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Additional information

In Vitro-Experiments:

Bacterial test in vitro

The mutagenicity of 2,4-pentanedione was investigated in a standard AMES test usingS. typhimuriumstrains TA98, 100, 1535, 1537 and 1538 both in the presence and absence of a metabolic activation system (liver S9 mix produced from rats pretreated with Aroclor 1254). The test was conducted according to currently valid guidelines. The test material did not produce a statistically significant increase in the revertants / plate (less than doubling) thereby demonstrating no potential of 2,4-pentanedione to induce gene mutations (Union Carbide Corp. Bushy Run Research Center 1985).

Another AMES test was conducted inS. typhimuriumstrains TA92, 98, 100 and 104 in the absence of a metabolic activation system. Water or DMSO served as solvent (negative) controls, potassium dichromate (10μg/plate), methylmethansulfonate (2μg/plate) and hycantone (20μg/plate) served as positive controls. No information was given as to the concentration ranges of 2,4-pentanedione used in strains TA92, 98 and 100 where no mutagenic effects were reported. Test concentrations in the strain TA104 were 1.9 – 48μmol/plate, substance was added in water or DMSO (not specified in the reference available) in a volume of 0.1 ml. According to the results observed and considering the rates of spontaneous revertants for this particular strain (400 – 700 revertants), 2,4-pentanedione has to be considered only slightly mutagenic in TA104 at concentrations ranging from 1.9 – 10μmol/plate, were the number of revertants/plate increased to its maximum of 1500, which is in contrast to the evaluation of the authors classifying the substance as “strongly mutagenic”. At concentrations > 10μmol/plate, no significant increase in the number of revertants compared to control values could be observed. Thus the increase of revertants/plate was not in a dose-response relationship (Gava et al. 1989).

Non-bacterial test(s) in vitro

The genotoxicity of 2,4-pentanedione was studied in a series of in vitro assays using mammalian cells (CHO cells) and investigating different endpoints such as sister chromatid exchanges (SCE), chromosomal aberrations (CA) and gene mutations (HGPRT-test). All tests performed correspond to current valid methodologies assessing the genotoxic potential of substances. In the SCE-assay 2,4-pentanedione produced a statistically significant increase in the number of SCE/cell at concentrations not causing overt cytotoxicity both in the presence and absence of a metabolic activation system (liver S9 mix produced from rats pretreated with Aroclor 1254). Highest test substance concentrations employed in this test were 0.1 and 0.3 mg/ml and exposure times were 5 h and 2 h in the absence and presence of a metabolic activation system, respectively. Additionally, it was found that 2,4-pentanedione was considered genotoxic particularly in the absence of metabolic activation since the magnitude of SCE induction was lower when activation by S9 mix was included (Union Carbide Corp. Bushy Run Research Center 1986).

In a forward-gene-mutation assay (HGPRT-test) 2,4-pentanedione did not cause any statistically significant increases in the incidence of mutations in CHO-cells at the HGPRT-locus both in the presence and absence of a metabolic activation system (liver S9 mix produced from rats pretreated with Aroclor 1254). The highest substance concentrations in this test were 1.5 and 1.0 mg/ml in the absence and presence of metabolic activation, respectively. The exposure time was 5 h with and without activation followed by a 9 – 12 days subculturing period to allow expression of the mutant phenotype. In addition, it was demonstrated that random cultures with increased mutant values were within the typical range of variability (Union Carbide Corp. Bushy Run Research Center 1986d). The ability of 2,4-pentanedione to induce chromosomal aberrations was investigated in cytogenetic tests using CHO-cells both in the presence and absence of a metabolic activation system (liver S9 mix produced from rats pretreated with Aroclor 1254). In the first study maximum substance concentrations employed were 0.03 mg/ml and 0.1 mg/ml with and without activation and did not cause excessive cytotoxicity or inhibition of mitotic cells. Cells were exposed to the test substance for 2 h with and 6 or 10 h without activation. Results obtained in this study were inconsistent and characterised by small increases in chromosomal aberrations (simple chromatid breakage) with and without activation by liver S9 mix (Union Carbide Corp. Bushy Run Research Center 1986).

Since the test substance could not definitively be classified as a clastogen a second study (repeat study) was performed for clarification. The highest test substance concentrations in the second investigation were 0.12 mg/ml and 0.14 mg/ml in the absence and presence of metabolic activation and did not produce excessive cytotoxicity. Exposure times of cells towards test substance were 2 h and 6 h with and without metabolic activation, respectively. In the absence of S9 activation a test substance related increase in the number of chromosomal aberration was observable as indicated by chromosome breakage while in the presence of activation thereby mimicking more physiological and realistic conditions no clastogenicity was found under the conditions of the experimental procedure (Union Carbide Corp. Bushy Run Research Center 1986).

In Vivo-Experiments:

2,4-pentanedione was studied for its potential to produce chromosomal aberrations and micronuclei in male and female ND4 Swiss Webster mice as well as male and female Sprague-Dawley rats after inhalation.

Male and female Swiss Webster mice were exposed to 0 (10 animals per sex), 100 (10 animals per sex), 400 (10 animals per sex) and 600 ppm (14 animals per sex) of 2,4-pentanedione vapour for five consecutive days, 6 h/day by whole body exposure. These concentrations correspond to 0; 417; 1,668 and 2,502 mg/m3. The highest dose of 600 ppm corresponded to about 50 % of the LC50- value determined in acute inhalation toxicity studies in rats (Union Carbide Corp. Bushy Run1984). Air-only controls (10 animals per sex) and a well established positive control (i.p. administration of cyclophosphamide monohydrate, five animals per sex) was included in this assay. Bone marrow from 2,4-pentanedione and air-control treated animals was collected 6 h and 24 h after the end of the exposure period while bone marrow from positive controls was collected after 24 h only. Colchicine was dosed by intraperitoneal injection (4 mg/kg) two to three hours prior to sacrifice. In the 600 ppm exposure groups only two females assigned to the bone marrow collection times of 6 h and 24 h survived until the scheduled sacrifice. Signs of toxicity in female animals of the 600 ppm exposure group were characterised by prostration. In all other doses groups no treatment related adverse effects were found. It could be demonstrated that 2,4- pentanedione did not increase the number of chromosomal aberrations in a statistically significant manner. Therefore, the material is not considered to be clastogenic under the conditions of the inhalative in vivo assay (Union Carbide Corp. Bushy Run Research Center 1994).

Ten animals (Sprague-Dawley rats) per sex and dose group were exposed to 0, 100, 400 ppm (corresponding to 0; 417 and 1,668 mg/m3) 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 (corresponding to 3,336 mg/m3). The doses were chosen based on the results of previous acute and repeated exposure studies. For positive control Cyclophosphamide was administered as a single injection to 5 male and 5 female rats. 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 (2,711 mg/m3) for the surviving male rats. Eleven out of the fourteen female rats exposed to 2,4-pentanedione at 800 ppm died after the second exposure and the three remaining moribund female rats were euthanized. An additional target concentration of 600 ppm (2,502 mg/m3) was added to the study and was administered to both male and female rats by whole body exposure to vapour 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 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. 2,4-Pentanedione produced one statistically significant increase in the incidence of chromosomal aberrations in 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. No statistically significant or concentration-related increases in the incidence of chromosomal aberrations were observed among 2,4-PD-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-PD was not considered to have biologically significant clastogenic activity in rats under the conditions of this test by the authors of the report (Union Carbide Corp. Bushy Run Research Center 1990).

When male and female Swiss Webster mice as well as male and female Sprague-Dawley rats (5/sex at the groups for air-only control, positive control, 100 ppm, 400 ppm, 600 ppm) were exposed to 2,4-pentanedione vapour under identical conditions (exposure concentrations 0, 100, 400 and 600 ppm; corresponding to 0; 417; 1,668 and 2,502 mg/m³, respectively; exposure period 5 days, 6 h/d) and bone marrow was collected 24 h after final exposure and examined for the formation of micronucleated polychromatic erythrocytes (PCEs) no statistically significant increases in the incidence of micronucleated PCEs could be found in any of the dose groups administered. In the highest exposure concentration of 600 ppm 3/5 female mice and 3/5 female rats died and substance related effects in this dose group were evident as hypoactivity, prostration, urogenital wetness, gasping, slow respiration and blepharospasm (Union Carbide Corp. Bushy Run Research Center 1993).

The potential of 2,4-pentanedione to induce micronuclei was investigated in male and female Swiss Webster mice after i.p. administration. Five mice per sex and dose group were used, doses administered were 0, 200, 400 and 650 mg/kg bw corresponding to 25, 50, and 80 % of the i.p. LD50, respectively. A negative (water) and a positive control (triethylenemelamine) was included in this assay. Blood samples were taken 30, 48 and 72 h after treatment with 2,4-pentanedione for the evaluation of micronucleated PCEs while blood samples from positive controls animals were subjected to PCE analysis after 30 h only. At 30 and 48 h, respectively, a statistically significant increase in the number of micronucleated PCEs was detectable in a dose dependent manner while the number of PCEs with micronuclei was not different from controls in the 72 h blood samples. Regardless of dose and time of blood collection no influence on the PCE/NCE ratio was observable while a significant decrease of the PCE/NCE ratio was found in the positive controls. In conclusion 2,4-pentanedione induces micronuclei in mice of both sexes after administration by the i.p. route (Union Carbide Corp.Bushy Run Research Center 1986).

The capability of 2,4-pentanedione to induce micronuclei was investigated in male and female Sprague-Dawley rats after i.p. administration, too. On the basis of the description available, the study was conducted according to current guidelines. Five animals per sex and dose were used in this study and a total of five dose groups (50, 100, 200, 400 and 650 mg/kg bw, corresponding to 6.5, 13, 26, 52, and 86 % of the oral LD50, respectively) was administered to the animals as a single i.p. injection. The two lowest dose groups of 50 and 100 mg/kg were included because of mortalities in the 400 and 650 mg/kg dose groups. Substance related signs of toxicity in the 400 and 650 mg/kg groups included hypoactivity, incoordination, prostration, whole body tremor, tonic convulsions, excessive vocalization, urogenital area wetness, labored respiration, gasping, perinasal and perioral wetness, nasal discharge, periocular encrustation and lacrimation. In the other dose groups no (50 and 100 mg/kg bw) or less pronounced (200 mg/kg bw) signs of toxicity were reported. Following a single administration by i.p. injection 2,4-pentanedione did not produce statistically significant, treatment related increases in the incidence of micronucleated polychromatic erythrocytes in male and female Sprague-Dawley rats as assessed at 6, 24 and 48 hours.

The capability of 2,4-pentanedione to induce germ cell mutations was studied in a dominant lethal assay in male F344 rats by the inhalation route of exposure. Dose ranges included 0, 100, 400 and 700 ppm (corresponding to 0; 417; 1,668 and 2,919 mg/m3), respectively, and 20 animals per dose group were exposed to test substance vapour for 5 consecutive days, 6 h/d. After the last exposure treated males were paired with naive females (two females with one male) of the same strain for eight consecutive weeks and observed for evidence of copulation. Females without evidence of breeding (copulation plug or vaginal smear) were removed and replaced weekly. After eight weeks brains, testes as well as thymus of males were removed for histopathological examination. Males exposed to 400 and 700 ppm test substance showed reduced body weights at week 1 and only males of the highest exposure group still showed reduced body weights one week after termination of exposure. Due to stress by inhalation exposure weight loss was evident in animals of all dose groups. No treatment related clinical signs of toxicity and no microscopic lesions were found on histological examination of brain, testes and thymus in any of the exposure groups. Signs of toxicity were restricted to males of the 700 ppm only and included aggression, red ocular discharge and red perioral encrustation. Reproductive parameters for males and females were affected only on week 3 where the number of pregnant females was slightly reduced at 400 and 700 ppm resulting in a lowering of the female fertility index. Gestational parameters were affected on weeks 2 and 4 of mating and characterised by a reduction of the corpora lutea per dam in week 2 and a reduction in the number of total and viable implants per dam both in week 2 and 4 at 700 ppm. In week 2 postimplantation loss was slightly but not statistically significantly increased at 400 and 700 ppm and preimplantation loss was significantly increased in week 4. Although there was weak statistical significance of the 700 ppm value, the very high s.d. in both cases indicates high variability of the data from individual animals. A clear evaluation of substance related dominant lethal effects is not possible on the basis of the results of the study (Tyl et al. 1989, Union Carbide Corp. Bushy1986). In a mouse spermatogonial assay 2,4-pentanedione was administered in deionised water to 6 male NMRI mice at a dose of 800 mg/kg bw and spermatogonial cells of 5 animals/dose prepared 24 and 48 hours after administration. The dose selected was close to the MTD as shown in a preceding range-finding test and caused signs of toxicity such as reduction of spontaneous activity, eyelid closure, apathy and tremor. The bioavailability of the material was ensured in a preceding study as well. The vehicle (deionised water) and adriblastin served as negative and positive controls, respectively. 100 cells per animal (i.e. 500 cells per time point) were examined for chromosomal aberrations. Neither a reduction in mitotic indices nor an increase in the number of numerical or structural chromosomal aberrations were detectable in the substance treated group when compared with vehicle treated controls while pronounced effects were caused by adriblastin. 2,4-pentanedione is considered non-clastogenic to germ cells under the conditions of the assay (RCC-CCR 2000) [modified from OECD SIDS Dossier]

In an oral administration test ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 6000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes.The following dose levels of the test item were investigated:Females: 24 h preparation interval: 125, 250, and 500 mg/kg b.w.; 48 h preparation interval: 500 mg/kg b.w.. Males: 24 h preparation interval: 250, 500, and 1000 mg/kg b.w.; 48 h preparation interval: 1000 mg/kg b.w.The highest doses (500 and 1000 mg/kg b.w. for females and males, respectively) was estimated by a pre-experiment to be suitable.The mean number of polychromatic erythrocytes was not decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control indicating that Acetylacetone did not have any cytotoxic properties in the bone marrow.

The initial analysis of the induced micronucleus frequencies showed an unclear picture. For this purpose the number of scored PCEs was increased to 6000 per animal. This, however, did not resolve the situation. In case of the females there was not any biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used. For the males a statistically significant increase of the micronucleus frequency (0.24%) was observed after treatment with the high dose at the 24 h preparation interval. This increase was also slightly above the historical control range (0.23%). This increase was not linked to a dose dependency. The values obtained for the low and mid dose were 0.21 and 0.15%. Obviously the females could not contribute to confirm the relevance of the effect, since they could only be treated with maximally half of the high dose used for the males. Since the increase was not clearly beyond the historical control range and a dose dependency was not observed the biological relevance of the effect observed in the males can not be addressed.

The test item 2,4-Pentanedione was assessed in the in vivoalkaline single cell gel electrophoresis analysis for its potential to induce primary DNA damage in cells prepared from the liver and small intestine treated rats. The doses to be applied in the main experiment were chosen as result of four preexperiments. The high dose was chosen in a way that clinical signs of toxicity such asruffled fur, reduced spontaneous activity, abdominal position, eyelid closure and were observed. In the next hgher dose (1000 mg/kg b.w.) the animals showed exceedingly strong clinical symptoms. In the main experiment application doses of 400 and 800 mg/kg b.w. were tested. Seven male rats (Wistar HsdCpb: WU) per group were treated twice orally with a dose of 400 and 800 mg 2,4-Pentanedione /kg b.w.. The test item was formulated in water and applicated 24 or 4 hours prior to preparation of the cells. In each experimental group including positive and solvent controls hepatocytes and cells form small intestine were assessed for the occurrence of DNA damage. For each dose group and at each treatment time 100 cells were evaluated in the COMET assay for primary DNA damage in terms of tail intensity. Compared to the corresponding vehicle controls none of the tested dose levels revealed a biologically relevant increase in DNA damage in one of the cell types. The mean tail intensities of the individual animals of 2,4-Pentanedione treated rats were within (cells of the small intestine) or close to (hepatocytes) the range of mean tail intensities of the corresponding vehicle controls. For liver and small intestine the vehicle control was in the range to ensure a valid performance of the study. An appropriate reference mutagen (MMS, 25 mg/kg b.w. oral) was used as a positive control. Treatment with the positive control substance led to a distinct and statistically significant increase of DNA damage as detected in tail intensity analysis. In conclusion, under the experimental conditions reported, oral administration of 400 and 800 mg/kg b.w. 2,4-Pentanedione did not induce any DNA-damage in thein vivoComet assay performed in liver cells and cells from the small intestine isolated from male rates. The rates were treated twice orally, 24 and 4 hours before preparation. Therefore, 2,4-Pentanedione is considered to be non-genotoxic in this in vivoalkaline single cell gel electrophoresis assay for liver cells and cells from the small intestine.


Short description of key information:
In Vitro-Experiments:
2,4-pentanedione was not mutagenic in bacterial test systems (except slightly mutagenic effect in Salmonella typhimurium TA104) and in mammalian test systems in vitro. It showed a weak clastogenic activity in mammalian cells in vitro in the absence of metabolic activation, but not in the presence of metabolic activation.
In Vivo-Experiments:
For the inhalation route no genotoxic effects were observed. In contrast, after intraperitoneal administration no consistent genotoxic responses were observable with the rat showing no genotoxicity while the results with mice were positive. After oral administration the results are inconclusive.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

For the inhalation route no genotoxic effects were observed. In contrast, after intraperitoneal administration no consistent genotoxic responses were observable with the rat showing no genotoxicity while the results with mice were positive. The results after oral administration with mice are inconclusive concerning the question of mutagenicity. This might be the result of a different metabolization of 2,4 -Pentanedione in the mice in contrast to the rat. As the IP route is an unphysiological one and the oral route did not show a clear result the substance is not classified as to the endpoint of genotoxicity.