aberrations. In addition, there is no dose related increase of chromosome aberrations. This might be understandable with regard to a similar lack of a dose relationship for cytotoxicity. However, in the present study there is a dose relationship for cytotoxicity (relative cell cycle increase at 0.03; 0.06 an d 0.10 mg/mL of 5, 69 and 98 %, respectively). Another unusual finding is, that there are very big differences in the number of aberrations per 50 cells (excluding gaps) within parallel cultures. A total of 146 aberrations is found in one sample of 50 cells while a total of only 54 aberrations is found in the cells of the parallel culture after treatment with 0.03 mg/mL test substance. A similar discrepancy is obvious in the data of the 0.12 mg/mL treatment group (42/137). Furthermore, it is very strange that in heavily damaged cells with 100% cells carrying aberrations (0.03 and 0.10 mg/mL) the number of gap-events remains in the negative control range.

Conclusions:

Interpretation of results (migrated information):
ambiguous

Reference 2

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Meets generally accepted scientific standards, well documented and acceptable for assessment.

Qualifier:

equivalent or similar to guideline

Guideline:

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

Principles of method if other than guideline:

Preliminary trials were performed to determine an appropriate range of test concentration in which the highest concentration would kill no more than 90 % of the treated CHO cells. In this preliminary experiment concentrations above 2.0 mg/mL in the presence of S9 and 3.0 mg/mL in the absence of S9 virtually killed all cells. All incubations were run in duplicate. Cells were exposed to at least five concentrations which allowed sufficient cell survival for assessment of survival and quantification of mutants. Cells were exposed for 5 h in tests both with and without metabolic activation. The mutant fraction was determined after a 9 to 12 day subculturing period to allow expression of the mutant phenotype. Metabolic activation was applied by S9 liver homogenate, prepared from Aroclor 1254-induced, Sprague-Dawley male rats.

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO-K1-BH4 (subclone D1)

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix from livers of Sprague-Dawley rats pretreated with Aroclor 1254

Test concentrations with justification for top dose:

Without S9-mix: 0.005-1.5 mg/mL
Wit S9-mix: 0.005-1.0 mg/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ethylenemethanesulfonate (200 µg/mL)

Details on test system and experimental conditions:

DURATION
- Exposure duration: 5 h
- Expression time (cells in growth medium): 9-12 days

NUMBER OF REPLICATIONS: incubations were run in duplicates

SURVIVING FRACTION:
Determination 18-24 hrs after removal of the test chemical using 4 plates/culture and 100 cells per plate.

MUTANT FRACTION:
200000 cells per plate in 5 plates per dose in selective medium.
Plating efficiency in non-selective medium: 4 plates/dosed culture with 100 cells/plate.

DETERMINATION OF CYTOTOXICITY
- Method:
other: Preliminary test where number of live cells was investigated (no data about applied method given)

Statistics:

After tranformation of the mutation frequencies (MF) according to the conversion method of Box and Cox (1964), CHO data were analyzed according to Snee and Irr (Snee, R .D. und J .D . Irr, Mutation Research . 85 (1981), 77-93).

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Only data from a preliminary test are available. The concentrations used should give at least 10% survival at the highest concentration used.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

2,4-Pentanedione did not produce any reproducible or statistically significant increases in the incidences of mutations of CHO cells at concentrations between 0.005 to 1.5 mg/mL in tests without an S9 metabolic activation system or from 0.005 to 1.0 mg/mL with S9-mix. Random cultures with increased mutant values were within the typical range of variability for this test in the investigating laboratory and the increases were not reproducible in the duplicate cultures/dose level. 2,4-Pentanedione was not considered to be an active gene mutagen under the conditions of the CHO test system.

Conclusions:

Under the evaluated conditions no increased mutation frequency was observed. The result was evaluated to be negative.

Executive summary:

2,4-Pentanedione did not produce any reproducible or statistically significant increases in the incidences of mutations of CHO cells at concentrations between 0.005 to 1.5 mg/mL in tests without an S9 metabolic activation system or from 0.005 to 1.0 mg/mL with S9-mix. Random cultures with increased mutant values were within the typical range of variability for this test in the investigating laboratory and the increases were not reproducible in the duplicate cultures/dose level. 2,4-Pentanedione was not considered to be an active gene mutagen under the conditions of the CHO test system.

Reference 3

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Conduction and documentation of study very acceptable. Study report available.

Principles of method if other than guideline:

In preliminary investigations it was shown that concentrations of 2,4-pentanedione above 2.0 mg/mL were lethal to CHO cells and a concentration of 0.3 mg/mL produced approximately a 28% inhibition of growth when tested with an S9 metabolic activation system and a 38% inhibition of growth in tests without S9-mix. For the main test the maximum concentrations chosen were 0.1 and 0.3 mg/mL in the absence and presence of a metabolic activation system. Cells were incubated in duplicate with at least 5 dose levels and SCE production was determined for the three highest doses which did not produce excessive cytoxic inhibition of cell division. In the absence of S9-mix 2,4-pentanedione was directly added to the culture medium and incubated for five hours. In the presence of S9-mix cells were exposed for two hours to the test substance. Bromodeoxyuridine (3 µg/mL) was present in the growth medium during exposure. Metabolic activation was achieved by S9 liver homogenate, prepared from Aroclor 1254-induced Sprague-Dawley male rats. A total of 25 cells/culture were examined for the induction of SCE. As an indicator of genotoxicity the number of SCE/cell as well as mean number of SCE/chromosome were determined. Positive (100 µg ethylmethanesulfonate/mL without S9-mix and 300 µg dimethylnitrosamine/mL with S9-mix), negative (culture medium) and solvent controls (H20) were included as well.

GLP compliance:

yes

Type of assay:

sister chromatid exchange assay in mammalian cells

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO-K1-BH4 (subclone D1)

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix from livers of Sprague-Dawley rats pretreated with Aroclor 1254

Test concentrations with justification for top dose:

0.02-0.1 mg/mL (without S9-Mix); 0.03-0.3 mg/mL (with S9-MIx)

Vehicle / solvent:

water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 100 µg ethylmethanesulfonate/mL without S9-mix and 300 µg dimethylnitrosamin/mL with S9-mix

Details on test system and experimental conditions:

Number of replicates: duplicate cultures
Exposure duration:
Without S9 mix: 5 h
With S9 mix: 2 h
Bromodeoxyuridine: 3 µg/mL (during treatment and culture period after treatment)

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

A statistically significant increase in the number of SCEs was produced by the three highest doses of the test substance evaluated for SCEs in the absence of a metabolic activation system. The 0.1 mg/mL dose produced a highly significant increase in the incidence of SCEs which was greater than a comparable concentration of the positive control agent EMS. 2,4-Pentanedione was therefore considered a highly active genotoxic agent in the SCE test without S9 activation.
In the presence of S9 activation a statistically significant increase in the SCE values was produced with all three doses of the test agent in
comparison to the untreated controls. The magnitude of the increase in SCEs was lower than in the test without S9 activation despite the use of a 3-fold greater amount of TS in this test. The positive increases in SCEs apparent in the test without S9 activation were also induced in the test with S9 activation. Although dose-response relationships were very steep in the test without S9-mix, and absent in the test with S9-mix, reproducible and statistically significant increases were detectable in both tests. 2,4-Pentanedione is therefore considered genotoxic particularly in the absence of S9-mix.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1: Production of SCEs without metabolic activation – 5 hour treatment:

Test chemicals	Total of chromosomes	Total of SCEs	SCEs per cell	Mean number SCEs per chromosome ± SD	Significance about solvent control
2,4-Pentanedione (mg/mL)
0.02	499	297	11.9	0.59±0.19	P<0.01
0.02	502	256	10.2	0.51±0.17
0.03	492	276	11.0	0.56±0.18	p<0.05
0.03	503	332	13.3	0.66±0.25	p<0.001
0.1	498	941	37.6	1.89±0.32	p<0.001
0.1	498	909	36.4	1.83±0.54	p<0.001
Solvent Control: H2O
5 µl/mL	500	231	9.2	0.46±0.18
5 µl/mL	498	203	8.1	0.41±0.15
Negative control
Medium	499	203	8.1	0.41±0.15	-
Positive control
(100 µg/mL)	496	695	27.8	1.40±0.37	p<0.001

Table 2: Production of SCEs with metabolic activation – 2 hour treatment:

Test chemicals	Total of chromosomes	Total of SCEs	SCEs per cell	Mean number SCEs per chromosome ± SD	Significance about solvent control
2,4-Pentanedione (mg/mL)
0.03	502	409	16.4	0.82±0.22	P<0.001
0.03	502	345	13.8	0.69±0.14	-
0.1	507	387	15.5	0.77±0.16	p<0.01
0.1	506	417	16.7	0.82±0.20	p<0.001
0.3	507	445	17.8	0.88±0.22	p<0.001
0.3	506	455	18.2	0.90±0.19	p<0.001
Solvent Control: H2O
5 µl/mL	499	305	12.2	0.61±0.21
5 µl/mL	499	307	12.3	0.61±0.16
Negative control
Medium	501	349	14.0	0.70±0.23
Positive control
DMN (300 µg/mL)	503	1644	65.8	3.27±0.92	p<0.001

Conclusions:

Interpretation of results: positive

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Conduction and documentation of study very acceptable. Literature reference and study report available. Restrictions: no use of TA102/E.coli; no second positive control substance for treatment with S9-mix.

Principles of method if other than guideline:

Test performed according to standard protocols by inclusion of a metabolic activation system (S9-Mix from livers of Sprague-Dawley rats pretreated with Aroclor 1254). In preliminary trials with strain TA 100 only, a concentration of 97.4 mg/plate proved to completely inhibit bacterial growth. The next lower concentration of 30 mg/plate showed some toxic effects. Accordingly, doses selected on the basis of these trials were 0.3, 1.0, 3.0, 10.0 and 30.0 mg/plate. Incubations were run in triplicate at 37°C for 24 to 48 hours. Plates were examined for the condition of their background lawns and growth was recorded as either confluent (non-toxic), sparse (moderately toxic) or absent (toxic). The solvent of choice was water. Concurrent solvent and positive controls were run in each test.
Positive control substances were without S9-mix 0.01 mg 4-nitro-o-phenylenediamine/ plate for TA98 and TA1538, 0.01 mg sodium azide for TA100, 0.06 mg 9-aminoacrididine/plate for TA1537 and with S9-mix 0.01 mg 2-aminoanthracene for all strains.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix from livers of Sprague-Dawley rats pretreated with Aroclor 1254

Test concentrations with justification for top dose:

0.3 - 30 mg/plate

Vehicle / solvent:

Water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without S9-mix: 0.01 mg 4-nitro-o-phenylenediamine/ plate for TA98 and TA1538, 0.01 mg sodium azide for TA100, 0.06 mg 9-aminoacrididine/plate for TA1537. With S9-mix 0.01 mg 2-aminoanthracene for all strains.

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS: triplicates
INCUBATION TIME: 24 to 48 h
METABOLIC ACTIVATION SYSTEM:
S9 liver homogenate, prepared from Aroclor 1254-induced, Sprague-Dawley male rats, was purchased from Microbiological Associates, Bethesda, MD. For tests with metabolic activation, 0.5 mL of S9 mix containing 50 µl of S9 was added per plate.

Evaluation criteria:

Doubling and a dose-response relationship of the number of revertants/plate in the Salmonella typhimurium strains.

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:

cytotoxicity

Remarks:

30 mg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

30 mg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Table 1: Number (arithmetic mean) of colonies of histidine-prototrophic back-mutants in experiments without microsomal activation.

Concentration (mg/plate)	TA98	TA100	TA1535	TA1537	TA1538
Solvent, 100	21	140	35	4	5
Positive, 0.01	897	1979	1809	46	984
0.3	22	145	25	3	6
1	25	133	34	5	8
3	17	123	31	3	5
10	9	50	13	4	3
30	toxic	toxic	toxic	toxic	toxic

Table 2: Number (arithmetic mean) of colonies of histidine-prototrophic back-mutants in experiments with microsomal activation.

Concentration (mg/plate)	TA98	TA100	TA1535	TA1537	TA1538
Solvent, 100	36	127	11	7	17
Positive, 0.01	1697	1295	79	88	422
0.3	29	134	13	9	12
1	32	129	11	7	14
3	24	100	8	3	12
10	17	57	10	5	15
30	0	0	0	toxic	toxic

 

Conclusions:

Interpretation of results:
negative

Executive summary:

Test performed according to standard protocols by inclusion of a metabolic activation system (S9-Mix from livers of Sprague-Dawley rats pretreated with Aroclor 1254). In preliminary trials with strain TA 100 only, a concentration of 97.4 mg/plate proved to completely inhibit bacterial growth. The next lower concentration of 30 mg/plate showed some toxic effects. Accordingly, doses selected on the basis of these trials were 0.3, 1.0, 3.0, 10.0 and 30.0 mg/plate. Incubations were run in triplicate at 37°C for 24 to 48 hours. Plates were examined for the condition of their background lawns and growth was recorded as either confluent (non-toxic), sparse (moderately toxic) or absent (toxic). The solvent of choice was water. Concurrent solvent and positive controls were run in each test. 2,4-pentanedione did not produce a doubling or a dose-response relationship of the number of revertants/plate in the Salmonella typhimurium strains used neither in the absence nor in the presence of a metabolic activation system. Dose selection appeared to be in a suitable range, because in tests both with and without metabolic activation, bacteriotoxicty was observed at 30 mg/plate (highest dose tested) with all strains. Well proven positive controls did produce mutagenic effects demonstrating the functionality of the test system. It can therefore be concluded that 2,4-pentanedione is not mutagenic under the conditions of the assay.

Genetic toxicity in vivo

Description of key information

CA, mouse, spermatogonial cells (OECD TG 483): negative
MNT, mouse, bone marrow, intraperitoneal: positive
MNT, mouse, bone marrow, inhalation: negative
MNT, rat, bone marrow, inhalation: negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Adopted according to OECD SIDS (publicly available peer reviewed source). Original document not available.

Principles of method if other than guideline:

Ten animals per sex and dose group were exposed to 0, 100, 400 ppm of 2,4-pentanedione vapour for five consecutive days, 6 h/day. Fourteen animals per sex (7 per harvest time) were exposed to 800 ppm. The doses were chosen based on the results of previous acute and repeated exposure studies. Due to unexpected mortalities among male and female rats exposed to the 800 ppm target concentration, that target concentration was lowered after the second exposure day to 650 ppm for the surviving male rats. An additional target concentration of 600 ppm was added to the study and was administered to both male and female rats by whole body exposure to vapor 6 hours per day for 5 consecutive days. Ten animals per sex (5 at each harvest time) were sacrificed 6 or 24 hours after the fifth exposure, the cyclophosphamide treated (positive control) animals were sacrificed at the same time as the 24 h post-2,4-PD treatment group. Bone marrow cells were harvested and evaluated for chromosomal damage. Colchicine was dosed by intraperitoneal injection (4 mg/kg) two to three hours prior to
sacrifice. Five hundred cells/animal/sacrifice were scored to determine mitotic index. Fifty metaphase cells/animal/sacrifice were scored for the induction of chromosomal aberrations. Evaluations were made on the chromosome number, specific type of chromosome- or chromatid-type aberrations and exchanges and further classified for deletions and exchanges. Gaps were noted but were not included as aberrations when computing the proportion of aberrant cells or for use in statistical analysis. Severely damaged cells (10 or more breakage events) and pulverized cells were scored as 10 aberrations/cell, but no attempt was made to classify the types of damage in such cells. Behaviour and appearance of animals were observed prior to, during and following each exposure. Body weights were measured prior to the first and prior to the fifth exposure.

GLP compliance:

yes

Type of assay:

chromosome aberration assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Route of administration:

inhalation: vapour

Details on exposure:

TYPE OF INHALATION EXPOSURE: whole body

TEST ATMOSPHERE
- Brief description of analytical method used:
Chamber concentrations of 2,4-pentanedione were analyzed by gas chromatography approximately 6 times during each 6-hour exposure period . The mean detected chamber concentrations of 2,4-pentanedione were 100.6, 414, 609 and 695 ppm for target concentrations of 100, 400,
600 and 800 ppm, respectively. The 800 ppm target concentration was lowered to 650 ppm after the second day of exposure due to animal deaths. No TS was found in the air-only (negative) control chamber.
- Samples taken from breathing zone: yes

Duration of treatment / exposure:

6 h

Frequency of treatment:

5 consecutive days

Remarks:

Doses / Concentrations:
100, 400, 600, 800 ppm
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
415, 1661, 2492, 3322 mg/m3
Basis:
nominal conc.

No. of animals per sex per dose:

Control: 10 animals per sex
100 ppm: 10 animals per sex
400 ppm: 10 animals per sex
600 ppm: 10 animals per sex
800 ppm: 10 animals per sex

Control animals:

yes, sham-exposed

Positive control(s):

Cyclophosphamide, 30 mg/kg bw (5 animals per sex)

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

CITERIA FOR DOSE SELECTION:
The highest concentrations of 600 ppm and 650 ppm were approximately 50 % below the LC50 values determined in female rats in studies on the acute toxicity after inhalation.

TREATMENT AND SAMPLING TIMES:
Ten animals per sex (5 at each harvest time) were sacrificed 6 or 24 hours after the fifth exposure, the cyclophosphamide treated (positive control) animals were sacrificed at the same time as the 24 h post-2,4-PD treatment group. Bone marrow cells were harvested and evaluated for chromosomal damage. Colchicine was dosed by intraperitoneal injection (4 mg/kg bw) two to three hours prior to sacrifice .

METHOD OF ANALYSIS:
Five hundred cells/animal/sacrifice were scored to determine mitotic index. Fifty metaphase cells/animal/sacrifice were scored for the induction of chromosomal aberrations. Evaluations were made on the chromosome number, specific type of chromosome-or chromatid-type aberrations and exchanges and further classified for deletions and exchanges. Gaps were noted but were not included as aberrations when computing the proportion of aberrant cells or for use in statistical analysis. Severely damaged cells (10 or more breakage events) and pulverized cells were scored as 10 aberrations/cell, but no attempt was made to classify the types of damage in such cells.

OTHER:
Less than 50 metaphase spreads per animal were evaluated on 2 of the test animals. One of these animals was a female in the 6 hr sacrifice group exposed to 600 ppm, the second was a female animal in the 24 hr sacrifice group exposed to 400 ppm. The mitotic index in both animals was less than 1%.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

16/28 animals of the 800 ppm group died or were sacrificed moribund.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not specified

Additional information on results:

All female rats and two out of fourteen male rats exposed to 800 ppm died or were sacrificed moribund between study day 1 and 3. Among the 14 male und 14 female rats exposed to 600 ppm, three female rats died during the exposure regime. Clinical signs prior to death were ataxia and/or prostration. Male rats exposed to 800 ppm and both male and female rats exposed to 600 ppm lost weight. In the 400 ppm exposure group both male and female rats had depressed body weight gains during the exposure regime. 2,4-Pentanedione produced one statistically significant increase in the incidence of chromosomal aberrations in the 6 h sacrifice group of male rats exposed at a target concentration of 100 ppm as compared to air-exposed (negative control). There were no statistically significant increases in the incidence of chromosomal aberrations among male rats exposed at target concentrations of 400, 600 or 800 ppm in the 6 h sacrifice group. No statistically significant or concentration-related increases in the incidence of chromosomal aberrations were observed among 2,4-pentanedione exposed male rats in the 24 h sacrifice group or among any of the 2,4-pentanedione exposed female rats. Because the statistically significant observation among male rats exposed at 100 ppm was small in magnitude (5.2 %) and did not persist at the 24 h sacrifice, 2,4-pentanedione was not considered to have biologically significant clastogenic activity in rats under the conditions of this test.

Conclusions:

Interpretation of results (migrated information): negative

Reference 2

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Adopted according to OECD SIDS (publicly available peer reviewed source). Original document not available.

Qualifier:

according to guideline

Guideline:

OECD Guideline 483 (Mammalian Spermatogonial Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

other: mouse spermatogonia chromosomal aberration test

Species:

mouse

Strain:

NMRI

Sex:

male

Route of administration:

oral: gavage

Vehicle:

- Vehicle: Water
- Amount of vehicle: 10 mL/kg bw (negative control)

Duration of treatment / exposure:

single application

Frequency of treatment:

single application

Remarks:

Doses / Concentrations:
800 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Adriblastin (5 mg/kg bw)

Tissues and cell types examined:

Spermatogonial cells

Details of tissue and slide preparation:

CITERIA FOR DOSE SELECTION:
In a preceding study the bioavailability of the test material was confirmed. It was determined that 800 mg/kg bw administered orally were close to the MTD as shown by signs of toxicity in the treated animals such as reduction of spontaneous activity, eyelid closure, apathy and tremor. In this previous test the systemic distribution of the test substance was also checked and it was found that after oral administration the test substance was detectable in blood serum up to four hours post-treatment.

TREATMENT AND SAMPLING TIMES:
For the investigation of chromosomal aberrations in germ cells, spermatogonial cells were prepared 24 and 48 hours after single test substance administration. Five male mice were examined at each time point.

METHOD OF ANALYSIS:
At least 100 metaphases per animal were scored for cytogenetic damage. Gaps, breaks, fragments, deletions, exchanges and chromosomal disintegrations were recognized as structural chromosome aberrations.

Statistics:

Statistical analysis of results observed was included and confirmed by the non-parametric Mann-Whitney test.

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

After preparation and examination of spread spermatogonial cells (100 cells of each animal, i.e. 500 per dose and time point were analysed) no reduction in the mitotic index could be observed, indicating that 2,4-pentanedione at the indicated dose and the indicated application route was not cytotoxic for spermatogonial cells. No statistically significant or biologically relevant increase in the number of numerical and structural aberration as compared to vehicle treated controls could be found. Aberration rates were 0.8 % and 1.0 % for the 24 h and the 48 h treatment, respectively, as compared to the vehicle control value of 0.6 % . The mean aberration frequencies observed after treatment with 2,4-pentanedione were consistently below 2% aberrant cells exclusive gaps, given as the upper limit of a tolerable vehicle control value. The positive control showed a statistically significant response (9 % aberration rate excluding gaps). In conclusion, 2,4-pentanedione is being considered non-mutagenic under the conditions of this assay.

Conclusions:

Interpretation of results: negative

Reference 3

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Conduction and documentation of study very acceptable. Study report available.

Principles of method if other than guideline:

As described in Union Carbide Corporation: Bushy Run Research Center Standard Operating Procedures:
Swiss Webster mice were exposed to 0, 100, 400 and 600 ppm of 2,4 - pentanedione vapour for five consecutive days, 6 h/day by whole body exposure. Bone marrow from test substance and air-only treated animals was collected from femurs 6 and 24 hours after final exposure. Bone marrow from positive control animals was collected approximately 24 hours after administration of the positive control substance since positive results have been consistently observed at this collection time. Colchicine was dosed by intraperitoneal injection (4 mg/kg) two to three hours prior to sacrifice. Five hundred cells/animal/sacrifice were scored to determine mitotic index. Hundred metaphase cells/animal/sacrifice were scored for the induction of chromosomal aberrations. Evaluations were made on the chromosome number, specific type of chromosome- or chromatid-type aberrations and exchanges. Gaps were noted but were not included as aberrations when computing the proportion of aberrant cells or for use in statistical analysis. Severely damaged cells (10 or more breakage events) and pulverized cells were scored as 10 aberrations/cell, but no attempt was made to classify the types of damage in such cells. All animals were observed individually for mortality and overt signs of toxicity twice each day on study days 1-5 and once (in the morning) on study day 6. During exposure, the animals were observed on a group basis. Body weight data were collected for all animals prior to initiation of the first exposure and prior to sacrifice.

GLP compliance:

yes

Type of assay:

chromosome aberration assay

Species:

mouse

Strain:

Swiss Webster

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Swiss webster mice (ND4) from Harlan Sprague-Dawley, Inc. (Indianapolis, IN)
- Age at study initiation: 36 days
- Housing: 1 per cage
- Diet: Agway Prolab animal diet (Rat, Mouse, Hamster 3000, Agway, Inc.), ad libitum
- Water: ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 40-70
- Photoperiod: 12 hrs dark / 12 hrs light

Route of administration:

inhalation: vapour

Details on exposure:

TYPE OF INHALATION EXPOSURE: whole body

TEST ATMOSPHERE
- Brief description of analytical method used:
Chamber concentrations of 2,4-pentanedione were analyzed by gas chromatography (FID) twice each hour during the 6-hour exposure periods.
- Samples taken from breathing zone: yes
-Air changes (per hour): 13-14
-Chamber volume: 900 L
-Air-flow: 200 L/min
-Generation of atmosphere:
Liquid test substance was metered from a piston pump into a heated glass evaporator. The evaporator temperature ranged from 32.2 to 43.7 °C.
The oxygen content of the chambers was 20.8%.

Duration of treatment / exposure:

6 h

Frequency of treatment:

5 consecutive days

Remarks:

Doses / Concentrations:
100, 400, 600 ppm
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
415, 1661, 2492 mg/m3
Basis:
nominal conc.

No. of animals per sex per dose:

Positive control: 5 animals per sex
Negative control: 10 animals per sex
100 ppm: 10 animals per sex
400 ppm: 10 animals per sex
600 ppm: 14 animals per sex

Control animals:

yes, sham-exposed

Positive control(s):

Cyclophosphamide monohydrate, 30 mg/kg bw, dissolved in distilled water (by i.p. administration)
Lot No.: 19F-0254

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The highest concentration of 600 ppm was approximately 50 % below the LC50 values determined in female rats in studies on the acute toxicity after inhalation.

TREATMENT AND SAMPLING TIMES:
Bone marrow from test substance and air-only treated animals was collected from femurs 6 and 24 hours after final exposure. Bone marrow from positive control animals was collected approximately 24 hours after administration of the positive control substance since positive results have been consistently observed at this collection time. Colchicine was dosed by intraperitoneal injection (4 mg/kg bw) two to three hours prior to sacrifice.

METHOD OF ANALYSIS:
Five hundred cells/animal/sacrifice were scored to determine mitotic index. Hundred metaphase cells/animal/sacrifice were scored for the induction of chromosomal aberrations. Evaluations were made on the chromosome number, specific type of chromosome- or chromatid-type aberrations and exchanges. Gaps were noted but were not included as aberrations when computing the proportion of aberrant cells or for use in statistical analysis. Severely damaged cells (10 or more breakage events) and pulverized cells were scored as 10 aberrations/cell, but no attempt was made to classify the types of damage in such cells.

Statistics:

The data for quantitative continuous variables were intercompared for the treatment groups and the control group by use of Levene's test for equality of variances, analysis of variance (ANOVA), and t-tests. The t-tests were used when the F value from the ANOVA was significant. When Levene's test indicated similar variances, and the ANOVA was significant, a pooled t-test was used for pairwise comparisons. When Levene's test indicated heterogeneous variances, all groups were compared by an ANOVA for unequal variances followed, when necessary, by a separate variance t-test for pairwise comparisons. Chromosomal aberration data were statistically evaluated using the Mann-Whitney U-Test. For all statistical tests the probability value of <0.05 (two-tailed) was used as the critical level of significance.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Ten females in the 600 ppm exposure group died between study day 2 and 5.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not specified

Additional information on results:

No mortalities, no noteworthy clinical signs and no significant effects on body weight changes were observable in treated animals of the 0, 100 and 400 ppm dose group. Prostration was observed in females of the 600 ppm exposure group. Ten females in the 600 ppm exposure group died between study day 2 and 5.
2,4-Pentanedione did not produce significant, exposure-related increases in the frequencies of chromosomal aberrations in the bone marrow of male and female Swiss Webster mice sampled 6 or 24 hours after the final exposure to 0, 100, 400 or 600 ppm. The test substance was therefore not considered to be clastogenic under the conditions of the in vivo assay performed.
Chamber concentrations of 2,4-pentanedione were analyzed by gas chromatography twice each hour during the 6-hour exposure periods. The mean detected chamber concentrations of 2,4-pentanedione were 98.7, 415, and 590 ppm for target concentrations of 100, 400, and 600 ppm, respectively. No concentration above the estimated minimum detection limit of 5 ppm was detected in the air-only control chamber atmosphere during the study.

Conclusions:

Interpretation of results : negative

Additional information

Mutagenicity in bacterial cells:

One study (GLP) is available where the mutagenic potential of 2,4 -pentanedione was investigated in an Ames assay (BRRC48 -140, 1985). Salmonella strains TA1535, 1537, 1538, 98, and 100 were treated with 0.3 - 30 mg test substance/plate, with and without metabolic activation with S9-Mix from livers of Sprague-Dawley rats pretreated with Aroclor 1254. 2,4-pentanedione did not produce a doubling or a dose-response relationship of the number of revertants/plate in the Salmonella typhimurium strains used neither in the absence nor in the presence of a metabolic activation system. Dose selection appeared to be in a suitable range, because in tests both with and without metabolic activation, bacteriotoxicty was observed at 30 mg/plate (highest dose tested) with all strains. Well proven positive controls did produce mutagenic effects demonstrating the functionality of the test system. It can therefore be concluded that 2,4-pentanedione is not mutagenic under the conditions of the assay.

 

Mutagenicity in mammalian cells:

In a GLP study the mutagenicity of 2,4 -pentanedione was investigated in CHO cells in a HGPRT test(BRRC, 48 -174, 1986). The cells were treated with 0.005 -1.5 mg/mL (without S9 -mix) and 0.005 -1.0 mg/mL (with S9 -Mix) for 5 hours. 2,4-Pentanedione did not produce any reproducible or statistically significant increases in the incidences of mutations of CHO cells at concentrations between 0.005 to 1.5 mg/mL in tests without an S9 metabolic activation system or from 0.005 to 1.0 mg/mL with S9 -mix. Random cultures with increased mutant values were within the typical range of variability for this test in the investigating laboratory and the increases were not reproducible in the duplicate cultures/dose level. 2,4-Pentanedione was not considered to be an active gene mutagen under the conditions of the CHO HGPRT test system.

 

Sister chromatid exchange in mammalian cells:

In a GLP study 2,4 -pentanedione was investigated for its ability to induce sister chromatid exchange (SCE) in CHO cells. After initial cytotoxicity test, concentrations chosen were 0.1 and 0.3 mg/mL in the absence and presence of a metabolic activation system. Cells were incubated in duplicate with at least 5 dose levels and SCE production was determined for the three highest doses which did not produce excessive cytoxic inhibition of cell division. A statistically significant increase in the number of SCEs was produced by the three highest doses of the test substance evaluated for SCEs in the absence of a metabolic activation system. The 0.1 mg/mL dose produced a highly significant increase in the incidence of SCEs which was greater than a comparable concentration of the positive control agent EMS. 2,4-Pentanedione was therefore considered a highly active genotoxic agent in the SCE test without S9 activation.

In the presence of S9 activation a statistically significant increase in the SCE values was produced with all three doses of the test agent in comparison to the untreated controls. The magnitude of the increase in SCEs was lower than in the test without S9 activation despite the use of a 3-fold greater amount of test substance in this test. The positive increases in SCEs apparent in the test without S9 activation were also induced in the test with S9 activation. Although dose-response relationships were very steep in the test without S9-mix, and absent in the test with S9-mix, reproducible and statistically significant increases were detectable in both tests. 2,4-Pentanedione is therefore considered genotoxic particularly in the absence of S9-mix.

 

Chromosomal aberrations in mammalian cells:

In a GLP study, CHO cells were exposed to 2,4-pentanedione and appropriate controls for a 6 hour period in the absence of metabolic activation. Indirect genotoxic potential, requiring metabolic activation by liver S9-homogenate, was studied with a 2 hour exposure period to the test substance and the S9 activation system. 2,4 -pentanedione was tested in concentrations from 0.04-0.12 mg/mL without S9-mix and from 0.06-0.14 mg/mL with S9-mix. Results were considered as ambiguous, due to peculiarities which raised doubts about the quality of the experimental performance of the study (lack of dose-response relationship, discrepancies in parallel cultures, discrepancies cytotoxicity/aberration spectrum).

 

Chromosomal aberration in vivo:

Three studies are available where 2,4 -pentanedione was investigated on its potential to induce chromosomal aberration (CA) in vivo. Two GLP inhalation studies are available, where mice (UCC, 1994) and rats (UCC, 1990) were exposed to 100 -600 and 100 -800 ppm (corresponding to 415-2492 and 415-3322mg/m³) 2,4 -pentanedione, respectively, and the bone marrow was examined for CA. In a third study according to OECD TG 483 and GLP, NMRI mice were dosed by gavage with 800 mg/kg bw and spermatogonial cells were examined for CA (RCC-CCR, 2000). 2,4-Pentanedione did not produce significant, exposure-related increases in the frequencies of chromosomal aberrations in the bone marrow of male and female Swiss Webster mice sampled 6 or 24 hours after the final exposure to 0, 100, 400 or 600 ppm. Ten of fourteen animals died in the 600 pm dose group, whereas no mortality occurred in the other dose groups. The test substance was therefore not considered to be clastogenic under the conditions of the in vivo assay performed (UCC, 1994).

Mortality of Sprague-Dawley rats was observed when they were exposed to 600 and 800 ppm six hours per day at five consecutive days (UCC, 1990). Five animals per sex were sacrificed after 6 and 24 h after the last exposure. 2,4-Pentanedione produced one statistically significant increase in the incidence of chromosomal aberrations in the 6 h sacrifice group of male rats exposed at a target concentration of 100 ppm as compared to air-exposed (negative control). There were no statistically significant increases in the incidence of chromosomal aberrations among male rats exposed at target concentrations of 400, 600 or 800 ppm in the 6 h sacrifice group. No statistically significant or concentration-related increases in the incidence of chromosomal aberrations were observed among 2,4-pentanedione exposed male rats in the 24 h sacrifice group or among any of the 2,4-pentanedione exposed female rats. Because the statistically significant observation among male rats exposed at 100 ppm was small in magnitude (5.2 %) and did not persist at the 24 h sacrifice, 2,4-pentanedione was not considered to have biologically significant clastogenic activity in rats under the conditions of this test.

In a mouse spermatogonial assay, the animals were dosed with 800 mg/kg bw, which has been shown to be close to the MTD in a preceding study, where also the systemic distribution of the test substance was checked and it was found that after oral administration the test substance was detectable in blood serum up to four hours post-treatment. For the investigation of chromosomal aberrations in germ cells, spermatogonial cells were prepared 24 and 48 hours after single test substance administration. Five male mice were examined at each time point. After preparation and examination of spread spermatogonial cells (100 cells of each animal, i.e. 500 per dose and time point were analysed) no reduction in the mitotic index could be observed, indicating that 2,4-pentanedione at the indicated dose and the indicated application route was not cytotoxic for spermatogonial cells. No statistically significant or biologically relevant increase in the number of numerical and structural aberration as compared to vehicle treated controls could be found. Aberration rates were 0.8 % and 1.0 % for the 24 h and the 48 h treatment, respectively, as compared to the vehicle control value of 0.6 % . The mean aberration frequencies observed after treatment with 2,4-pentanedione were consistently below 2% aberrant cells exclusive gaps, given as the upper limit of a tolerable vehicle control value. The positive control showed a statistically significant response (9 % aberration rate excluding gaps). In conclusion, 2,4-pentanedione is being considered non-mutagenic under the conditions of this assay.

 

Micronucleus test in vivo:

Five GLP in vivo micronucleus tests are available. Two of them were conducted in rats with inhalation and intraperitoneal exposure (UCC, 1986; UCC, 1993), while the other 3 studies were performed in mice after inhalation (UCC, 1993) and intraperitoneal administration (UCC, 1986; UCC, 1993). Mice were treated with 2,4 -pentanedione from 200 -650 mg/kg bw (intraperitoneal) and 97, 405, 592 ppm (inhalation), and rats with 50 -400 mg/kg bw (intraperitoneal) and 97, 405, 592 ppm (corresponding to 403, 1682, 2458 mg/m3). An induction of micronuclei by 2,4 -pentanedione was observed after treatment of mice intraperitoneal. Treatment by the inhalation route had no influence on the frequency of micronuclei. Furthermore, intraperitoneal treatment of rats did not result in an increased frequency of micronuclei, indicating that the potency of 2,4-pentanedione to induce micronuclei in vivo depends on species and exposure route.

Justification for classification or non-classification

No classification is warranted for genetic toxicity of pentane-2,4-dione. The majority of studies in relevant test systems and the most relevant administration routes in vivo indicate a negative potential for genetic toxicity.