Tris(pentane-2,4-dionato-O,O')vanadium

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: peer-reviewed data

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

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

Test concentrations with justification for top dose:

0.3, 1.0, 3.0, 10, and 30 mg/plate. Test concentrations were chosen based on preliminary cytotoxicity testing in strain TA100 (data not given in publication).

Vehicle / solvent:

distilled water

Negative solvent / vehicle controls:

yes

Remarks:

water

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: without S9: 4-nitro-o-phenylenediamine (TA98, TA1538), with S9: 2-aminoanthracene (all 5 strains)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
DURATION: Exposure duration: 48-72 hours
NUMBER OF REPLICATIONS: 3/dose group/strain/treatment set

Evaluation criteria:

Number of revertant colonies.

Statistics:

Mean revertant colony count and standard deviation were determined for each dose point.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

the test substance was cytotoxic at 10 mg/plate to strain TA1538, and cytotoxic to all strains at 30 mg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

The acetylacetonate test substance was cytotoxic to all Salmonella strains, both in the absence and in the presence of S9 at 30 mg/plate, and also at 10 mg/plate to strain TA1538 (-S9). No dose-related increases in the number of mutant colonies were observed in Salmonella strains TA98, TA100, TA1535, TA1537, or TA1538, either in the absence or in the presence S9 metabolic activation system. Solvent and positive control cultures demonstrated appropriate activity in the test system.

Conclusions:

The study was conducted scientifically reasonable equivalent to OECD 471. Positive and negative controls gave the appropriate response. Hence, the results are considered sufficiently reliable to assess the mutagenic potential of the test item in bacteria.
The acetylacetonate test material was not mutagenic to Salmonella and E. Coli strains with and without metabolic activation.

Executive summary:

In a reverse gene mutation assay in bacteria, strains TA98, TA100, TA1535, TA 1537 and TA1538 of S. typhimurium were exposed to acetylacetonate at concentrations of 0.3, 1.0, 3.0, 10.0 and 30.0 mg/plate in the presence and absence of mammalian metabolic activation (Ames Test according to OECD 471). A dose range-finding study was conducted using tester strain TA100, and dose levels of the acetylacetonate test material ranging from 0.3 to 10 mg/plate were used. In the main study there were two treatment sets for each tester strain, with (+S9) and without (-S9) metabolic activation. Each of the tester strains was dosed with five concentrations of test substance, vehicle controls, and a positive control. Three plates/dose group/strain/treatment set were evaluated. Plates were incubated for 48 -72 hours at 37°C.

The acetylacetonate test material was cytotoxic to all tester strains at 30 mg/plate, and at 10 mg/plate to strain TA1538 in the absence of the metabolic activation system (-S9). No dose-related increase in the number of mutant colonies were observed in all tester strains, either in the absence or in the presence of S9 metabolic activation system. The positive control for each respective test strain exhibited at least a 3-fold increase (with or without S9) over the mean value of the vehicle control for a given strain, confirming the expected positive control response.

Therefore, the acetylacetonate test substance was considered to be non-mutagenic without and with S9 mix in the bacterial reverse mutation test.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: peer-reviewed data

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

not specified

Type of assay:

other: in vitro 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, obtained from Abraham Hsie, Oak Ridge, TN, USA.

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

0.01, 0.05, 0.1, 0.5, and 1.0 mg/ml with and without S9 metabolic activation system, and 1.5 mg/ml without S9 metabolic activation system.
Test concentration 1.0 mg/ml reduced relative survival to <10 % in the absence of S9 and to <50 % in the presence of S9.
The top dose 1.5 mg/ml was cytotoxic to CHO cells.

Vehicle / solvent:

distilled water

Untreated negative controls:

yes

Remarks:

medium

Negative solvent / vehicle controls:

yes

Remarks:

water

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

ethylmethanesulphonate

Details on test system and experimental conditions:

Cultured CHO cells (CHO-K1-BH4, Abraham Hsie, Oak Ridge, TN, USA) were treated in duplicate, both in the absence and in the presence of S9, with the vehicle control substance (water), an appropriate positive control substance, and various concentrations of the acetylacetonate test substance.

Evaluation criteria:

Number of mutant colonies/plate.

Statistics:

Data were analyzed by the method of Irr and Snee (1979, Proc. Cold Spring Harbor-Bamburg Conf. vol II, pp. 263-274) after Box-Cox transformation (Box & Cox, 1964, J. R. Stat. Soc. 26:211-252).

Key result

Species / strain:

Chinese hamster Ovary (CHO)

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:

Acetylacetonate treatment at 1.0 mg/ml reduced relative survival to <10 % in the absence of S9 and to <50 % in the presence of S9.
Corrected mutation frequencies were 2-3.9 x 10^-6 for solvent controls, 6.0 x 10^-6 for medium controls, and 0-17.3 x 10^-6 for acetylacetonate-treated cultures in the absence of S9. In the presence of S9, corrected mutation frequencies were 0-1.4 x 10^-6, 0, and 0-3.0 x 10^-6 for vehicle, medium, and acetylacetonate-treated cultures.
There was no significant increase in gene mutation in CHO cells treated with acetylacetonate, either in the absence or in the presence of S9.
Increased mutation frequencies were obtained in the test system on treatment with positive control substances.

Conclusions:

Interpretation of results: negative
The acetylacetonate test material was not genotoxic under the conditions of the test.

Executive summary:

In a mammalian cell gene mutation assay conducted according to OECD Guideline 476, cultured CHO cells were exposed to the test substance acetylacetonate at concentrations of  0.01, 0.05, 0.1, 0.5, 1.0 and 1.5 mg/ml in the presence and absence of a mammalian metabolic activation system. The positive controls induced the appropriate response.  There was no evidence of induced mutant colonies over background, therefore the acetylacetonate test material was not genotoxic under the conditions of the test.

Cross-reference 3

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

sister chromatid exchange

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: peer-reviewed data

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)

GLP compliance:

not specified

Type of assay:

sister chromatid exchange assay in mammalian cells

Specific details on test material used for the study:

no details given

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO-K1-BH4 cells were obtained from Abraham Hsie, Oak Ridge, TN, USA.

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

0.1, 0.2, 0.3 mg/L

Vehicle / solvent:

distilled water

Untreated negative controls:

yes

Remarks:

medium

Negative solvent / vehicle controls:

yes

Remarks:

water

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period:
- Exposure duration: 5 h (without S9), 2 h (with S9)
- Expression time (cells in growth medium): 24 - 28 h
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells): 28 - 33 (without S9), 31 - 36 (with S9)

SPINDLE INHIBITOR (cytogenetic assays): treatment with colchichin (0.5 µg/mL) for 2 - 3 h

STAIN (for cytogenetic assays): Hoechst 33258, Gurr's Giemsa

NUMBER OF REPLICATIONS: 2 replicates

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: cells were harvested, fixed, and spread on microscope slides. After staining with Hoechst 33258, slides were rinsed i n Sorenson's buffer and exposed to a sunlamp for 15-30 min. Slides were then stained in Gurr's Giemsa and coded to prevent scoring bias.

NUMBER OF CELLS EVALUATED: min 25 cells/culture

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): Only metaphases with 20 ± 2 chromosomes were scored

Rationale for test conditions:

Test concentrations were selected on the basis of a preliminary cytotoxicity study

Evaluation criteria:

A minimum of 25 cells/culture was scored. Only metaphases with 20 ± 2 chromosomes were scored. Centromeric switch of label was not scored as an SCE.

Statistics:

The SCE data were analyzed statistically after Box-Cox transformation (Box & Cox, 1964). Data for treatment and solvent control groups were intercompared for equality of variance by Levene's test, analysis of variance (ANOVA), and t tests. The t tests were used when the F value from 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 ANOVA for unequal variances, followed when necessary by a separate variance t test for pairwise comparisons; p < 0.05 (two-tailed) was used as the critical level of significance.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Significant increases in the frequencies of SCE were observed both in the absence and in the presence of S9 activation. In the absence of S9, the increases were 1.2-fold at the low dose (0.02 mg/ml), 1.4-fold at the middose (0.03 mg/ml), and 4.2-fold at the high dose (0.10 mg/ml). In the presence of S9 activation, the increases were 1.2-fold at the low dose (0.03 mg/ml), 1.3-fold at the mid-dose (0.10 mg/ml), and 1.5-fold at the high dose (0.3 mg/ml).

Conclusions:

Acetylacetone was positive in vitro in Sister Chromatide Exchange Assay in CHO cells.

Executive summary:

Acetylacetone was tested in Sister Chromatid Exchange Assay similar to OECD 479. Significant increases in the frequencies of SCE were observed both in the absence and in the presence of S9 activation. In the absence of S9, the increases were 1.2-fold at the low dose (0.02 mg/ml), 1.4-fold at the middose (0.03 mg/ml), and 4.2-fold at the high dose (0.10 mg/ml). In the presence of S9 activation, the increases were 1.2-fold at the low dose (0.03 mg/ml), 1.3-fold at the mid-dose (0.10 mg/ml), and 1.5-fold at the high dose (0.3 mg/ml).

Cross-reference 4

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

no

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

2,4-PD samples from several production lots were provided by Union Carbide Corporation (South Charleston, WV, USA) between 1985 and 1994 for use in these studies. Compositional analyses indicated that the samples were greater than 99 % pure.

Species:

other: Sprague-Dawley rats and ND4 Swiss Webster mice

Strain:

other: Sprague-Dawley rats and ND4 Swiss Webster mice

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: ND4 Swiss Webster mice were purchased from Harlan Sprague Dawley (Indianapolis, IN, USA) or Charles River Laboratories (Portage, MI, USA).
- Age at study initiation: 6 to 8 weeks (mice), 7 weeks (rats)
- Housing: The animals were housed individually in stainless steel, wire mesh cages
- Diet (e.g. ad libitum): pelleted, certified AGWAY® PRO LAB® animal Diet, Rat, Mouse, Hamster 3000 was available ad libitum, except during treatment.
- Water (e.g. ad libitum): tap water was available ad libitum, except during treatment.
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 25
- Humidity (%): 40 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: vapour

Vehicle:

- Vehicle(s)/solvent(s) used: air
- Justification for choice of solvent/vehicle: The 0 (control), 100, 400, and 600 ppm target concentrations were chosen because mortalities occurred at 800 ppm in the bone marrow chromosomal aberrations study in the rat.
- Concentration of test material in vehicle: 0 (control), 100, 400, 600 ppm

Details on exposure:

TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Stainless steel inhalation chambers with glass windows for animal observation were used.
Liquid 2,4-PD was metered from a piston pump (Fluid Metering, Oyster Bay, NY, USA) into a heated glass evaporator. The temperature in the evaporator was maintained at the level sufficient to vaporize the liquid 2,4-PD. The evaporator temperature ranged from 32-47°C. The oxygen content of each chamber was measured using an MSA Oxygen Indicator (Mine Safety Appliance, Pittsburgh, PA, USA).

TEST ATMOSPHERE
- Brief description of analytical method used: Each exposure chamber atmosphere was analyzed for 2,4-PD a minimum of once each hour by flame ionization gas chromatography. Chamber temperature and relative humidity were recorded approximately 2 times each hour using a Fisherbrand® dial type thermometer (Fisher Scientific, Pittsburgh, PA, USA) and an Airguide humidity indicator (Airguide Instrument, Chicago, IL, USA)

Duration of treatment / exposure:

6 h per day for 5 consecutive days

Frequency of treatment:

daily

Post exposure period:

Bone marrow was collected 24 hr after the final vapor

Dose / conc.:

0 ppm (nominal)

Remarks:

rats and mice

Dose / conc.:

100 ppm (nominal)

Remarks:

rats and mice

Dose / conc.:

400 ppm (nominal)

Remarks:

rats and mice

Dose / conc.:

600 ppm (nominal)

Remarks:

rats and mice

Dose / conc.:

800 ppm (nominal)

Remarks:

only rats

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

triethylenemelamine
- Route of administration: i.p.
- Doses / concentrations: 0.3 mg/kg

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

Rat bone marrow cells were passed over a cellulose column to remove nucleated cells. Bone marrow cells were pelleted by centrifugation of either the column eluate (rat) or the whole aspirated marrow sample (mouse). Bone marrow cells were smeared on a microscope slide and then stained with Gurr's R-66 Giemsa diluted in phosphate buffer. The proportion of PCE in a total of 1000 cells/animal was determined.

Evaluation criteria:

A minimum of 2000 PCE/animal was scored for the presence of micronuclei.

Statistics:

Micronucleus data were evaluated statistically using the Mann-Whitney U-test; p < 0.05 (two-tailed) was used as the critical level of significance.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

please refer to 'Any other information on results'

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

No significant differences i n the proportion of PCE were observed i n any exposure group. The mean PCE/1000 erythrocytes ranged from a low of 418.4 for the 400 ppm male mice to a high of 612.4 for the 400 ppm female mice. The mean PCE/1000 erythrocytes ranged from a low of 258.6 for the air-exposed male rats to a high of 420.4 for the 100 ppm female rats. The mean percentages of MNPCE in mice were 0.34 ± 0.20, 0.28 ± 0.18, 0.20 ± 0.10, 0.20 ± 0.16, and 2.66 ± 0.54 for males and 0.14 ± 0.11, 0.10 ± 0, 0.22 ± 0.08, 0.20 ± 0.14, and 3.18 ± 1.50 for females for the air-exposed, low, mid, high, and positive control groups. The mean percentages of MNPCE in rats were 0.30 ± 0.16, 0.38 ± 0.13, 0.24 ± 0.25, 0.46 ± 0.15, and 3.92 ± 1.50 for males and 0.34 ± 0.29, 0.24 ± 0.23,0.11 ± 0.07,0.20 ± 0.07, and 2.86 ± 1.20 for females for the air-exposed, low, mid, high, and positive control groups. 2,4-PD did not produce significant exposurerelated increases in the incidence of MNPCE in bone marrow samples taken 24 hr after the 5th day of exposure in either species.

Toxicity

mice

There were no noteworthy signs of toxicity at 100 or 400 ppm in either male or female mice. Of 5 female mice in the 600 ppm exposure group, 3 died on exposure days 3 or 4. Prior to death, hypoactivity, prostration gasping, slow respiration, and blepharospasm were observed in one or more of the mice. There were no significant body weight changes in mice during the study.

rats

There were no noteworthy clinical signs of toxicity in male or female rats exposed to 100 or 400 ppm 2,4-PD. Of 5 female rats in the 600 ppm group, 3 died on exposure days 2—4. Significant body weight losses were observed in male and female rats at 600 ppm. Females also had significant body weight losses at 400 ppm, whereas males had significantly lowered body weight gains.

Table 11 Effects of 2,4-PD on micronucleus frequencies in the bone marrow of Swiss Webster mice and Sprague Dawley® rats (whole body vapor exposure)

2,4-PD (ppm)	Sex	Mean PCE/1000 erythrocytes	#PCE evaluated	#MNPCE	Mean %MNPCE (±S.D.)
24-Hr sample—Mice
0	M	486.0±150.08	5,000	17	0.34±0.20
	F	559.8±54.22	5,000	7	0.14±0.11
100	M	518.6±110.42	5,000	14	0.28±0.18
	F	624.0 ±66.19	5,000	5	0.10±0
400	M	418.4±91.25	5,000	10	0.20±0.10
	F	612.4±63.28	5,000	11	0.22±0.08
600	M	433.0±106.56	5,000	10	0.20±0.16
	F	543.0±111.72	2,000	4	0.20±0.14
TEM^a(0.3mg/kg)	M	283.4±73.94	5,000	133	2.66± 0.54^b
	F	456.0±69.59	5,000	159	3.18±1.50^b
24-Hr sample—Rats
0	M	258.6±2.17	5,000	15	0.30±0.16
	F	319.4±69.31	5,000	17	0.34±0.29
100	M	379.4±99.60	5,000	19	0.38±0.13
	F	420.4±104.06	5,000	12	0.24±0.23
400	M	381.2±52.69	5,000	12	0.24±0.25
	F	248.8±118.07	4,536	5	0.11 ±0.07
600	M	419.6±95.66	5,000	23	0.46±0.15
	F	308.0±74.95	2,000	4	0.20±0
TEM^a(0.3mg/kg)	M	307.0 ±86.33	5,000	196	3.92± 1.50^b
	F	335.0±65.80	5,000	143	2.86± 1.20^b

^b significant at p < 0.01

^aTEM-triethylenemelamine

Conclusions:

2,4-pentanedione was not clastogenic in rat and mouse bone marrow cells in micronucleus assay after inhalation exposure for 5 days at 6h/day.

Executive summary:

The study was similar to OECD Guideline 474. When rats and mice were exposed to 2,4-PD vapour for 6 h/day for 5 consecutive days at target concentrations up to 600 ppm, there were no significant exposure-related increases in the incidences of micronucleated PCE in bone marrow samples taken 24 h after the 5th day of exposure in either species.

Data source

Reference

Reference Type:

publication

Title:

2,4-Pentanedione: Evaluation of the genotoxic potential in vitro and in vivo

Author:

Vergnes JS

Year:

2000

Bibliographic source:

Toxic Substance Mechanisms, 19:151-175

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Version / remarks:

According to OECD 475 Test substances are preferably administered as a single treatment. Test substances may also be administered as a split dose, i.e. two treatments on the same day separated by no more than a few hours, to facilitate administering a large volume of material. Other dose regimens should be scientifically justified. Here dose is administered daily for five consecutive days.

Deviations:

yes

Remarks:

please refer to 'Remarks'

GLP compliance:

no

Type of assay:

mammalian bone marrow chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Pentane-2,4-dione

EC Number:

204-634-0

EC Name:

Pentane-2,4-dione

Cas Number:

123-54-6

Molecular formula:

C5H8O2

IUPAC Name:

pentane-2,4-dione

Specific details on test material used for the study:

2,4-PD samples from several production lots were provided by Union Carbide Corporation (South Charleston, WV, USA) between 1985 and 1994 for use in these studies. Compositional analyses indicated that the samples were greater than 99 % pure.

Test animals

Species:

other: Sprague-Dawley rats and ND4 Swiss Webster mice

Strain:

other: Sprague-Dawley rats and ND4 Swiss Webster mice

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: ND4 Swiss Webster mice were purchased from Harlan Sprague Dawley (Indianapolis, IN, USA) or Charles River Laboratories (Portage, MI, USA).
- Age at study initiation: 6 to 8 weeks (mice), 7 weeks (rats)
- Assigned to test groups randomly: yes, under following basis: Only those animals with body weights ± 20 % of the population mean for each sex were included in the randomization.
- Housing: The animals were housed individually in stainless steel, wire mesh cages
- Diet (e.g. ad libitum): pelleted, certified AGWAY® PRO LAB® animal Diet, Rat, Mouse, Hamster 3000 was available ad libitum, except during treatment.
- Water (e.g. ad libitum): tap water was available ad libitum, except during treatment.
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 25
- Humidity (%): 40 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

inhalation: vapour

Vehicle:

- Vehicle(s)/solvent(s) used: air
- Justification for choice of solvent/vehicle: concentrations were chosen on the basis of previous acute toxicity (Ballantyne et aL, 1986) and repeated exposure studies (Dodd et al., 1986) in the rat.
- Concentration of test material in vehicle: 0 (control), 100, 400, 600 and 800 ppm
Due to unexpected mortalities in both male and female rats exposed at the 800 ppm target concentration, an additional target concentration of 600 ppm was added.

Details on exposure:

TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Stainless steel inhalation chambers with glass windows for animal observation were used.
Liquid 2,4-PD was metered from a piston pump (Fluid Metering, Oyster Bay, NY, USA) into a heated glass evaporator. The temperature in the evaporator was maintained at the level sufficient to vaporize the liquid 2,4-PD. The evaporator temperature ranged from 32-47°C. The oxygen content of each chamber was measured using an MSA Oxygen Indicator (Mine Safety Appliance, Pittsburgh, PA, USA).

TEST ATMOSPHERE
- Brief description of analytical method used: Each exposure chamber atmosphere was analyzed for 2,4-PD a minimum of once each hour by flame ionization gas chromatography. Chamber temperature and relative humidity were recorded approximately 2 times each hour using a Fisherbrand® dial type thermometer (Fisher Scientific, Pittsburgh, PA, USA) and an Airguide humidity indicator (Airguide Instrument, Chicago, IL, USA)

Duration of treatment / exposure:

6 h per day for 5 consecutive days

Frequency of treatment:

daily

Post exposure period:

6 h and 24 h

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm (nominal)

Remarks:

rats and mice

Doses / concentrations 2

Dose / conc.:

100 ppm (nominal)

Remarks:

rats and mice

Doses / concentrations 3

Dose / conc.:

400 ppm (nominal)

Remarks:

rats and mice

Doses / concentrations 4

Dose / conc.:

600 ppm (nominal)

Remarks:

rats and mice

Doses / concentrations 5

Dose / conc.:

800 ppm (nominal)

Remarks:

only rats

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: i.p.
- Doses / concentrations: 30 mg/kg bw

Examinations

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The 0 (control), 100, 400, and 800 ppm target concentrations were chosen on the basis of previous acute toxicity (Ballantyne et aL, 1986) and repeated exposure studies (Dodd et al., 1986) in the rat. Due to unexpected mortalities in both male and female rats exposed at the 800 ppm target concentration, an additional target concentration of 600 ppm was added.

DETAILS OF SLIDE PREPARATION: Bone marrow was flushed from a femur into a centrifuge tube containing 10-15 ml Hank's Balanced Salt Solution (pH 7.0). Cells were collected by centrifu-gation, fixed, spread on clean microscope slides, and stained with dilute Giemsa. Slides were coded to prevent scoring bias. Whenever possible, 50 metaphase cells/animal were scored.

METHOD OF ANALYSIS: Both chromosome and chromatid aberrations were scored, including breaks, fragments, rings, minutes, quadriradi-als, triradials, translocations, and dicentrics. Gaps were scored but not included as aberrations in calculations. Severely damaged cells (>10 breaks) and pulverized cells were recorded as severely damaged and scored as 10 breaks/cell without classifying the nature of the damage.

Statistics:

Chromosomal aberrations data were statistically evaluated using the Mann-Whitney U-test (Kliesch et al., 1981); p < 0.05 (two-tailed) was used as the critical level of significance.

Results and discussion

Test results

Test results 1

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Significant body weight losses were observed among the males exposed at 800 ppm and both males and females exposed at 600 ppm. Both males and females in the 400 ppm group had depressed body weight gains over the 5-day exposure period

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: rats

Test results 2

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: mice

Additional information on results:

rats
The only noteworthy clinical signs observed prior to death were ataxia and/or prostration. Significant body weight losses were observed among the males exposed at 800 ppm and both males and females exposed at 600 ppm. Both males and females in the 400 ppm group had depressed body weight gains over the 5-day exposure period.
2,4-PD did not produce significant exposure-related increases in the incidence of chromosomal aberrations in the bone marrow of Sprague Dawley® rats exposed, whole body, to 2,4-PD vapour 6 hr/day for 5 consecutive days at target concentrations of 0, 100, 400, and 800 ppm.
The mean percentages of aberrant cells were 1.6 ± 2.6, 5.2 ± 4.6, 1.2 ± 1.8, 2.4 ± 3.3, and 4.4 ± 2.6 for the air-exposed, 100, 400, 600, and 800 ppm males; and 2.4 ± 1.7, 1.6 ± 1.7, 2.0 ± 1.4, and 3.0 ± 3.3 for the air-exposed, 100, 400, and 600 ppm females sampled 6 hr after the 5th exposure. At the 24-hr sampling time, the mean percentages of aberrant cells were 7.6 ± 12.8, 4.0 ± 3.7, 2.0 ± 2.0, 0.4 ± 0.9, 1.2 ± 1.8, and 19.2 ± 3.0 for the air-exposed, 100, 400, 600, 800, and positive control males; and 2.4 ± 2.6, 2.4 ± 0.9, 1.1 ± 1.6, 1.6 ± 0.9, and 28.0 ± 7.9 for the air-exposed, 100, 400, 600, and positive control females.

mice
2,4-PD did not produce significant exposure-related increases in the incidence of chromosomal aberrations in the bone marrow of Swiss Webster mice exposed, whole body, to 2,4-PD vapor 6 hr/day for 5 consecutive days at target concentrations of 0, 100, 400, and 600 ppm. The mean percentages of aberrant cells were 3.6 ± 2.6, 1.6 ± 0.9, 2.0 ± 2.8, and 1.4 ± 2.2 for the air-exposed, 100, 400, 600, and 800 ppm males; and 0.8 ± 1.1, 0, 0.8 ± 1.1, and 0 for the air-exposed, 100, 400, and 600 ppm females sampled 6 hr after the 5th exposure. At the 24-hr sampling time, the mean percentages of aberrant cells were 1.2 ± 1.1, 1.6 ± 1.7, 0.4 ± 0.9, 1.1 ± 1.6, and 28.4 ± 13.0 for the air-exposed, 100, 400, 600, 800, and positive control males; and 2.0 ± 1.4, 0.4 ± 0.9, 1.2 ± 1.8, 1.0 ± 1.4, and 22.8 ± 10.4 for the air-exposed, 100, 400, 600, and positive control females.

Any other information on results incl. tables

Table 12 Effects of 2,4-PD on chromosomal aberration frequencies in the bone marrow of Sprague Dawley® rats (whole body vapour exposure)

2,4-PD (ppm)	Sex	# Cells evaluated	Total* aberrantcells	Mean % aberrant cells ±S.D.)
6-Hr sample
0	M	250	4	1.6±2.6
	F	250	6	2.4±1.7
100	M	250	13	5.2±4.6^c
	F	250	4	1.6±1.7
400	M	250	3	1.2±1.8
	F	250	5	2.0±1.4
600	M	250	6	2.4±3.3
	F	232	7	3.0±3.3
800^a	M	250	11	4.4±2.6
24-Hr sample
0	M	250	19	7.6±12.8
	F	250	6	2.4±2.6
100	M	250	10	4.0±3.7
	F	250	6	2.4±0.9
400	M	250	5	2.0±2.0
	F	228	2	1.1±1.6
600	M	250	1	0.4±0.9
	F	250	4	1.6±0.9
800^a	M	250	3	1.2±1.8
CP^b(30mg/kg)	M	250	48	19.2±3.0^d
	F	250	70	28.0± 7.9^d

^aTarget concentration was lowered to 650 ppm after 2nd exposure day due to unexpected mortalities.

^bCP = cyclophosphamide monohydrate.

^cSignificant at p<0.05.

^dSignificant at p <0.01.

Table 13 Effects of 2,4-PD on chromosomal aberration frequencies in the bone marrow of SwissWebster mice (whole body vapour exposure)

2,4-PD (ppm)	Sex	# Cells evaluated	Total # aberrant cells	Mean % aberrant cells (±S.D.)
6-Hr sample
0	M	250	9	3.6±2.6
	F	250	2	0.8±1.1
100	M	250	4	1.6±0.9
	F	250	0	0
400	M	250	5	2.0 ±2.8
	F	250	2	0.8±1.1
600	M	350	5	1.4±2.2
	F	100	0	0
24-Hr sample
0	M	250	3	1.2±1.1
	F	250	5	2.0±1.4
100	M	250	4	1.6±1.7
	F	250	1	0.4±0.9
400	M	250	1	0.4±0.9
	F	250	3	1.2±1.8
600	M	350	4	1.1 ±1.6
	F	100	1	1.0±1.4
CP^a(40mg/kg)	M	214	53	28.4± 13.0^b
	F	250	57	22.8± 10.4^b

^aCP=cyclophosphamidemonohydrate.

^bSignificant at p < 0.01.

Applicant's summary and conclusion

Conclusions:

2,4-pentanedione was not clastogenic in rat and mouse bone marrow cells in chromosome aberration assay after inhalation exposure for 5 days at 6h/day

Executive summary:

The study was similar to OECD Guideline 475. When rats and mice were exposed to 2,4-PD vapour for 6 h/day for 5 consecutive days at target concentrations up to 800 ppm, there were no significant exposure-related increases in the incidences of chromosomal aberrations in bone marrow samples taken 6 and 24 h after the 5th day of exposure in either species.