State Institution “Ukrainian Scientific and Research Institute of Industrial Medicine”, Kryvyi Rih, Ukraine
ABSTRACT. Objective. Based on the analysis and compilation of the literature data and own studies on toxicological characteristics of diisodecyl phthalate plasticizer, substantiation of its tentative safe exposure level in the air of the working zone was performed. Materials and Methods. Regulation of diisodecyl phthalate in the air of the working zone was performed based on the analytical review of the scientific publications, reference database of scientific libraries and PubMed text database of publication, as well as own results of studies of the toxicological properties of diisodecyl phthalate and its homologs under different ways of penetration of experimental animals in conditions of acute, subacute, subchronic, chronic experiments, carcinogenicity and mutagenicity, effect on reproductive function and embryogenesis, as well as investigation of its toxicodynamics and toxicokinetics was used. The resulting experimental findings were confirmed by the epidemiological studies.
Results. This work provides substantiation of tentative safe exposure level (TSEL) of diisodecyl phthalate in the air of the working zone.
Conclusion. For prevention of the possible negative effect on the body of workers, TSEL of diisodecyl phthalate in the air of working zone is 1.0 mg/m3, physical state — vapour + aerosol, with a mark + (Order of the Ministry of Health of Ukraine No. 1257 as of 13.10.2017 registered with the Ministry of Justice of Ukraine under No. 1353/21221 as of07.11.2017).
Key Words: TSEL, working zone, plasticizer, diisodecyl phthalate, phthalates, toxicity, reproductive toxicity, mutagenicity, carcinogenicity.
Introduction. Diisodecyl phthalate (DIDP) is used as the primary plasticizer of polyvinyl chloride and epoxide resins, in the manufacture of polyvinyl chloride plasticates, film and sheet material, synthetic leather, industrial rubber goods. DIDP is the main substitute of dioctylphthalate, its benefits are the following: stability, ability to provide finished products with wear resistance and high electroinsulating quality, as well as stability under high temperatures that is topical for the manufacture of cable plasticate.
Materials and methods. Regulation of DIDP in the air of working zone was performed based on the data analysis from literature on the study of toxic properties of substances and their homologues under different ways of penetration of experimental animals in conditions of acute, subacute, subchronic, chronic experiments, carcinogenicity and mutagenicity, effect on reproductive function and embryogenesis, as well as investigation of its toxicodynamics and toxicokinetics. Irritating action of DIDP on the skin and mucous membranes in the experiment was studied according to the methodology guidelines [1] with adherence to the international rules and bioethics standards.
According to the MG No. 4000-85 [3], justification of DIDP TSEL in the air of working zone was performed using interpolation and extrapolation in the range of substances close by chemical structure, physical and chemical properties, and the nature of biological action. Determination of the physical state of DIDP in the air of the working zone was performed according to the recommendations [4]. The resulting experimental findings were confirmed by the epidemiological studies.
Results and discussion. Among o-phthalic acid ethers, the closet rated homologues of DIDP in the air of working zone are: di-(2-ethylhexyl)-phthalate (DEHP), dinonyl phthalate (DnNP), didodecyl phthalate (DDP) used as the plasticizers in the manufacture of polyvinyl chlorides for provision of their elasticity, flexibility, transparency, strength, and durability. They have the following established MPC (maximum permissible concentration) in the air of working zone: DEHP — 1 mg/m3 (v+а) (vapour + aerosol), hazard class II; DnNP — 1 mg/m3 (v+а), hazard class II; DDP — 1 mg/m 3 (v+а), hazard class III according to GOST 12.1.005-88.
Structural formulas and physicochemical properties of phthalates are provided in Table 1 and 2. By appearance, DIDP and its rated homologues are viscous oily liquids with faint odour; they are colourless to yellow-brown. In the homologous range of o-phthalic acid ethers, boiling point increases with the increase of the molecular weight; density varies within 0.95 to 0.98 g/cm3 (20 °С). DIDP and its rated homologues are virtually insoluble in water. Distribution factor in n-octanol/water system increases with the increase of molecular weight of phthalates.
Table 1
Structural formulas of ortho-phthalic acid ethers
Table 2
Comparative characteristics of the physicochemical properties of phthalic acid ethers
Toxicity of phthalates for a living organism is stipulated by the number of carbon atoms in the alkyl group, depending on this, phthalates are divided into low-molecular (carbon content ≤6 atoms) and high-molecular phthalates, where a number of carbon atoms exceeds 6. Among rated DIDP homologues, DEHP belongs to the first group, and the second includes — DIDP, DnNP and DDP. The biological activity of phthalates is also directly depended on their solubility in water and inversely depends on the molecular weight.
Toxicological characteristics of DIDP is provided in [5]. According to the results of multiple studies, it has been established that DIDP has local irritating action on the skin of rabbits at the level of 1–2 points under different exposure; the reaction is reversible with the effect of peeling skin [6]. If other phthalates are applied to the skin of rabbits, irritating action is absent or mild [7, 8].
After application of phthalates in the conjunctival sac of the rabbit eye, hyperaemia of the mucous membranes is registered: at the level of 2 points for DIDP [6], 1–2 points for DEHP; reaction disappeared in 48–72 hours [7]. No pronounced signs of damage were found following contact of other phthalates on eye mucous membranes [8].
Acute toxicity of the above phthalates has been studied in different species upon different ways of contact (Table 3). As per experts’ assessment, phthalates are characterised by low oral toxicity; the highest toxicity was established for DnNP: LD50 for rats — 18,000 mg/kg. For other phthalates LD50 in rats is: DIDP — 64,000 mg/kg, DDP > 50,000 mg/kg, DEHP > 20,000 mg/kg [8–10]. Clinical manifestations of intoxication in rats under acute action of DEHP — a reduction of motor activity, no animal death is observed. Also, rat death was not detected at the dose of DIDP up to 29,100 mg/kg; body weight reduction and diarrhoea were registered in the animals. Species sensitivity in terms of mice and rats is low (species sensitivity factor is < 3) [8].
Table 3
Parameters of phthalate toxicity under conditions of acute action
Due to the high boiling point of phthalates and low volatility, there are difficulties in creating conditions for determining LC50. Animal death has not been established under the inhalation of DEHP in the concentration of 10.62 mg/L (the highest dose of aerosol formation from the technical point of view) during 4 days [7]. For DIDP inhalation in rats during 4 hours, LC50 exceeds 12.54 mg/L. In animals exposed to the inhalation of DEHP and DIDP, a similar pattern of intoxication was found: body weight reduction that during 14 days came up to control values; histological examination found inflammation foci in the lungs; pulmonary weight factor virtually did not differ from the control values [6].
In acute and repeated experiments, the ability of phthalates to penetrate the skin was found. Even high phthalate doses (3,000–10,000 mg/kg) did not result in the death of experimental animals, however, changes in the most sensitive parameters of the nervous system condition were registered. Following single DIDP application at the dose of 3,160 mg/kg during 24 hours on the rabbit skin, moderate skin resorptive action manifested as anorexia and moderate depression in all animals was observed. After termination of exposure to the substance, the condition of the animals became normalized, the autopsy did not show significant abnormal changes of the rabbit skin [9, 11]. Application of DnNP on the rat skin in the concentration of 5 mL/kg during 7 days led to the suppression in animals that suggests neurotoxicity of the substance [8].
Іcum for DEHP is calculated at the level of 2.3–2.7; there is no significant difference in the manifestations of cumulative effect depending on the initial fractionality of administration (1/5 LD50, 1/10 LD50, 1/ 20 LD50) suggesting its high hazard under chronic action. Іcum DnNP and DDP (10.0 and > 12, respectively) suggests their low cumulative ability [8].
According to the results of the acute action of phthalates, the highest sensitivity of the nervous system was found. Single action limit Lim ac by changes in the nervous system parameters (sub-threshold interval and behavioural reaction) is 105 mg/m3 for DEHP, 94 mg/m3 for DDP and 72 mg/m3 for DnNP. By the calculation method, Limch for DEHP was established at the level of 10.0 mg/m3, for DDP — 9.0 mg/m3, for DnNP — 8.0 mg/m3 [8].
By the results of Magnusson and Kligman test and Buehler test in guinea pigs, no signs of sensitization action of DEHP and DIDP were found. In the modified Buehler test, positive reaction to DIDP was found, and this is associated by experts with the effect of admixtures since DIDP composition has not been completely established. Patch tests in volunteers did not find positive reactions, and literature sources did not describe cases of sensitization action of DIDP in workers (only one case of dermatitis was reported), therefore, DIDP sensitization is unlikely [6, 7].
Following DEHP inhalation in rat body during 4 days, no negative effect on gonads and fertility of males was found, NOAEL is 1,000 mg/m3 [7]. Under inhalation exposure to DIDP in the concentration of 505 ± 7 mg/m 3 (6 hours daily, 5 times per week) during 2 weeks, no external signs of intoxication were reported. Histopathological changes were found in the lungs: increase in the width of alveolar septa, inflammatory processes of interstitial tissue were observed [6].
Subchronic toxicity studies of DEHP following oral administration with feed were conducted in rats, mice, rabbits, cats, monkeys; studies with DIDP were conducted in rats and dogs. DIDP has hepatotoxic action under conditions of subchronic toxicity. The most sensitive species of laboratory animals is dogs: oral NOAEL during 13 days is 15 mg/kg. For rats fed with DIDP during 90 days, NOAEL has been established at the level of 60 mg/kg (females), 200 mg/kg (males); limiting parameter — increase in the absolute liver weight (Table 4). Abnormal changes in the testicles, ovaries, liver and kidneys were not registered [6, 11].
Table 4
NOAEL of phthalates under conditions of subchronic and chronic action
When rats were fed with DEHP during 13 weeks, males showed dose-dependent gonadotoxicity: concentration of 37.6 mg/kg resulted in vacuolization of Sertoli cells (7/9) that is early morphological sign of testicle involvement, concentration of 375.2 mg/kg results in the reduction of the absolute and relative weight of testicles, increase in the rate of vacuolization of Sertoli cells (9/10), impaired spermatogenesis. Under the parameter of vacuolization of Sertoli cells, NOAEL was observed at the level of 3.7 mg/kg [7].
DEHP action under chronic exposure was studied in rats, mice, guinea pigs, dogs. Under the results of two-year studies, it has been established that DEHP has nephrotoxic action, and severity of symptoms is dose-dependent: fromthe increase of the absolute and relative weight of kidneys, mineralization of renal papilla to the development and progression of nephropathy upon the highest dose of 789 mg/kg for males and 938.5 mg/kg for females. NOAEL for males is 29 mg/kg and for females — 36 mg/kg (increase in the absolute and relative weight of kidneys) [7].
According to the results of multiple studies of different duration, it has been established that phthalates, in particular, DEHP, DnNP and DIDP induce proliferation of peroxisomes in the liver of experimental animals. Authors highlight higher hepatotoxicity of phthalates for rats and mice compared with other experimental animals due to the features of their metabolism. Among phthalates, the highest hepatotoxicity for rodent body has been established for DEHP, the effect is dose-dependent and manifests with hepatomegaly due to the proliferation of hepatocytes, increase in the number of peroxisomes, development of hepatocellular tumours [6, 7, 12].
The most severe reproductive toxicity among specified phthalates is typical for DEHP. In the test of two generations, dose-dependent gonadotoxicity was found: DEHP at the dose of 14 mg/kg results in testicle atrophy in rats, 359 mg/kg — in their complete atrophy and aspermia. It was proved that DEHP has anti-androgenic, anti-oestrogenic effects, impairs sexual differentiation, forms reproductive abnormalities, accelerates terms of sexual maturation in females. [7, 13-14]. Immature rats are more sensitive to DEHP compared with adult animals. For characterisation of DEHP risks, NOAEL was established at the level of 4.8 mg/kg (by the parameters of gonadotoxicity) [7].
According to the test results in two generations, the negative effect of DIDP on the indices of survival rate in both generations of rats was established; for risk assessment, NOAEL was established at the level of 33 mg/kg [6].
It was found that DIDP has embryotoxicity. Exposure of pregnant rats to DIDP from Day 6 to 15 of gestation induces development of skeletal abnormalities in foetuses (occurrence of cervical and lumbar embryonic ribs), NOAEL is 500 mg/kg [6].
The test of two generations established that DIDP has a negative effect on the number of normal sperm cells in rats expressed as a percentage ratio that in general does not affect animal fertility. Histological examinations did not find abnormal changes in the reproductive organs of mature rats of both genders (2-year studies) [15]. Comparative assessment of the reproductive toxicity of DEHP and DIDP showed that at the level of the same dose and similar study conditions, DEHP results in testicle atrophy in 2.5 % of cases, whereas DIDP did not show any histological changes in gonads. Under the parameter of body weight reduction in two generations, NOAEL is 253 mg/kg [6].
As opposed to DIDP, low DEHP doses show teratogenic action. When mice are fed with DEHP (doses of 0; 44; 91; 191; 293 mg/kg) during 0–17 days of gestation, this results in the development of skeletal, visceral, external developmental abnormalities, reduction of foetal weight, an increase of prenatal death. NOAEL for the maternal body and foetus is 44 mg/kg [7, 16].
Oestrogenic activity of DIDP has not been proved in vitro and in vivo, and study of the anti-androgenic activity of DIDP is ongoing in vitro [6, 9].
DIDP in the sufficient number of in vitro (Ames test with and without metabolic activation; mutation test in L5178Y mice lymphoma cells and Balb/c-3T3 cells with and without metabolic activation) and in vivo (micronuclear test with mice bone marrow) test did not show mutagenicity [6, 9].
Literature sources did not provide information on carcinogenic properties of DIDP under conditions of chronic action. Results of two-year studies have proved that DEHP is a hepatic carcinogen for mice and rats of both genders, it induces leukaemia (MCL) in male rats, and NOAEL for induction of hepatic tumours and MCL: in male rats was established at the level of 29 mg/kg. Scientists associate hepatic carcinogenicity of DEHP for rats and mice with activation of proliferation of peroxisomes (PPARα activation), the evidence is available that human are less sensitive to hepatotoxicity induced by peroxisome proliferation [7]. International Agency for Research on Cancer (IARC) has declared that DEHP belongs to the group B2 — probable carcinogen for human [17]. DIDP and other high-molecular phthalates are not classified by IARC.
Based on the obtained data, it was established that DIDP by the parameters of acute toxicity following oral, inhalation or skin exposure belongs to low-toxic substances. Considering clinical manifestations of intoxication, morphological changes in the internal organs and parameters studied in the chronic studies, biological action is similar for the entire homologous range of phthalates.
Among rated homologues of DIDP, close by chemical structure, physical and chemical properties and nature of biological action under conditions of subchronic and chronic exposure, DEHP has pronounced systemic and reproductive toxic properties on the animal body. No carcinogenicity, mutagenicity and reproductive toxicity were predominantly registered for DIDP, DnNP and DDP.
Considering physicochemical parameters, toxic properties of DIDP and comparing them with toxic parameters of the rated phthalate homologues under different ways of exposure to the animal body in the conditions of acute, subacute, subchronic and chronic experiments, when remote effects (mutagenic, carcinogenic, teratogenic, embryotoxic, effect on reproductive function) were studied, recommended TSEL in the working zone air is 1.0 mg/m3, physical state — vapours + aerosol, with a mark +.
This value of TSEL is confirmed by the results of epidemiological studies on the investigation of the effect of plasticizer on workers of different professions under conditions of polyvinyl chloride manufacturing industry. During the clinical examination of 54 workers (working concentrations of DIDP in the production premises are within 0.02 to 2 mg/m3), no signs of the negative effect on the respiratory and peripheral nervous system were found, except for detection of the mild amount of metabolic substances in urine [18].
Conclusion
1. DIDP ant its homologues by the parameters of acute toxicity belong to the low-toxic substances; they are characterised by the mild irritating effect on the mucous membranes and skin, and by the moderate skin resorptive action. DEHP and DIDP, as the majority of phthalates, have no sensitizing properties.
2. Among the homologous range of phthalates in the conditions of subchronic and chronic action, DEHP has pronounced toxic properties under long-term exposure to the animal body. Biological action of phthalates studied in the chronic experiments is equal to the entire homologous range, these substances are characterised by hepatotoxicity and nephrotoxicity.
3. Mutagenic, carcinogenic, teratogenic, embryotoxic activity and reproductive toxicity of DIDP is not limiting upon an assessment of its safety. Hepatotoxicity is the main factor in the nature of DIDP toxic action under long-term exposure on the body.
4. Based on interpolation and extrapolation in the range of phthalates close by chemical structure, chemical and physical properties and nature of biological action, justified and approved TSEL in the working zone air is 1.0 mg/m3, physical state - vapours + aerosol with a mark + (Order of the MoH of Ukraine No. 1257 as of 13.10.2017, registered with the Ministry of Justice of Ukraine under No. 1353/21221 as of 07.11.2017).
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