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In vitro studies

Gene mutation

Bacteria

DEHP and MEHP have been tested for mutagenic potential in a number of Ames-type assays. Unfortunately, several of these studies have been marred by inadequate reporting, for instance the omission of details of the types of tester strains used and phthalateconcentrations; many have only been published in abstractform.

In studies comparable to a guideline study, DEHP was tested inS. typhimurium(TA98, TA100, TA1535 and TA1537 or TA97, TA98, TA100 and TA1535 or TA97, TA98, TA100, TA102) with quantities of 100-10,000 μg DEHP/plate (purity > 99%), with and without metabolic activation with S9-mix from rat and/or hamster (Zeiger et al., 1982, 1985a, 1985b; Baker and Bonin, 1985). DEHP exhibited no mutagenicity in any of the tester strains neither with nor without metabolic activation.

In a study comparable to a guideline study, DEHP was tested inS. typhimurium(TA98, TA100, TA1535, TA1537, TA1538) at concentrations from 0.1-10 μl DEHP/plate (purity: 99.9%) (98-9,800 μg/plate) with and without metabolic activation with Aroclor-induced rat liver S9 (Kirby et al., 1983). No significant mutagenic activity was observed.

In a study comparable to a guideline study, DEHP was tested inS. typhimurium(TA97, TA98, TA100, TA102) with quantities of 100-5,000 μg DEHP/plate (purity > 99%), with and without metabolic activation with S9-mix from rat and hamster (Matsushima et al., 1985). DEHP exhibited no mutagenicity in any of the tester strains neither with nor without metabolic activation.

In a study comparable to a guideline study, DEHP was tested for the induction of bacterial mutation using five histidine auxotrophs ofSalmonella typhimurium(TA98, TA100, TA1535, TA1537, TA1538) according to the method of Ames (Rexroat and probst, 1985). The test was conducted at concentrations of 50-5000 µg/plate with and without metabolic activation using an S9 fraction prepared from the livers of Aroclor-1254-induced rats. N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG), 2-nitrofluorene (2NF) and 9-aminoacridine (9AmAc) served as the positive controls for the non-activated test, while 2-aminoanthracene (2AA) served as the positive control for the activated test. Either with or without metabolic activation no revertants were induced by treatment with DEHP.

DEHP and MEHP were tested inSalmonella typhimurium(TA98, TA100) andEscherichia colitrp- (uvr+, uvr-) at concentrations from 50 to 2,000 μg/plate with and without metabolic activation with rat S9-mix (Yoshikawa et al., 1983). DEHP and MEHP did not show any mutagenic activity but some lethality towards theS. typhimuriumstrains.

DEHP (0.5-1000 µg/ml) was tested in the Ames test onSalmonella typhimuriumTA7001, TA7002, TA7003, TA7004, TA7005, TA7006, and a mix of 7000-strains, TA1537, TA98 with metabolic activation (Gee et al., 1998). Each of theSalmonella typhimuriumstrains TA7001-7006 carry a target missense mutation in biosynthetic operon that reverts to phototrophy by base-substitution events unique to each strain. TA7001-3 detect point substitutions at A:T and TA7004-6 at G:C. TA98 and TA1537 detect frameshift mutations. DEHP exhibited no mutagenicity in any of the tester strains with metabolic activation.

The potential of Diethylhexylphthalate DEHP to induce reverse mutation inSalmonella typhimuriumwas evaluated in strain TA 102 (Jung et al., 1992). DEHP was tested in two independent experiments in three different laboratories, with and without a metabolic activation system. Bacteria cultures were exposed DEHP at five dose-levels (three plates/dose-level) up to 5000µg/plate in DMSO or ethanol. After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored. DEHP did not induce any noteworthy increase in the number of revertants, both with and without S9 mix. DEHP did not show any mutagenic activity in the bacterial reverse mutation test withSalmonella typhimurium.

In a study comparable to a guideline study, MEHP was tested inS. typhimurium(TA 1535, TA 1537, TA1538, TA 98 and TA 100) with quantities of 250-2,000 μg MEHP/plate (purity > 99%), with and without metabolic activation with S9-mix from rat (Ruddick et al., 1981). MEHP exhibited no mutagenicity in any of the tester strains neither with nor without metabolic activation.

DEHP was tested at concentrations of 0, 313, 625, 1250, 2500, 5000 μg/plate onSalmonella typhimuriumstrains: TA102 and TA2638 andEscherichia ColiWP2 uvrA/pKM101 and WP2/pKM101 with and without metabolic activation (Watanabe et al., 1998). DEHP exhibited no mutagenicity in any of the tester strains neither with nor without metabolic activation.

DEHP (purity: > 99%) was tested inSalmonella typhimuriumstrain TA102, a strain sensitive to mutations arising as a cause of oxidative DNA damage, in concentrations from 1.0 to 20.0 μmol/plate (391-7,812 μg/plate) in the presence of enzymes proposed to be responsible for the metabolic activation of DEHP (Schmezer et al., 1988). No mutagenic response was observed. Similarly, MEHP (purity not stated) was not mutagenic inS. typhimurium(TA100, TA102) when tested in concentrations from 0.16 to 1.25 μmol/plate (45-348 μg/plate) with and without metabolic activation with S9 (rat).

The mutagenicity of urine from rats treated with DEHP was examined (DiVincenzo et al., 1985). Male Sprague-Dawley rats were administered a daily dose of 2,000 mg/kg of DEHP in corn oil by gavage for 15 days. Urine was collected and pooled and tested undiluted by a direct plating procedure (0.02, 0.06, 0.20, 0.66, 2.0 ml urine per plate) inS. typhimurium(TA98, TA100, TA1535, TA1537, TA1538) with and without metabolic activation with rat S9-mix. There was no evidence that mutagenic substances were excreted in the urine following administration of DEHP.

Yeast

DEHP (purity: not stated) was tested inSaccharomyces cerevisiaefor point mutations in strains XV185-14C and RM52 using two different cell culture conditions at concentrations from 1,541 to 12,325 nl/ml (1,510-12,080 μg/ml) with and without metabolic activation with rat S9 (Mehta and van Borstel, 1985). DEHP induced point mutations his+with S9 in strain XV185-14C cultured in buffer pH 7.0 being significant only in the lowest and the highest concentration.

When cultured in YEPD medium pH 6.3, DEHP induced point mutations his+in strain RM52 (-S9), arg+/his+(+/-S9), trp+(+S9) in strain XV185-14C. Mutation frequencies were 2-fold increased but not dose-dependent.

DEHP was tested in theadeforward-mutation system inSchizosaccharomyces pombeP1 in concentrations from 369 to 5,870 μg/ml with and without metabolic activation with S9 (rat) (Loprieno et al., 1985). According to the authors, 3-fold increases in mutant frequency were obtained over 3 consecutive doses in the assay without metabolic activation, but in a second experiment this finding was not confirmed. The results were therefore regarded as equivocal.

DEHP was tested in a gene mutation test in the yeastSccharomyces cerevisiaeat doses of 0, 40, 200, 1000, 5000 µg/ml (Arni, 1985). DEHP induced mitotic gene conversion with and without activation at the concentration of 5000 µg/ml.

DEHP was tested for mitotic gene conversion, point mutation and mitotic segregation using the yeast Saccharomyces cerevisiae strain D7 at doses of 200, 500, 1000, 2000, 3000, 5000 µg/ml (Parry and Eckardt, 1985). No genotoxic effect has been observed with and without metabolic activation.

Mammalian cells

DEHP was tested in anin vitrogene mutation assay using mammalian cells cultures both in the absence and presence of metabolic activation (S9 mix), according to a protocol similar to the OECD n° 476 Guideline (Kirby et al., 1983). Mouse lymphoma L5178Y cells cultured in vitro were exposed to DEHP (99.9 %) at concentrations from 0.016 to 0.21 µg/ml (non-activated cultures) and from 0.089 to 1.2 µg/ml (activated cultures), in ethanol. Appropriate positive controls were used.After a 48 rest period, cells were then incubated mutagenicity evaluation with trifluorothymidine during 10-12 days.None of the cultures treated with DEHP at any dose-level, exhibited mutation frequencies that were significantly greater (two-fold greater than background) than that of the corresponding ethanol solvent control. The positive control chemicals, on the other hand, demonstrated significant increases in mutation frequencies for both S9 activated and non-activated cultures.DEHP was not mutagenic in the L5178Y TK+/- mammalian mutagenicity assay.

DEHP was tested for the ability to induce mammalian cell mutation in the L5178Y TK+/- mouse lymphoma cell forward-mutation assay with and without metabolic activation with rat S9 at concentrations from 10 to 400 μg/ml (-S9) or 1.0-80 μg/ml (+S9) (Oberly et al., 1985). A dose dependent decrease in values for total survival was obtained in the absence of S9 ranging from 5 to 73% and a 2-fold or greater increase in mutation frequency was seen at 2 dose levels, however, survival was only 12 and 5% at these dose levels. In the presence of S9, a dose-related decrease in total survival was observed ranging from 5 to 90% and a 2-fold or greater increase in mutant frequency was seen at three dose levels. According to the authors, DEHP was mutagenic in the assay with metabolic activation.

DEHP was tested in anin vitrogene mutation assay using mammalian cells cultures both in the absence and presence of metabolic activation (S9 mix), according to the a protocol similar to the OECD n° 476 Guideline (Myhr et al., 1985). Mouse lymphoma L5178Y cellsculturedin vitrowere exposed to DEHP in ethanol for 4 hours at concentrations between 125 and 5000 nl/ml in the presence and absence of metabolic activation. Appropriate positive controls were used and showed a statistical increase in mutant colonies. After a 48h rest period, cells were then incubated for 11-12 days for mutagenicity evaluation with trifluorothymidine 3µg/ml. The evaluation of DEHP should be based on soluble concentrations less than 25 nl/ml, and it was assumed from the current data that such treatments would be relatively non-toxic and not mutagenic. Under these experimental conditions, DEHP did not induce any increase in mutant colonies and is not considered as mutagenic.

DEHP was tested for mutagenicity in mouse lymphoma L5178Y and L5178Y clone 372+/+ cells at concentrations of 0, 78, 392, 1,962, 9,810μg/ml, with and without metabolic activation (Styles et al., 1985). DEHP did not induce any increase in mutant colonies and is not considered as mutagenic.

DEHP was tested for gene mutation in a human lymphoblasts TK6 and AHH-1 assay at concentrations of 0, 200, 250, 400, 600, 750, 800, 1,000 μg/ml with and without metabolic activation (Crespi et al., 1985). DEHP was found negative for gene-locus mutations.

DEHP (purity: 98%) was tested in mouse embryo cells Balb/c-3T3 for gene mutations at concentrations of 0, 79, 250, 791, 2,000, 7,910 nl/ml (77 -7,752 μg/ml) with metabolic activation (Matthews et al., 1985). DEHP induced greater than a 2-fold increase in the appearance of Oua R mutants at one intermediate dose. However, this activity was not statistically significant; and, therefore, its mutagenic activity in this study was classified as questionable.

DNA damages

Mammalian cells

DEHP (purity 99%) was tested for the capacity to enhance the adenovirus (SA7) transformation of Syrian hamster embryo cells (SHE) at concentrations from 0, 0.2, 0.3, 0.6, 1.3 and 2.6 mM (78-1,016 μg/ml) (Hatch and Anderson, 1985). No enhancement of virus transformation was observed in a first experiment. A second experiment detected activity at the two highest concentrations that was independent of cytotoxicity for significance.

The potential of DEHP to induce sister-chromatid exchanges in Chinese hamster ovary cells was evaluated with and without a metabolic activation system according a protocol similar to the OECD 479 (Gulati et al., 1985, 1989). Using the standard protocol, without delay in the cell harvest time, all results were negative. However, in the absence of S9, extending the harvest time to 40 hours to compensate for DEHP-induced cell cycle delay permitted the detection of a significant dose-related increase in SCE. The frequency of SCE/cell increased with increasing concentrations of DEHP over a range of 20-100µg/ml. The magnitude of the increase being between 30% to 40% above the solvent control (or about three induced SCE/cell). Although the effect was small, they were reproducible, dose-related, and statistically significant. The autors considered that the observed effects were not DEHP dependant.

DEHP was tested for genotoxicity in a sister chromatic exchange assay on chinese hamster ovarian cells at doses of 3.9, 19.5, 39, 195, 390, 1170, 2340, 3900 μg/ml (Douglas et al., 1985, 1986). No genotoxicity has been observed.

DEHP was tested for genotoxicity in a sister chromatic exchange assay on rat liver cells RL4 at doses of 0, 125, 250, 500, 1,000 μg/ml (Priston and Dean, 1985). No genotoxicity has been observed.

DEHP was tested at concentrations of 391, 1172, 3907 µg/ml for single strand breaks in rat primary hepatocytes (Bradley, 1985). No genotoxic activity was observed.

The ability of DEHP to induce DNA damage or repair was examined in rat and human hepatocytes in vitro (Butterworth et al., 1984). Unscheduled DNA synthesis was measured by incorporation of [3H]thymidine into primary hepatocyte cultures immediately isolated from hepatocyte cultures incubated directly with DEHP. DNA damage was measured by alkaline elution of cellular DNA from the same cultures. In vitro conditions were 0.1, 1.0 and 10.0 mM DEHP in the cultures for 18 h. Primary cultures of human hepatocytes were prepared from freshly discarded surgical material and exposed to the same concentration of DEHP. Concentrations up to 0.5 mM mono(2-ethylhexyl)phthalate, a principal metabolite of DEHP, were also examined in the human hepatocyte assay. No chemically induced DNA damage or repair was observed in vitro in rat or human hepatocytes under any of the conditions employed. However, an increase in the percentage of cells in S-phase in the animals given DEHP was observed. These data indicate that DEHP does not exhibit direct genotoxic activity in the animals even with a treatment regimen which eventually produced tumors in a long term bioassay, and that both rat and human hepatocytes are similar in their lack of a genotoxic response to DEHP exposure in culture.

DEHP was tested for induction of unscheduled DNA synthesis (UDS) in primary rat hepatocytes at concentrations from 0.01 to 10 mM DEHP (purity: 99.8%) (Kornbrust et al., 1984). Incubation of DEHP with rat hepatocytes did not produce any evidence of unscheduled DNA synthesis, as assessed by the autoradiographic determination of [3H]thymidine incorporated during the incubation.

DEHP was tested for the induction of unscheduled DNA synthesis in cultured hepatocytes (Probst and Hill, 1985). Primary cultures of adult rat hepatocytes were incubated for 20 h with 8 concentrations (0.19, 0.39, 1.95, 3.9, 19.5, 39, 195, 390, 1,950, 3,900 μg/ml). N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and 2-acetylaminofluorene (2AAF) were also tested and served as the positive controls. Unscheduled DNA synthesis was measured by autoradiography, and the study was replicated using 2 independent hepatocyte preparations. A concentration-dependent induction of DNA-repair synthesis was observed in hepatocytes exposed to the carcinogens MNNG and 2AAF. No induction of DNA-repair synthesis was observed in any of the cultures treated with DEHP.

DEHP was tested for the induction of unscheduled DNA synthesis in cultured hepatocytes (Williams et al., 1985). Primary cultures of adult rat hepatocytes were incubated for 18-20 h with 6 concentrations (0.1, 1, 10, 100, 1,000, 10,000 μg/ml). Benzo(a)pyrene was also tested and served as the positive controls. Unscheduled DNA synthesis was measured by autoradiography. A concentration-dependent induction of DNA-repair synthesis was observed in hepatocytes exposed to benzo(a)pyrene. No induction of DNA-repair synthesis was observed in any of the cultures treated with DEHP.

Chromosomal effects

Yeast

DEHP was tested at doses of 0, 3000, 10000, 30000, 100000, 200000 μg/ml for mitotic recombination inSaccharomyces cerevisiae(Carls and Schiestl, 1994). No genotoxic effect has been observed.

Mammalian cells

DEHP was tested in anin vitromicronucleus test on Chinese hamster V79 cells in compliance with the Principles of Good Laboratory Practice (von der Hude et al., 2000). Cells were treated with and without metabolic activation at doses of 0.1, 0.25, 1, 2.5, 10, 25, 100, 250, 1000 µg/ml. In the absence of S9 the cells were incubated for 24 h, and for 3 h in the presence of S9. Thereafter cells were washed, fixed and Giemsa stained. Appropriate negative and positive control substances were tested. DEHP was tested in 3 laboratories. The common result was that DEHP did not increase the incidence of micronuclei, neither in the presence nor in the absence of metabolic activation. DEHP was not considered to be genotoxic in the Chinese hamster V79in-vitromicronucleus assay.

The potential of DEHP to induce structural chromosome aberrations in Chinese hamster ovary cells was evaluated with and without a metabolic activation system according a protocol similar to the OECD 473 (Gulati et al., 1985, 1989). Cells were treated with 0, 5, 16, 50, 600, 1600, 2000, 3000, 4000, 5000 µg DEHP/ml for about 10-11 h and colcemid was added 2-3 hr prior to tell harvest by mitotic shake-off. The chromosome number was recorded for each cell and chromosome or chromatid type aberrations were classified into three categories: simple (breaks, fragments, double minutes), complex (interchanges, rearrangements), and other (pulverized, more than ten aberrations/cell). DEHP did not induce any increase in the number of cells with structural chromosome aberration, both with and without S9 mix.

DEHP (purity: not stated) was tested in a primary liver cell line (CH1-L) from Chinese hamster at concentrations up to 50 μl/ml (49 mg/ml) without metabolic activation (Parry et al., 1984). DEHP produced increases in hyperploidy cells (chromosome number > 22) but was negative for hypoploidy and polyploidy cells.

DEHP (purity > 99%) was tested in primary liver cells (CH1-L) from Chinese hamster at concentrations from 5 to 50 mg/ml (Parry, 1985). The mitotic index was reduced at the top dose, the AT/M ratio was reduced in a dose related manner, and the chromosome cluster group and the abnormal division stage was increased by treatment. According to the author, these observations indicate a positive spindle effect. There was no effect on chromosome dislocation, multipolar spindles, or chromosome lagging and bridge formation.

DEHP was tested in a cytogenetic assay in Chinese hamster lung fibroblasts (CHL) (Ishidate and Sofuni, 1985). The cells were treated for 24 and 48 h with 1375, 2750, 4130 µg DEHP/ml with and without metabolic activation. DEHP did not show any cytotoxicity at even the maximum dose used here, and this finding may relate to the insolubility of this chemical. DEHP showed negative results on structural and numerical chromosome aberrations.

DEHP was tested in micronucleus assay on primary cultures of rat hepatocytes (Müller-Tegethoff et al., 1995). The cells were treated for 48 h with 0.039, 0.39, 3.9, 39, 390, 3900 µg DEHP/ml. DEHP had no influence on the incidence of micronuclei. No cytotoxicity was noted in tested concentration range.

In vivo studies

Gene mutation:

Male and femalelacZ-plasmid based transgenic mice were treated at 4 months of age with 6 doses of 2333 mg DEHP per kg over a two-week period (Boerrigter, 2004). Control animals were treated with the vehicle only (35% propyl glycol). The mutant frequency in liver, kidney and spleen DNA was determined as the proportion of retrieved mutant and wild-typelacZplasmids expressed in Escherichia Coli C host cells employing a positive selection system for mutant plasmids. Exposure to DEHP significantly increased the mutant frequency in liver, but not in kidney or spleen, of both female and male mice. The results indicate that some peroxisome proliferators display an organ-specific mutagenicity inlacZplasmid-based transgenic mice consistent with historical observations of organ- and compound-specific carcinogenicity.

In vivomutagenicity and mutation spectra of DEHP was investigated in guanine phosphoribosyltransferase (gpt) delta transgenic rats (Kanki et al., 2005). After 13-wk treatment, in the DEHP-treated rats (12000 ppm in diet), marked hepatomegaly with centrilobular hypertrophy of hepatocytes occurred, although GST-P staining was consistently negative. (MFs) in the liver DNA were 188.0 x 10e-6 and 56.5 x 10e-6, approximately 35-fold and 10-fold higher, respectively, than that of non-treatment control rats (5.5 x 10e-6). There were no increases in mutant frequencies in the DEHP-treated rats as compared to the non-treatment control value. These data provided support for the conclusion that DEHP exerts its influence via a nongenotoxic promotional pathway.

DNA damage

To evaluate the relationship between hydrogen peroxide generation and subsequent DNA damage caused by peroxisome proliferation, DNA damage and changes in peroxisomal ß-oxidation activity in rat liver were examined (Tamura et al., 1991). In male F-344 rats (4 rats/group) fed 2% DEHP in the diet for 52 or 78 weeks, hepatocarcinomas or neoplastic nodules were seen in 1/4 or 2/4 rats, respectively. The hepatic DNA from tumour-bearing rats showed a 5-fold increase in single strand DNA breaks whereas no increase was observed in non tumour-bearing rats. According to the authors, the results show that although prolonged treatment with peroxisome proliferators induces markedly peroxisomal ß-oxidation activity, the active oxygen species from peroxisomal ß-oxidation are not enough to give rise to significant DNA damage.

To elucidate the relationship between hepatic peroxisome proliferation and oxidative DNA damage induced by hepatocarcinogenic peroxisome proliferators, DEHP were fed at doses of 1.2% to male F-344 rats for up to 1 year (Takagi et al., 1990). Evidence of hepatic peroxisome proliferation and 8-hydroxydeoxyguanosine (8-OH-dG) formation in liver and kidney DNA were assessed at 1, 2, 3, 6, 9 and 12 months. Peroxisomal (beta-oxidation enzyme activities were increased 3- to 8-fold and catalase was elevated to 1.4- to 2.2-fold the control level by DEHP from months 1 to 12 of the treatment. 8-OH-dG levels in liver DNA of DEHP-fed rats were increased approximately 2-fold after 1 month, the tendency for elevation also being observed in the liner DNA at 2, 3, 9 and 12 months. The results thus clearly demonstrate that persistent peroxisome proliferation in the liver leads to continued specific oxidative DNA damage.

The ability of DEHP to induce DNA damage or repair was examined in rat hepatocytes in vivo (Butterworth et al., 1984). Unscheduled DNA synthesis was measured by incorporation of [3H]thymidine into primary hepatocyte cultures immediately isolated from treated animals. DNA damage was measured by alkaline elution of cellular DNA from the same cultures. In vivo-in vitro treatment regimens were: (i) female rats, 12 000 p.p.m. DEHP in the diet for 30 days; (ii) female rats, 12 000 p.p.m. in the diet for 30 days, followed by 500 mg/kg DEHP by gavage 2 h before sacrifice; (iii) male rats, 500 mg/kg DEHP by gavage 2, 12, 24, or 48 h before sacrifice; and (iv) male rats, 150 mg/kg/day by gavage for 14 days. No chemically induced DNA damage or repair was observed in vivo in rat hepatocytes under any of the conditions employed. However, an increase in the percentage of cells in S-phase in the animals given DEHP was observed. These data indicate that DEHP does not exhibit ,direct genotoxic activity in the animals even with a treatment regimen which eventually produced tumors in a long term bioassay, and that both rat and human hepatocytes are similar in their lack of a genotoxic response to DEHP exposure in culture.

DEHP was tested for mutagenic potential in an UDS assay in vivo. Sprague Dawley rats were treated daily by gavage during 4 or 8 weeks at 5000mg/kg or were fed in diet at 2% during 4 or 8 weeks followed by a single gavage of 5000mg/kg (Kornbrust et al., 1984). In vivo administration of 5 g DEHP/kg body weight by gavage did not elicit DNA repair in hepatocytes, isolated 2, 15, or 24 h following the administration of DEHP. When rats were treated with DEHP at a dosed-feed level of 2% for 4 or 8 wk and administered a dose of 5 g DEHP/kg body weight by gavage 15 h prior to isolation of hepatocytes, DNA repair was still not detected. DNA repair was not induced in hepatocytes isolated from treated rats.

Genotoxicity as DNA repair or unscheduled DNA synthesis (UDS) and cell replication as the percentage of cells undergoing scheduled DNA synthesis (SDS or S phase) were determined in mouse hepatocytes in vivo in response to DEHP (Smith-Oliver and Butterworth, 1987). UDS and SDS were determined bv autoradiographic quantitation of [3H]-thymidine incorporation in primary hepatocyte cultures isolated from B6C3F1 male mice treated in vivo. No DNA repair was observed in cultures from mice treated with up to 500 mg/kg DEHP 12, 24 or 48 h previously or from animals treated up to 28 days with 6000 ppm DEHP in the diet. At 24 h following treatment with 500 mg/kg DEHP, 3.1% of the hepatocytes were in S phase compared to control values of 0.27%. Administration of DEHP in the diet at 6000 ppm produced 9.2% of the cells in S phase at day 7 with the value returning to control levels by day 14. On day 28 of the feeding study the liver to body weight ratios had almost doubled in the group treated with DEHP compared to controls. No increase in the liver-specific enzyme alanine aminotransferase was seen in the serum following treatment with 500 mg/kg DEHP, indicating that the hyperplasia was due to mitogenic stimulation rather than regenerative hyperplasia in response to cytotoxicity.

8-OH-dG levels in liver DNA of DEHP-fed rats were increased approximately 2-fold after 1 month, the tendency for elevation also being observed in the liner DNA at 2, 3, 9 and 12 months. The results thus clearly demonstrate that persistent peroxisome proliferation in the liver leads to continued specific oxidative DNA damage.

Male mice were exposed to 0, 2000 or 8000 mg DEHP/kg bw 3 times a week for 8 weeks and then mated with untreated females. Additional groups were investigated after 4 weeks of exposure or 8 weeks of exposure plus 4 weeks recovery. Exposure did not cause DNA damage in the testis (Comet Assay) of parental animals or offspring (Dobrzynska et al., 2012).

Dominant lethal mutations

Male mice were exposed to 0, 2000 or 8000 mg DEHP/kg bw 3 times a week for 8 weeks and then mated with untreated females. Additional groups were investigated after 4 weeks of exposure or 8 weeks of exposure plus 4 weeks recovery. Exposure resulted in a slight increase in dominant lethal mutations (0, 6 and 13 % DLM at 0, 2000 and 8000 mg/kg, respectively). The number of fertile males and pregnant females was reduced in the treatment groups, however this effect was not dose dependent. DEHP treatment did not cause consistent effects on body weight, but resulted in decreased testis and epididymidis weight as well as decreased sperm count and sperm motility (only at 8000 mg/kg) and increased number of abnormal spermatozoa. The only effects observed in male F1 offspring were an increased number of skeletal malformations and a delay in fur development in the high dose group (Dobrzynska et al., 2012). Validity of these findings is limited, as effects were observed at doses which already induced general toxicity. Furthermore no positive control was investigated.

Chromosomal effects

Drosophila

DEHP was tested for mutagenicity in the sex-linked recessive lethal (SLRL) mutation assay after a single injection of 0 or 20 ppm to Drosophila melanogaster larvae (Yoon et al., 1985). Adult males emerging from the treatment (18600 ppm in diet) were mated at approximately 24 hr of age with two successive harems of three to five Basc females to establish two single-day broods. The percentage of lethals was 3% compared to 5% in the control group. No genotoxic effect has been observed.

DEHP was tested for mutagenicity in the sex-linked recessive lethal (SLRL) mutation assay after being fed toDrosophila melanogasterlarvae (Zimmering et al., 1989). Adult males emerging from the treatment (18600 ppm in diet) were mated at approximately 24 hr of age with two successive harems of three to five Basc females to establish two single-day broods. Males were then discarded, and the conventional SLRL assay carried out. The percentage of lethals was 7% compared to 11% in the control group. No genotoxic effect has been observed.

Mammals

In a study performed according to GLP principles, DEHP, MEHP and 2-EH were tested for their ability to induce chromosomal damage in male Fischer rats after oral administration (Putman et al., 1983). Five rats per group were given by gavage in corn oil 0.5, 1.7, 5.0 ml/kg/day of DEHP (purity: 99.9%), 0.01, 0.05, 0.14 ml/kg/day of MEHP (purity: 94.7%), or 0.02, 0.07, 0.21 ml/kg/day of 2-EH (purity: 99.7%) for 5 consecutive days. A positive control group was included. No significant increase in chromatid and chromosome breaks or structural rearrangements were noted and the mitotic index was also unaffected.

DEHP was tested in a Mammalian Erythrocyte Micronucleus Test, according to the OECD n° 474 Guideline and in compliance with the Principles of Good Laboratory Practice (Morita et al., 1997). Groups of 6 males CD-1 mouse were treated twice intraperitoneally with DEHP (purity >98%) at doses of 0, 500, 1000, 2000 mg/kg.  One group of 6 males received the vehicle under the same experimental conditions, and acted as control group. One group of 6 males received the positive control substance (mitomycin C 0.5 mg/kg) once by intraperitoneal route. For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 1000 polychromatic erythrocytes. Mitomycin C induced a highly significant increase in the frequency of MPE, indicating the sensitivity of the test system under our experimental conditions. At the high dose (2000mg/kg), the mortality was 1/6. DEHP did not induce any noteworthy increase in the number of micronucleated with structural chromosome aberration, both with and without S9 mix, in any experiment or at any harvest time. In conclusion, DEHP did not induce an increase in micronucleus mouse bone marrow when tested at concentrations up to 2000 mg/kg.

DEHP was tested for mutagenicity in anin vivomicronucleus test on mouse. Groups of 15 mice were injected intraperitoneously DEHP at concentrations of 600, 3000 and 6000 mg/kg and peripherical blood was examined for micronucleus immediately after the last injection, two and four we'eks after the last injection (Douglas et al., 1986). The positive control showed a significantly elevated number of micronuclei on the day following the last treatment but levels returned to control values by the second week. No increase in micronuclei was detected at any of the doses or times following DEHP exposure, nor with the solvent or untouched control.

Simultaneous liver and peripheral blood micronucleus assays in young rats was performed with DEHP. DEHP was administered by gavage at dose levels of 0, 1000 and 2000 mg/kg bw to groups of 4 rats (Suzuki et al., 2005).Positive control animals received DEN at 40 mg/kg (liver micronucleus assay) or CP at 10 mg/kg (peripheral blood micronucleus assay). DEHP was negative for micronucleus induction in both liver and peripheral blood.

Other in vitro and in vivo studies related to mutagenicity and/or carcinogenicity

DEHP (purity not stated) was tested inSaccharomyces cerevisiaefor gene conversion in strain D7-144 using two different cell culture conditions at concentrations from 1,541 to 12,325 nl/ml (1,510-12,080 μg/ml) with and without metabolic activation with rat S9 (Mehta and van Borstel, 1985). When cultured in YEPD medium pH 6.3, DEHP induced gene conversion with and without metabolic activation but not when cultured in buffer pH 7.0. According to the authors, overall test results were positive.

DEHP was examined for activity in the C3H/10T½ murine fibroblast cell transformation system in concentrations from 0 to 100 μM DEHP (purity: 99.8%) (0-39 μg/ml) (Sanchez et al., 1987). DEHP did not produce cell transformation, initiate transformation in cultures treated with a tumour promoter or promote transformation in cultures pre-treated with a chemical carcinogen.

DEHP was evaluated in the Syrian hamster embryo (SHE) cell transformation assay in three different laboratories using the same basic experimental protocol with minor modifications at concentrations from 13 to 4,000 μg/ml (Jones et al., 1988). In one laboratory, DEHP induced a high level of transformation in two assays but gave only one transformed colony at a single dose (> 1,000 μg/ml) in a third assay. In a second laboratory, transformation was observed generally at concentrations above 1,000 μg/ml. In a third laboratory, a low number of transformed colonies were observed in two assays. It was concluded by the authors that DEHP was positive for induction of morphological transformation in this assay.

DEHP (purity > 99%) induced morphological cell transformations when evaluated in the Syrian hamster embryo (SHE) cell transformation assay at concentrations from 0.8 to 300 μg/ml (Sanner and Rivedal, 1985).

Short description of key information:

The possible genotoxic effect of DEHP has been thoroughly investigated in several different short-term tests. Most of the studies are performed according to GLP principles and are comparable to guideline studies.

The results have been negative in the majority of the in vitro and in vivo studies on DEHP for detection of gene mutation, DNA damage, and chromosomal effects. The more conclusive positive results were obtained on cell transformation, induction of aneuploidy and cell proliferation. These test systems are, however, also sensitive to several non-genotoxic substances such as tumour promoters and/or peroxisome proliferators. Taken together all the results, both negative and positive, DEHP is considered to be non-genotoxic

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

According to the criteria edicted in REGULATION (EC) No 1272/2008 OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 16 December 2008, no classification is warranted for germ cell mutagen.